The pathophysiology of Parkinson’s disease (PD) involves gene-environment interactions that impair various cellular processes including mitochondrial dysfunction. Mitochondria-associated mutations increase PD risk, respiration is altered in the PD brain, and mitochondria-damaging toxicants cause PD-like motor and gastrointestinal symptoms in animal models. The gut microbiome is altered in PD, representing an environmental risk, however a relationship between mitochondrial function and the microbiome in PD has not been previously established. Herein, we discover that dysregulation of mitochondria-associated genes and hyperactive striatal mitochondria are induced by the microbiome in α-synuclein-overexpressing (Thy1-ASO) mice. Thy1-ASO mice elaborate increased reactive oxygen species in the striatum whereas germ-free counterparts express increased oxygen scavenging proteins. Indeed, treatment with an antioxidant drug improves motor performance in Thy1-ASO mice and blocking oxidant scavenging in germ-free mice enhances motor deficits in an α-synuclein dependent manner. Thus, the gut microbiome promotes motor symptoms in a mouse model of PD via increased mitochondrial respiration and oxidative stress in the brain.
", "doi": "10.1038/s41531-025-01142-5", "pmcid": "PMC12537952", "issn": "2373-8057", "publisher": "Nature Publishing Group", "publication": "npj Parkinson's Disease", "publication_date": "2025-10-20", "volume": "11", "pages": "301" }, { "id": "authors:sgvtb-faf72", "collection": "authors", "collection_id": "sgvtb-faf72", "cite_using_url": "https://authors.library.caltech.edu/records/sgvtb-faf72", "type": "article", "title": "Spatial genomics of AAV vectors reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo", "author": [ { "family_name": "Coughlin", "given_name": "Gerard M.", "orcid": "0000-0003-0644-4721", "clpid": "Coughlin-Gerard-M" }, { "family_name": "Borsos", "given_name": "M\u00e1t\u00e9", "orcid": "0000-0002-2801-8910", "clpid": "Borsos-M\u00e1t\u00e9" }, { "family_name": "Barcelona", "given_name": "Bre'Anna H.", "orcid": "0009-0007-1897-1461", "clpid": "Barcelona-Bre'Anna-H" }, { "family_name": "Appling", "given_name": "Nathan", "orcid": "0009-0007-9488-6621" }, { "family_name": "Mayfield", "given_name": "Acacia M. H.", "orcid": "0000-0001-7308-6480", "clpid": "Mayfield-Acacia-M-H" }, { "family_name": "Mackey", "given_name": "Elisha D.", "clpid": "Mackey-Elisha-D" }, { "family_name": "Eser", "given_name": "Rana A.", "orcid": "0000-0003-4078-1502", "clpid": "Eser-Rana-A" }, { "family_name": "Jackson", "given_name": "Cameron R.", "clpid": "Jackson-Cameron-R" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Kumar", "given_name": "Sripriya Ravindra", "orcid": "0000-0001-6033-7631", "clpid": "Kumar-Sripriya-Ravindra" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Cell-type-specific regulatory elements such as enhancers can direct expression of recombinant adeno-associated viruses (AAVs) to specific cell types, but this approach is limited by the relatively small packaging capacity of AAVs. In this study, we used spatial genomics to show that transcriptional crosstalk between individual AAV genomes provides a general method for cell-type-specific expression of large cargo by separating distally acting regulatory elements into a second AAV genome. We identified and profiled transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. We developed spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue, and we demonstrate here that transcriptional crosstalk is dependent upon concatemer formation. Finally, we leveraged transcriptional crosstalk to drive expression of a 3.2-kb Cas9 cargo in a cell-type-specific manner with systemically administered engineered AAVs, and we demonstrate AAV-delivered, minimally invasive, cell-type-specific gene editing in wild-type mice that recapitulates known disease phenotypes.
", "doi": "10.1038/s41587-025-02565-4", "issn": "1087-0156", "publisher": "Nature Publishing Group", "publication": "Nature Biotechnology", "publication_date": "2025-03-20", "volume": "43" }, { "id": "authors:mbrmj-h6e60", "collection": "authors", "collection_id": "mbrmj-h6e60", "cite_using_url": "https://authors.library.caltech.edu/records/mbrmj-h6e60", "type": "article", "title": "Peripheral neuronal activation shapes the microbiome and alters gut physiology", "author": [ { "family_name": "Griffiths", "given_name": "Jessica A.", "orcid": "0000-0002-5586-1567", "clpid": "Griffiths-Jessica-A" }, { "family_name": "Yoo", "given_name": "Bryan B.", "orcid": "0000-0003-1450-2696", "clpid": "Yoo-Bryan-B" }, { "family_name": "Thuy-Boun", "given_name": "Peter", "orcid": "0000-0003-3285-4273", "clpid": "Thuy-Boun-Peter" }, { "family_name": "Cantu", "given_name": "Victor J.", "clpid": "Cantu-Victor-J" }, { "family_name": "Weldon", "given_name": "Kelly C.", "orcid": "0000-0003-1064-8153", "clpid": "Weldon-Kelly-C" }, { "family_name": "Challis", "given_name": "Collin", "orcid": "0000-0003-4716-6086", "clpid": "Challis-Collin" }, { "family_name": "Sweredoski", "given_name": "Michael J.", "orcid": "0000-0003-0878-3831", "clpid": "Sweredoski-Michael-J" }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Thron", "given_name": "Taren M.", "orcid": "0000-0001-9577-2617", "clpid": "Thron-Taren-M" }, { "family_name": "Sharon", "given_name": "Gil", "orcid": "0000-0002-4605-9943", "clpid": "Sharon-Gil" }, { "family_name": "Moradian", "given_name": "Annie", "orcid": "0000-0002-0407-2031", "clpid": "Moradian-Annie" }, { "family_name": "Humphrey", "given_name": "Gregory" }, { "family_name": "Zhu", "given_name": "Qiyun", "orcid": "0000-0002-3568-6271", "clpid": "Zhu-Qiyun" }, { "family_name": "Shaffer", "given_name": "Justin P.", "orcid": "0000-0002-9371-6336", "clpid": "Shaffer-Justin-P" }, { "family_name": "Wolan", "given_name": "Dennis W.", "orcid": "0000-0001-9879-8353", "clpid": "Wolan-Dennis-W" }, { "family_name": "Dorrestein", "given_name": "Pieter C.", "orcid": "0000-0002-3003-1030", "clpid": "Dorrestein-Pieter-C" }, { "family_name": "Knight", "given_name": "Rob", "orcid": "0000-0002-0975-9019" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "abstract": "The gastrointestinal (GI) tract is innervated by intrinsic neurons of the enteric nervous system (ENS) and extrinsic neurons of the central nervous system and peripheral ganglia. The GI tract also harbors a diverse microbiome, but interactions between the ENS and the microbiome remain poorly understood. Here, we activate choline acetyltransferase (ChAT)-expressing or tyrosine hydroxylase (TH)-expressing gut-associated neurons in mice to determine effects on intestinal microbial communities and their metabolites as well as on host physiology. The resulting multi-omics datasets support broad roles for discrete peripheral neuronal subtypes in shaping microbiome structure, including modulating bile acid profiles and fungal colonization. Physiologically, activation of either ChAT+ or TH+ neurons increases fecal output, while only ChAT+ activation results in increased colonic contractility and diarrhea-like fluid secretion. These findings suggest that specific subsets of peripherally activated neurons differentially regulate the gut microbiome and GI physiology in mice without involvement of signals from the brain.
\nDeep-learning-based methods for protein structure prediction have achieved unprecedented accuracy, yet their utility in the engineering of protein-based binders remains constrained due to a gap between the ability to predict the structures of candidate proteins and the ability toprioritize proteins by their potential to bind to a target. To bridge this gap, we introduce Automated Pairwise Peptide-Receptor Analysis for Screening Engineered proteins (APPRAISE), a method for predicting the target-binding propensity of engineered proteins. After generating structural models of engineered proteins competing for binding to a target using an established structure prediction tool such as AlphaFold-Multimer or ESMFold, APPRAISE performs a rapid (under 1 CPU second per model) scoring analysis that takes into account biophysical and geometrical constraints. As proof-of-concept cases, we demonstrate that APPRAISE can accurately classify receptor-dependent vs. receptor-independent adeno-associated viral vectors and diverse classes of engineered proteins such as miniproteins targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, nanobodies targeting a G-protein-coupled receptor, and peptides that specifically bind to transferrin receptor or programmed death-ligand 1 (PD-L1). APPRAISE is accessible through a web-based notebook interface using Google Colaboratory (https://tiny.cc/APPRAISE). With its accuracy, interpretability, and generalizability, APPRAISE promises to expand the utility of protein structure prediction and accelerate protein engineering for biomedical applications.
\nThe circadian rhythm pacemaker, the suprachiasmatic nucleus (SCN), mediates light entrainment via vasoactive intestinal peptide (VIP) neurons (SCN^(VIP)). Yet, how these neurons uniquely respond and connect to intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing melanopsin (Opn4) has not been determined functionally in freely behaving animals. To address this, we first used monosynaptic tracing from SCN^(VIP) neurons in mice and identified two SCN^(VIP) subpopulations. Second, we recorded calcium changes in response to ambient light, at both bulk and single-cell levels, and found two unique activity patterns in response to high- and low-intensity blue light. The activity patterns of both subpopulations could be manipulated by application of an Opn4 antagonist. These results suggest that the two SCN^(VIP) subpopulations connect to two types of Opn4-expressing ipRGCs, likely M1 and M2, but only one is responsive to red light. These findings have important implications for our basic understanding of non\u2013image-forming circadian light processing.
", "doi": "10.1016/j.isci.2023.107865", "pmcid": "PMC10520357", "issn": "2589-0042", "publisher": "Cell Press", "publication": "iScience", "publication_date": "2023-10-20", "series_number": "10", "volume": "26", "issue": "10", "pages": "107865" }, { "id": "authors:6qa0d-19h32", "collection": "authors", "collection_id": "6qa0d-19h32", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230725-857203000.51", "type": "article", "title": "Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain", "author": [ { "family_name": "Chuapoco", "given_name": "Miguel R.", "orcid": "0000-0001-5397-996X", "clpid": "Chuapoco-Miguel-R" }, { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-Nicholas-C" }, { "family_name": "Goeden", "given_name": "Nick", "clpid": "Goeden-Nicholas" }, { "family_name": "Octeau", "given_name": "J. Christopher", "orcid": "0000-0001-6462-9439" }, { "family_name": "Roxas", "given_name": "Kristina M." }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Scherrer", "given_name": "Jon", "orcid": "0009-0008-8040-4860" }, { "family_name": "Winchester", "given_name": "Janet", "orcid": "0000-0002-8521-9153" }, { "family_name": "Blackburn", "given_name": "Roy J." }, { "family_name": "Campos", "given_name": "Lillian J.", "orcid": "0000-0003-0839-0288", "clpid": "Campos-Lillian-J" }, { "family_name": "Man", "given_name": "Kwun Nok Mimi", "orcid": "0000-0002-0132-9129" }, { "family_name": "Sun", "given_name": "Junqing" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Lefevre", "given_name": "Arthur", "orcid": "0000-0003-2847-0859" }, { "family_name": "Singh", "given_name": "Vikram Pal" }, { "family_name": "Arokiaraj", "given_name": "Cynthia M.", "orcid": "0000-0003-3201-9868", "clpid": "Arokiaraj-Cynthia-M" }, { "family_name": "Shay", "given_name": "Timothy F.", "orcid": "0000-0001-6591-3271", "clpid": "Shay-Timothy-F" }, { "family_name": "Vendemiatti", "given_name": "Julia", "orcid": "0000-0002-6547-9601", "clpid": "Vendemiatti-Julia" }, { "family_name": "Jang", "given_name": "Min J.", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Mich", "given_name": "John K.", "orcid": "0000-0002-1626-1139" }, { "family_name": "Bishaw", "given_name": "Yemeserach" }, { "family_name": "Gore", "given_name": "Bryan B.", "orcid": "0000-0003-1721-4235" }, { "family_name": "Omstead", "given_name": "Victoria", "orcid": "0000-0002-3796-970X", "clpid": "Omstead-Victoria" }, { "family_name": "Taskin", "given_name": "Naz", "orcid": "0009-0003-2417-8428" }, { "family_name": "Weed", "given_name": "Natalie", "orcid": "0000-0003-0891-0327", "clpid": "Weed-Natalie" }, { "family_name": "Levi", "given_name": "Boaz P.", "orcid": "0000-0002-8346-872X" }, { "family_name": "Ting", "given_name": "Jonathan T.", "orcid": "0000-0001-8266-0392" }, { "family_name": "Miller", "given_name": "Cory T.", "orcid": "0000-0001-8345-2720", "clpid": "Miller-Cory-T" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-Benjamin-E" }, { "family_name": "Pickel", "given_name": "James", "orcid": "0000-0002-3617-3072" }, { "family_name": "Tian", "given_name": "Lin", "orcid": "0000-0001-7012-6926" }, { "family_name": "Fox", "given_name": "Andrew S.", "orcid": "0000-0003-0695-3323", "clpid": "Fox-Andrew-S" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Crossing the blood\u2013brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood\u2013brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.", "doi": "10.1038/s41565-023-01419-x", "pmcid": "PMC10575780", "issn": "1748-3387", "publisher": "Nature Publishing Group", "publication": "Nature Nanotechnology", "publication_date": "2023-10", "series_number": "10", "volume": "18", "issue": "10", "pages": "1241-1251" }, { "id": "authors:vytfy-emg42", "collection": "authors", "collection_id": "vytfy-emg42", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230301-701033500.22", "type": "article", "title": "Spatial transcriptomics for profiling the tropism of viral vectors in tissues", "author": [ { "family_name": "Jang", "given_name": "Min J.", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Coughlin", "given_name": "Gerard M.", "orcid": "0000-0003-0644-4721", "clpid": "Coughlin-Gerard-M" }, { "family_name": "Jackson", "given_name": "Cameron R.", "clpid": "Jackson-Cameron-R" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Chuapoco", "given_name": "Miguel R.", "orcid": "0000-0001-5397-996X", "clpid": "Chuapoco-Miguel-R" }, { "family_name": "Vendemiatti", "given_name": "Julia L.", "clpid": "Vendemiatti-Julia-L" }, { "family_name": "Wang", "given_name": "Alexander Z.", "orcid": "0000-0001-7375-5445", "clpid": "Wang-Alexander-Z" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "A barrier to advancing engineered adeno-associated viral vectors (AAVs) for precision access to cell subtypes is a lack of high-throughput, high-resolution assays to characterize in vivo transduction profiles. In this study, we developed an ultrasensitive, sequential fluorescence in situ hybridization (USeqFISH) method for spatial transcriptomic profiling of endogenous and viral RNA with a short barcode in intact tissue volumes by integrating hydrogel-based tissue clearing, enhanced signal amplification and multiplexing using sequential labeling. Using USeqFISH, we investigated the transduction and cell subtype tropisms across mouse brain regions of six systemic AAVs, including AAV-PHP.AX, a new variant that transduces robustly and efficiently across neurons and astrocytes. Here we reveal distinct cell subtype biases of each AAV variant, including a bias of AAV-PHP.N toward excitatory neurons. USeqFISH also enables profiling of pooled regulatory cargos, as we show for a 13-variant pool of microRNA target sites in AAV genomes. Lastly, we demonstrate potential applications of USeqFISH for in situ AAV profiling and multimodal single-cell analysis in non-human primates.", "doi": "10.1038/s41587-022-01648-w", "pmcid": "PMC10443732", "issn": "1087-0156", "publisher": "Nature Publishing Group", "publication": "Nature Biotechnology", "publication_date": "2023-09", "series_number": "9", "volume": "41", "issue": "9", "pages": "1272-1286" }, { "id": "authors:sxje4-sj785", "collection": "authors", "collection_id": "sxje4-sj785", "cite_using_url": "https://authors.library.caltech.edu/records/sxje4-sj785", "type": "article", "title": "Characterization and quantification of adeno-associated virus capsid-loading states by multi-wavelength analytical ultracentrifugation with UltraScan", "author": [ { "family_name": "Henrickson", "given_name": "Amy", "orcid": "0000-0003-3266-5202", "clpid": "Henrickson-Amy" }, { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe" }, { "family_name": "Seal", "given_name": "Austin G.", "orcid": "0009-0003-3417-1908", "clpid": "Seal-Austin-G" }, { "family_name": "Qu", "given_name": "Zhe", "orcid": "0009-0001-6323-9305", "clpid": "Qu-Zhe" }, { "family_name": "Tomlinson", "given_name": "Lauren", "orcid": "0000-0001-6936-1881", "clpid": "Tomlinson-Lauren-J" }, { "family_name": "Forsey", "given_name": "John", "orcid": "0009-0001-5557-4381", "clpid": "Forsey-John" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Oka", "given_name": "Kazuhiro", "orcid": "0000-0003-2159-3637", "clpid": "Oka-Kazuhiro" }, { "family_name": "Demeler", "given_name": "Borries", "orcid": "0000-0002-2414-9518", "clpid": "Demeler-Borries" } ], "abstract": "Aim: We present multi-wavelength (MW) analytical ultracentrifugation (AUC) methods offering superior accuracy for adeno-associated virus characterization and quantification.
Methods: Experimental design guidelines are presented for MW sedimentation velocity and analytical buoyant density equilibrium AUC.
Results: Our results were compared with dual-wavelength AUC, transmission electron microscopy and mass photometry. In contrast to dual-wavelength AUC, MW-AUC correctly quantifies adeno-associated virus capsid ratios and identifies contaminants. In contrast to transmission electron microscopy, partially filled capsids can also be detected and quantified. In contrast to mass photometry, first-principle results are obtained.
Conclusion: Our study demonstrates the improved information provided by MW-AUC, highlighting the utility of several recently integrated UltraScan programs, and reinforces AUC as the gold-standard analysis for viral vectors.
", "doi": "10.2217/nnm-2023-0156", "pmcid": "PMC10652292", "issn": "1743-5889", "publisher": "Future Medicine Ltd", "publication": "Nanomedicine", "publication_date": "2023-09", "series_number": "22", "volume": "18", "issue": "22", "pages": "1519-1534" }, { "id": "authors:6g99y-t0377", "collection": "authors", "collection_id": "6g99y-t0377", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230615-129198000.12", "type": "article", "title": "Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates", "author": [ { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Wolfe", "given_name": "Damien A.", "orcid": "0000-0003-3005-6788", "clpid": "Wolfe-Damien-A" }, { "family_name": "Bindu", "given_name": "Dhanesh Sivadasan", "clpid": "Bindu-Dhanesh-Sivadasan" }, { "family_name": "Zhang", "given_name": "Mengying", "orcid": "0000-0003-0674-9437", "clpid": "Zhang-Mengying" }, { "family_name": "Taskin", "given_name": "Naz", "orcid": "0009-0003-2417-8428", "clpid": "Taskin-Naz" }, { "family_name": "Goertsen", "given_name": "David", "orcid": "0000-0001-7138-1697", "clpid": "Goertsen-David" }, { "family_name": "Shay", "given_name": "Timothy F.", "orcid": "0000-0001-6591-3271", "clpid": "Shay-Timothy-F" }, { "family_name": "Sullivan", "given_name": "Erin E.", "orcid": "0000-0002-1724-6520", "clpid": "Sullivan-Erin-E" }, { "family_name": "Huang", "given_name": "Sheng-Fu", "orcid": "0000-0002-6409-5470", "clpid": "Huang-Sheng-Fu" }, { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-Sripriya" }, { "family_name": "Arokiaraj", "given_name": "Cynthia M.", "orcid": "0000-0003-3201-9868", "clpid": "Arokiaraj-Cynthia-M" }, { "family_name": "Plattner", "given_name": "Viktor M.", "orcid": "0000-0002-0740-2905", "clpid": "Plattner-Viktor-M" }, { "family_name": "Campos", "given_name": "Lillian J.", "orcid": "0000-0003-0839-0288", "clpid": "Campos-Lillian-J" }, { "family_name": "Mich", "given_name": "John K.", "orcid": "0000-0002-1626-1139", "clpid": "Mich-John-K" }, { "family_name": "Monet", "given_name": "Deja", "clpid": "Monet-Deja" }, { "family_name": "Ngo", "given_name": "Victoria", "orcid": "0000-0001-9973-8379", "clpid": "Ngo-Victoria" }, { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Omstead", "given_name": "Victoria", "orcid": "0000-0002-3796-970X", "clpid": "Omstead-Victoria" }, { "family_name": "Weed", "given_name": "Natalie", "orcid": "0000-0003-0891-0327", "clpid": "Weed-Natalie" }, { "family_name": "Bishaw", "given_name": "Yeme", "clpid": "Bishaw-Yeme" }, { "family_name": "Gore", "given_name": "Bryan B.", "orcid": "0000-0003-1721-4235", "clpid": "Gore-Bryan-B" }, { "family_name": "Lein", "given_name": "Ed S.", "orcid": "0000-0001-9012-6552", "clpid": "Lein-Ed-S" }, { "family_name": "Akrami", "given_name": "Athena", "orcid": "0000-0001-5711-0903", "clpid": "Akrami-Athena" }, { "family_name": "Miller", "given_name": "Cory T.", "orcid": "0000-0001-8345-2720", "clpid": "Miller-Cory-T" }, { "family_name": "Levi", "given_name": "Boaz P.", "orcid": "0000-0002-8346-872X", "clpid": "Levi-Boaz-P" }, { "family_name": "Keller", "given_name": "Annika", "orcid": "0000-0003-1466-3633", "clpid": "Keller-Annika" }, { "family_name": "Ting", "given_name": "Jonathan T.", "orcid": "0000-0001-8266-0392", "clpid": "Ting-Jonathan-T" }, { "family_name": "Fox", "given_name": "Andrew S.", "orcid": "0000-0003-0695-3323", "clpid": "Fox-Andrew-S" }, { "family_name": "Eroglu", "given_name": "Cagla", "orcid": "0000-0002-7204-0218", "clpid": "Eroglu-Cagla" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.", "doi": "10.1038/s41467-023-38582-7", "pmcid": "PMC10250345", "issn": "2041-1723", "publisher": "Nature Publishing Group", "publication": "Nature Communications", "publication_date": "2023-06-08", "volume": "14", "pages": "Art. No. 3345" }, { "id": "authors:jsmm1-2ye23", "collection": "authors", "collection_id": "jsmm1-2ye23", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230221-20005500.75", "type": "article", "title": "Spatial tropism profiling of AAV vectors by ultrasensitive sequential FISH in tissue", "author": [ { "family_name": "Jang", "given_name": "Min Jee", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Ultrasensitive sequential fluorescence in situ hybridization (USeqFISH) enables multiplexed detection of the expression of endogenous and exogenous genes delivered by adeno-associated virus (AAV) vectors in intact tissue. USeqFISH provides a spatial map of AAV tropism with high throughput and resolution.", "doi": "10.1038/s41587-023-01681-3", "pmcid": "PMC9885914", "issn": "1087-0156", "publisher": "Nature Publishing Group", "publication": "Nature Biotechnology", "publication_date": "2023-04-26" }, { "id": "authors:rsg2f-2nj92", "collection": "authors", "collection_id": "rsg2f-2nj92", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230602-251993000.57", "type": "article", "title": "Primate-conserved carbonic anhydrase IV and murine-restricted LY6C1 enable blood-brain barrier crossing by engineered viral vectors", "author": [ { "family_name": "Shay", "given_name": "Timothy F.", "orcid": "0000-0001-6591-3271", "clpid": "Shay-Timothy-F" }, { "family_name": "Sullivan", "given_name": "Erin E.", "orcid": "0000-0002-1724-6520", "clpid": "Sullivan-Erin-E" }, { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-Sripriya" }, { "family_name": "Goertsen", "given_name": "David", "orcid": "0000-0001-7138-1697", "clpid": "Goertsen-David" }, { "family_name": "Brown", "given_name": "David", "orcid": "0000-0002-9757-1744", "clpid": "Brown-David" }, { "family_name": "Crosby", "given_name": "Anaya", "clpid": "Crosby-Anaya" }, { "family_name": "Vielmetter", "given_name": "Jost", "orcid": "0000-0002-4314-7163", "clpid": "Vielmetter-Jost" }, { "family_name": "Borsos", "given_name": "Mate", "orcid": "0000-0002-2801-8910", "clpid": "Borsos-M\u00e1t\u00e9" }, { "family_name": "Wolfe", "given_name": "Damien A.", "orcid": "0000-0003-3005-6788", "clpid": "Wolfe-Damien-A" }, { "family_name": "Lam", "given_name": "Annie W.", "orcid": "0009-0008-6982-1452", "clpid": "Lam-Annie-W" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "The blood-brain barrier (BBB) presents a major challenge for delivering large molecules to study and treat the central nervous system. This is due in part to the scarcity of targets known to mediate BBB crossing. To identify novel targets, we leverage a panel of adeno-associated viruses (AAVs) previously identified through mechanism-agnostic directed evolution for improved BBB transcytosis. Screening potential cognate receptors for enhanced BBB crossing, we identify two targets: murine-restricted LY6C1 and widely conserved carbonic anhydrase IV (CA-IV). We apply AlphaFold-based in silico methods to generate capsid-receptor binding models to predict the affinity of AAVs for these identified receptors. Demonstrating how these tools can unlock target-focused engineering strategies, we create an enhanced LY6C1-binding vector, AAV-PHP.eC, that, unlike our prior PHP.eB, also works in Ly6a-deficient mouse strains such as BALB/cJ. Combined with structural insights from computational modeling, the identification of primate-conserved CA-IV enables the design of more specific and potent human brain\u2013penetrant chemicals and biologicals, including gene delivery vectors.", "doi": "10.1126/sciadv.adg6618", "pmcid": "PMC10115422", "issn": "2375-2548", "publisher": "American Association for the Advancement of Science", "publication": "Science Advances", "publication_date": "2023-04-21", "series_number": "16", "volume": "9", "issue": "16", "pages": "Art. No. eadg6618" }, { "id": "authors:j3b0b-jfb61", "collection": "authors", "collection_id": "j3b0b-jfb61", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230725-706045000.29", "type": "article", "title": "Advances in AAV technology for delivering genetically encoded cargo to the nonhuman primate nervous system", "author": [ { "family_name": "Campos", "given_name": "Lillian J.", "orcid": "0000-0003-0839-0288", "clpid": "Campos-Lillian-J" }, { "family_name": "Arokiaraj", "given_name": "Cynthia M.", "orcid": "0000-0003-3201-9868", "clpid": "Arokiaraj-Cynthia-M" }, { "family_name": "Chuapoco", "given_name": "Miguel R.", "orcid": "0000-0001-5397-996X", "clpid": "Chuapoco-Miguel-R" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Goeden", "given_name": "Nick", "clpid": "Goeden-Nicholas" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Fox", "given_name": "Andrew S.", "orcid": "0000-0003-0695-3323", "clpid": "Fox-Andrew-S" } ], "abstract": "Modern neuroscience approaches including optogenetics, calcium imaging, and other genetic manipulations have facilitated our ability to dissect specific circuits in rodent models to study their role in neurological disease. These approaches regularly use viral vectors to deliver genetic cargo (e.g., opsins) to specific tissues and genetically-engineered rodents to achieve cell-type specificity. However, the translatability of these rodent models, cross-species validation of identified targets, and translational efficacy of potential therapeutics in larger animal models like nonhuman primates remains difficult due to the lack of efficient primate viral vectors. A refined understanding of the nonhuman primate nervous system promises to deliver insights that can guide the development of treatments for neurological and neurodegenerative diseases. Here, we outline recent advances in the development of adeno-associated viral vectors for optimized use in nonhuman primates. These tools promise to help open new avenues for study in translational neuroscience and further our understanding of the primate brain.", "doi": "10.1016/j.crneur.2023.100086", "pmcid": "PMC10313870", "issn": "2665-945X", "publisher": "Elsevier", "publication": "Current Research in Neurobiology", "publication_date": "2023-04-12", "volume": "4", "pages": "Art. No. 100086" }, { "id": "authors:c9mfw-wg325", "collection": "authors", "collection_id": "c9mfw-wg325", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230525-772152300.6", "type": "article", "title": "CRISPR-clear imaging of melanin-rich B16-derived solid tumors", "author": [ { "family_name": "Schubert", "given_name": "Rajib", "orcid": "0000-0002-7071-0134", "clpid": "Schubert-Rajib" }, { "family_name": "Bae", "given_name": "Taegeun", "clpid": "Bae-Taegeun" }, { "family_name": "Simic", "given_name": "Branko", "orcid": "0000-0002-7314-3913", "clpid": "Simic-Branko" }, { "family_name": "Smith", "given_name": "Sheena N.", "orcid": "0000-0002-2162-7107", "clpid": "Smith-Sheena-N" }, { "family_name": "Park", "given_name": "Seong-Ho", "orcid": "0000-0002-1257-8315", "clpid": "Park-Seong-Ho" }, { "family_name": "Nagy-Davidescu", "given_name": "Gabriela", "clpid": "Nagy-Davidescu-Gabriela" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Pl\u00fcckthun", "given_name": "Andreas", "orcid": "0000-0003-4191-5306", "clpid": "Pl\u00fcckthun-Andreas" }, { "family_name": "Hur", "given_name": "Junho K.", "orcid": "0000-0003-3794-1149", "clpid": "Hur-Junho-K" } ], "abstract": "Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast,\u00a0melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging. To address this challenge, we have developed a CRISPR-based gene editing approach that is easily incorporated into existing tissue-clearing workflows such the PACT clearing method. We term this method CRISPR-Clear. We demonstrate its applicability to highly melanin-rich B16-derived solid tumors, including one made transgenic for HER2, constituting one of very few syngeneic mouse tumors that can be used in immunocompetent models. We demonstrate the utility in detailed tumor characterization by staining for targeting antibodies and nanoparticles, as well as expressed fluorescent proteins. With CRISPR-Clear we have unprecedented access to optical interrogation in considerable portions of intact melanoma tissue for stained surface markers, expressed fluorescent proteins, of subcellular compartments, and of the vasculature.", "doi": "10.1038/s42003-023-04614-7", "pmcid": "PMC10073193", "issn": "2399-3642", "publisher": "Nature Publishing Group", "publication": "Communications Biology", "publication_date": "2023-04-04", "volume": "6", "pages": "Art. No. 370" }, { "id": "authors:ndk7v-fhz93", "collection": "authors", "collection_id": "ndk7v-fhz93", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20221202-906989500.8", "type": "article", "title": "A prebiotic diet modulates microglial states and motor deficits in \u03b1-synuclein overexpressing mice", "author": [ { "family_name": "Abdel-Haq", "given_name": "Reem", "orcid": "0000-0002-7418-5736", "clpid": "Abdel-Haq-Reem" }, { "family_name": "Schlachetzki", "given_name": "Johannes C. M.", "orcid": "0000-0002-7801-9743", "clpid": "Schlachetzki-Johannes-C-M" }, { "family_name": "Boktor", "given_name": "Joseph C.", "orcid": "0000-0003-2456-1913", "clpid": "Boktor-Joseph-C" }, { "family_name": "Cantu-Jungles", "given_name": "Thaisa M.", "orcid": "0000-0001-8928-9717", "clpid": "Cantu-Jungles-Thaisa-M" }, { "family_name": "Thron", "given_name": "Taren", "orcid": "0000-0001-9577-2617", "clpid": "Thron-Taren-M" }, { "family_name": "Zhang", "given_name": "Mengying", "clpid": "Zhang-Mengying" }, { "family_name": "Bostick", "given_name": "John W.", "orcid": "0000-0001-8925-2447", "clpid": "Bostick-John-W" }, { "family_name": "Khazaei", "given_name": "Tahmineh", "orcid": "0000-0002-4743-2383", "clpid": "Khazaei-Tahmineh" }, { "family_name": "Chilakala", "given_name": "Sujatha", "orcid": "0000-0003-1581-3381", "clpid": "Chilakala-Sujatha" }, { "family_name": "Morais", "given_name": "Livia H.", "orcid": "0000-0002-5738-2658", "clpid": "Morais-Livia-H" }, { "family_name": "Humphrey", "given_name": "Greg", "clpid": "Humphrey-Gregory" }, { "family_name": "Keshavarzian", "given_name": "Ali", "orcid": "0000-0002-7969-3369", "clpid": "Keshavarzian-Ali" }, { "family_name": "Katz", "given_name": "Jonathan E.", "clpid": "Katz-Jonathan-E" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Knight", "given_name": "Rob", "orcid": "0000-0002-0975-9019", "clpid": "Knight-Rob" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Hamaker", "given_name": "Bruce R.", "orcid": "0000-0001-6591-942X", "clpid": "Hamaker-Bruce-R" }, { "family_name": "Glass", "given_name": "Christopher K.", "orcid": "0000-0003-4344-3592", "clpid": "Glass-Christopher-K" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "abstract": "Parkinson's disease (PD) is a movement disorder characterized by neuroinflammation, \u03b1-synuclein pathology, and neurodegeneration. Most cases of PD are non-hereditary, suggesting a strong role for environmental factors, and it has been speculated that disease may originate in peripheral tissues such as the gastrointestinal (GI) tract before affecting the brain. The gut microbiome is altered in PD and may impact motor and GI symptoms as indicated by animal studies, although mechanisms of gut-brain interactions remain incompletely defined. Intestinal bacteria ferment dietary fibers into short-chain fatty acids, with fecal levels of these molecules differing between PD and healthy controls and in mouse models. Among other effects, dietary microbial metabolites can modulate activation of microglia, brain-resident immune cells implicated in PD. We therefore investigated whether a fiber-rich diet influences microglial function in \u03b1-synuclein overexpressing (ASO) mice, a preclinical model with PD-like symptoms and pathology. Feeding a prebiotic high-fiber diet attenuates motor deficits and reduces \u03b1-synuclein aggregation in the substantia nigra of mice. Concomitantly, the gut microbiome of ASO mice adopts a profile correlated with health upon prebiotic treatment, which also reduces microglial activation. Single-cell RNA-seq analysis of microglia from the substantia nigra and striatum uncovers increased pro-inflammatory signaling and reduced homeostatic responses in ASO mice compared to wild-type counterparts on standard diets. However, prebiotic feeding reverses pathogenic microglial states in ASO mice and promotes expansion of protective disease-associated macrophage (DAM) subsets of microglia. Notably, depletion of microglia using a CSF1R inhibitor eliminates the beneficial effects of prebiotics by restoring motor deficits to ASO mice despite feeding a prebiotic diet. These studies uncover a novel microglia-dependent interaction between diet and motor symptoms in mice, findings that may have implications for neuroinflammation and PD.", "doi": "10.7554/elife.81453", "pmcid": "PMC9668333", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2022-11-08", "volume": "11", "pages": "Art. No. e81453" }, { "id": "authors:1w82f-14a30", "collection": "authors", "collection_id": "1w82f-14a30", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20221209-478595000.5", "type": "article", "title": "Molecular basis of astrocyte diversity and morphology across the CNS in health and disease", "author": [ { "family_name": "Endo", "given_name": "Fumito", "orcid": "0000-0003-4445-6707", "clpid": "Endo-Fumito" }, { "family_name": "Kasai", "given_name": "Atsushi", "orcid": "0000-0002-1612-3078", "clpid": "Kasai-Atsushi" }, { "family_name": "Soto", "given_name": "Joselyn S.", "orcid": "0000-0002-5869-9091", "clpid": "Soto-Joselyn-S" }, { "family_name": "Yu", "given_name": "Xinzhu", "orcid": "0000-0003-1244-0110", "clpid": "Yu-Xinzhu" }, { "family_name": "Qu", "given_name": "Zhe", "clpid": "Qu-Zhe" }, { "family_name": "Hashimoto", "given_name": "Hitoshi", "orcid": "0000-0001-6548-4016", "clpid": "Hashimoto-Hitoshi" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Kawaguchi", "given_name": "Riki", "orcid": "0000-0002-2489-4825", "clpid": "Kawaguchi-Riki" }, { "family_name": "Khakh", "given_name": "Baljit S.", "orcid": "0000-0002-0939-1218", "clpid": "Khakh-Baljit-S" } ], "abstract": "Astrocytes, a type of glia, are abundant and morphologically complex cells. Here, we report astrocyte molecular profiles, diversity, and morphology across the mouse central nervous system (CNS). We identified shared and region-specific astrocytic genes and functions and explored the cellular origins of their regional diversity. We identified gene networks correlated with astrocyte morphology, several of which unexpectedly contained Alzheimer's disease (AD) risk genes. CRISPR/Cas9\u2013mediated reduction of candidate genes reduced astrocyte morphological complexity and resulted in cognitive deficits. The same genes were down-regulated in human AD, in an AD mouse model that displayed reduced astrocyte morphology, and in other human brain disorders. We thus provide comprehensive molecular data on astrocyte diversity and mechanisms across the CNS and on the molecular basis of astrocyte morphology in health and disease.", "doi": "10.1126/science.adc9020", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2022-11-04", "series_number": "6619", "volume": "378", "issue": "6619", "pages": "Art. No. adc9020" }, { "id": "authors:ynwa0-bnv65", "collection": "authors", "collection_id": "ynwa0-bnv65", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220801-544880000", "type": "article", "title": "Structural basis of receptor usage by the engineered capsid AAV-PHP.eB", "author": [ { "family_name": "Jang", "given_name": "Seongmin", "clpid": "Jang-Seongmin" }, { "family_name": "Shen", "given_name": "Hao K.", "orcid": "0000-0003-2687-0736", "clpid": "Shen-Hao-K" }, { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Miles", "given_name": "Timothy F.", "orcid": "0000-0001-6591-3271", "clpid": "Miles-T-F" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Adeno-associated virus serotype 9 (AAV9) is a promising gene therapy vector for treating neurodegenerative diseases due to its ability to penetrate the blood-brain barrier. PHP.eB was engineered from AAV9 by insertion of a 7-amino acid peptide and point mutation of neighboring residues, thereby enhancing potency in the central nervous system. Here, we report a 2.24-\u00c5 resolution cryo-electron microscopy structure of PHP.eB, revealing conformational differences from other 7-mer insertion capsid variants. In PHP.eB, the 7-mer loop adopts a bent conformation, mediated by an interaction between engineered lysine and aspartate residues. Further, we identify PKD2 as the main AAV receptor (AAVR) domain recognizing both AAV9 and PHP.eB and find that the PHP.eB 7-mer partially destabilizes this interaction. Analysis of previously reported AAV structures together with our pull-down data demonstrate that the 7-mer topology determined by the lysine-aspartate interaction dictates AAVR binding strength. Our results suggest that PHP.eB's altered tropism may arise from both an additional interaction with LY6A and weakening of its AAVR interaction. Changing the insertion length, but not sequence, modifies PKD2 binding affinity, suggesting that a steric clash impedes AAVR binding. This research suggests improved library designs for future AAV selections to identify non-LY6A-dependent vectors and modulate AAVR interaction strength.", "doi": "10.1016/j.omtm.2022.07.011", "pmcid": "PMC9382559", "issn": "2329-0501", "publisher": "Cell Press", "publication": "Molecular Therapy - Methods & Clinical Development", "publication_date": "2022-09-08", "volume": "26", "pages": "343-354" }, { "id": "authors:atz4j-4ag97", "collection": "authors", "collection_id": "atz4j-4ag97", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220816-373601000", "type": "article", "title": "Targeting the lung epithelium after intravenous delivery by directed evolution of underexplored sites on the AAV capsid", "author": [ { "family_name": "Goertsen", "given_name": "David", "orcid": "0000-0001-7138-1697", "clpid": "Goertsen-David" }, { "family_name": "Goeden", "given_name": "Nick", "clpid": "Goeden-Nicholas" }, { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-Nicholas-C" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Advances in adeno-associated virus (AAV) engineering have provided exciting new tools for research and potential solutions for gene therapy. However, the lung has not received the same tailored engineering as other major targets of debilitating genetic disorders. To address this, here we engineered the surface-exposed residues AA452-458 of AAV9 capsid proteins at the three-fold axis of symmetry and employed a Cre-transgenic-based screening platform to identify AAV capsids targeted to the lung after intravenous delivery in mice. Using a custom image processing pipeline to quantify transgene expression across whole tissue images, we found that one engineered variant, AAV9.452sub.LUNG1, displays dramatically improved transgene expression in lung tissue after systemic delivery in mice. This improved transduction extends to alveolar epithelial type II cells, expanding the toolbox for gene therapy research for diseases specific to the lung.", "doi": "10.1016/j.omtm.2022.07.010", "pmcid": "PMC9372736", "issn": "2329-0501", "publisher": "Cell Press", "publication": "Molecular Therapy - Methods & Clinical Development", "publication_date": "2022-09-08", "volume": "26", "pages": "331-342" }, { "id": "authors:tb77e-ca060", "collection": "authors", "collection_id": "tb77e-ca060", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20221116-602375500.16", "type": "article", "title": "Multimodal imaging of capsid and cargo reveals differential brain targeting and liver detargeting of systemically-administered AAVs", "author": [ { "family_name": "Seo", "given_name": "Jai Woong", "orcid": "0000-0002-2732-7498", "clpid": "Seo-Jai-Woong" }, { "family_name": "Ajenjo", "given_name": "Javier", "clpid": "Ajenjo-Javier" }, { "family_name": "Wu", "given_name": "Bo", "orcid": "0000-0002-7199-6525", "clpid": "Wu-Bo" }, { "family_name": "Robinson", "given_name": "Elise", "orcid": "0000-0002-6527-901X", "clpid": "Robinson-Elise" }, { "family_name": "Raie", "given_name": "Marina Nura", "orcid": "0000-0002-5549-404X", "clpid": "Raie-Marina-Nura" }, { "family_name": "Wang", "given_name": "James", "orcid": "0000-0002-6363-4014", "clpid": "Wang-James" }, { "family_name": "Tumbale", "given_name": "Spencer K.", "clpid": "Tumbale-Spencer-K" }, { "family_name": "Buccino", "given_name": "Pablo", "clpid": "Buccino-Pablo" }, { "family_name": "Anders", "given_name": "David Alexander", "orcid": "0000-0001-6315-3010", "clpid": "Anders-David-Alexander" }, { "family_name": "Shen", "given_name": "Bin", "clpid": "Shen-Bin" }, { "family_name": "Habte", "given_name": "Frezghi G.", "clpid": "Habte-Frezghi-G" }, { "family_name": "Beinat", "given_name": "Corinne", "clpid": "Beinat-Corinne" }, { "family_name": "James", "given_name": "Michelle L.", "clpid": "James-Michelle-L" }, { "family_name": "Reyes", "given_name": "Samantha Taylor", "clpid": "Reyes-Samantha-Taylor" }, { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-Sripriya" }, { "family_name": "Miles", "given_name": "Timothy F.", "orcid": "0000-0001-6591-3271", "clpid": "Miles-T-F" }, { "family_name": "Lee", "given_name": "Jason T.", "orcid": "0000-0003-2590-2011", "clpid": "Lee-Jason-T" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Ferrara", "given_name": "Katherine W.", "orcid": "0000-0002-4976-9107", "clpid": "Ferrara-Katherine-W" } ], "abstract": "The development of gene delivery vehicles with high organ specificity when administered systemically is a critical goal for gene therapy. We combine optical and positron emission tomography (PET) imaging of 1) reporter genes and 2) capsid tags to assess the temporal and spatial distribution and transduction of adeno-associated viruses (AAVs). AAV9 and two engineered AAV vectors (PHP.eB and CAP-B10) that are noteworthy for maximizing blood-brain barrier transport were compared. CAP-B10 shares a modification in the 588 loop with PHP.eB, but also has a modification in the 455 loop, added with the goal of reducing off-target transduction. PET and optical imaging revealed that the additional modifications retained brain receptor affinity. In the liver, the accumulation of AAV9 and the engineered AAV capsids was similar (\u223c15% of the injected dose per cc and not significantly different between capsids at 21 h). However, the engineered capsids were primarily internalized by Kupffer cells rather than hepatocytes, and liver transduction was greatly reduced. PET reporter gene imaging after engineered AAV systemic injection provided a non-invasive method to monitor AAV-mediated protein expression over time. Through comparison with capsid tagging, differences between brain localization and transduction were revealed. In summary, AAV capsids bearing imaging tags and reporter gene payloads create a unique and powerful platform to assay the pharmacokinetics, cellular specificity and protein expression kinetics of AAV vectors in vivo, a key enabler for the field of gene therapy.", "doi": "10.1016/j.biomaterials.2022.121701", "pmcid": "PMC9621732", "issn": "0142-9612", "publisher": "Elsevier", "publication": "Biomaterials", "publication_date": "2022-09", "volume": "288", "pages": "Art. No. 121701" }, { "id": "authors:aj0e9-00c08", "collection": "authors", "collection_id": "aj0e9-00c08", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220606-735990000", "type": "article", "title": "Engineered AAVs for non-invasive gene delivery to rodent and non-human primate nervous systems", "author": [ { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-Sripriya" }, { "family_name": "Adams", "given_name": "Cameron D.", "orcid": "0000-0002-9945-2140", "clpid": "Adams-Cameron-D" }, { "family_name": "Yang", "given_name": "Daping", "orcid": "0000-0002-3104-1943", "clpid": "Yang-Daping" }, { "family_name": "Wang", "given_name": "Tongtong", "clpid": "Wang-Tongtong" }, { "family_name": "Wolfe", "given_name": "Damien A.", "clpid": "Wolfe-Damien-A" }, { "family_name": "Arokiaraj", "given_name": "Cynthia M.", "orcid": "0000-0003-3201-9868", "clpid": "Arokiaraj-Cynthia-M" }, { "family_name": "Ngo", "given_name": "Victoria", "orcid": "0000-0001-9973-8379", "clpid": "Ngo-Victoria" }, { "family_name": "Campos", "given_name": "Lillian J.", "clpid": "Campos-Lillian-J" }, { "family_name": "Griffiths", "given_name": "Jessica A.", "orcid": "0000-0002-5586-1567", "clpid": "Griffiths-Jessica-A" }, { "family_name": "Ichiki", "given_name": "Takako", "orcid": "0000-0002-8670-9541", "clpid": "Ichiki-Takako" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Osborne", "given_name": "Peregrine B.", "orcid": "0000-0002-8458-4159", "clpid": "Osborne-Peregrine-B" }, { "family_name": "Keast", "given_name": "Janet R.", "orcid": "0000-0002-4341-3265", "clpid": "Keast-Janet-R" }, { "family_name": "Miller", "given_name": "Cory T.", "orcid": "0000-0001-8345-2720", "clpid": "Miller-Cory-T" }, { "family_name": "Fox", "given_name": "Andrew S.", "orcid": "0000-0003-0695-3323", "clpid": "Fox-Andrew-S" }, { "family_name": "Chiu", "given_name": "Isaac M.", "orcid": "0000-0002-0723-4841", "clpid": "Chiu-Isaac-M" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Gene therapy offers great promise in addressing neuropathologies associated with the central and peripheral nervous systems (CNS and PNS). However, genetic access remains difficult, reflecting the critical need for the development of effective and non-invasive gene delivery vectors across species. To that end, we evolved adeno-associated virus serotype 9 (AAV9) capsid in mice and validated two capsids, AAV-MaCPNS1 and AAV-MaCPNS2, across rodent species (mice and rats) and non-human primate (NHP) species (marmosets and rhesus macaques). Intravenous administration of either AAV efficiently transduced the PNS in rodents and both the PNS and CNS in NHPs. Furthermore, we used AAV-MaCPNS1 in mice to systemically deliver the following: (1) the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia and (2) the neuronal actuator DREADD to dorsal root ganglia to mediate pain. This conclusively demonstrates the translatability of these two systemic AAVs across four species and their functional utility through proof-of-concept studies in mice.", "doi": "10.1016/j.neuron.2022.05.003", "pmcid": "PMC9308721", "issn": "0896-6273", "publisher": "Cell Press", "publication": "Neuron", "publication_date": "2022-07-20", "series_number": "14", "volume": "110", "issue": "14", "pages": "2242-2257" }, { "id": "authors:0h03p-c6230", "collection": "authors", "collection_id": "0h03p-c6230", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210709-224144084", "type": "article", "title": "Age-dependent alterations in key components of the nigrostriatal dopaminergic system and distinct motor phenotypes", "author": [ { "family_name": "Fan", "given_name": "Jiang-peng", "clpid": "Fan-Jiang-Peng" }, { "family_name": "Geng", "given_name": "Hui-zhen", "clpid": "Geng-Hui-Zhen" }, { "family_name": "Ji", "given_name": "Ya-wei", "clpid": "Ji-Ya-Wei" }, { "family_name": "Jia", "given_name": "Tao", "clpid": "Jia-Tao" }, { "family_name": "Treweek", "given_name": "Jennifer B.", "orcid": "0000-0002-5601-9646", "clpid": "Treweek-Jennifer-B" }, { "family_name": "Li", "given_name": "An-an", "clpid": "Li-An-An" }, { "family_name": "Zhou", "given_name": "Chun-yi", "clpid": "Zhou-Chun-Yi" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Xiao", "given_name": "Cheng", "orcid": "0000-0001-9649-7450", "clpid": "Xiao-Cheng" } ], "abstract": "The nigrostriatal dopaminergic (DA) system, which includes DA neurons in the ventral and dorsal tiers of the substantia nigra pars compacta (vSNc, dSNc) and DA terminals in the dorsal striatum, is critically implicated in motor control. Accumulating studies demonstrate that both the nigrostriatal DA system and motor function are impaired in aged subjects. However, it is unknown whether dSNc and vSNc DA neurons and striatal DA terminals age in similar patterns, and whether these changes parallel motor deficits. To address this, we performed ex vivo patch-clamp recordings in dSNc and vSNc DA neurons, measured striatal dopamine release, and analyzed motor behaviors in rodents. Spontaneous firing in dSNc and vSNc DA neurons and depolarization-evoked firing in dSNc DA neurons showed inverse V-shaped changes with age. But depolarization-evoked firing in vSNc DA neurons increased with age. In the dorsal striatum, dopamine release declined with age. In locomotor tests, 12-month-old rodents showed hyperactive exploration, relative to 6- and 24-month-old rodents. Additionally, aged rodents showed significant deficits in coordination. Elevating dopamine levels with a dopamine transporter inhibitor improved both locomotion and coordination. Therefore, key components in the nigrostriatal DA system exhibit distinct aging patterns and may contribute to age-related alterations in locomotion and coordination.", "doi": "10.1038/s41401-021-00713-5", "pmcid": "PMC8975991", "issn": "1671-4083", "publisher": "Nature Publishing Group", "publication": "Acta Pharmacologica Sinica", "publication_date": "2022-04", "series_number": "4", "volume": "43", "issue": "4", "pages": "862-875" }, { "id": "authors:g463t-rp078", "collection": "authors", "collection_id": "g463t-rp078", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220104-157923000", "type": "article", "title": "Glutamate in primary afferents is required for itch transmission", "author": [ { "family_name": "Cui", "given_name": "Lian", "clpid": "Cui-Lian" }, { "family_name": "Guo", "given_name": "Jeff", "clpid": "Guo-Jeffrey" }, { "family_name": "Cranfill", "given_name": "Suna L.", "clpid": "Cranfill-Suna-L" }, { "family_name": "Gautam", "given_name": "Mayank", "orcid": "0000-0002-7257-5837", "clpid": "Gautam-Mayank" }, { "family_name": "Bhattarai", "given_name": "Janardhan", "orcid": "0000-0002-4102-1380", "clpid": "Bhattarai-Janardhan" }, { "family_name": "Olson", "given_name": "William", "clpid": "Olson-William-S" }, { "family_name": "Beattie", "given_name": "Katherine", "orcid": "0000-0002-1055-4547", "clpid": "Beattie-Katherine" }, { "family_name": "Challis", "given_name": "Rosemary C.", "orcid": "0000-0003-3086-6553", "clpid": "Challis-Rosemary-C" }, { "family_name": "Wu", "given_name": "Qinxue", "clpid": "Wu-Qinxue" }, { "family_name": "Song", "given_name": "Xue", "clpid": "Song-Xue" }, { "family_name": "Raabe", "given_name": "Tobias", "orcid": "0000-0001-8944-3278", "clpid": "Raabe-Tobias" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Ma", "given_name": "Minghong", "clpid": "Ma-Minghong" }, { "family_name": "Liu", "given_name": "Qin", "clpid": "Liu-Qin" }, { "family_name": "Luo", "given_name": "Wenqin", "clpid": "Luo-Wenqin" } ], "abstract": "Whether glutamate or itch-selective neurotransmitters are used to confer itch specificity is still under debate. We focused on an itch-selective population of primary afferents expressing MRGPRA3, which highly expresses Vglut2 and the neuropeptide neuromedin B (Nmb), to investigate this question. Optogenetic stimulation of MRGPRA3+ afferents triggers scratching and other itch-related avoidance behaviors. Using a combination of optogenetics, spinal cord slice recordings, Vglut2 conditional knockout mice, and behavior assays, we showed that glutamate is essential for MRGPRA3+ afferents to transmit itch. We further demonstrated that MRGPRA3\u207a afferents form monosynaptic connections with both NMBR\u207a and NMBR\u207b neurons and that NMB and glutamate together can enhance the activity of NMBR\u207a spinal DH neurons. Moreover, Nmb in MRGPRA3\u207a afferents and NMBR\u207a DH neurons are required for chloroquine-induced scratching. Together, our results establish a new model in which glutamate is an essential neurotransmitter in primary afferents for itch transmission, whereas NMB signaling enhances its activities.", "doi": "10.1016/j.neuron.2021.12.007", "pmcid": "PMC8898340", "issn": "0896-6273", "publisher": "Cell Press", "publication": "Neuron", "publication_date": "2022-03-02", "series_number": "5", "volume": "110", "issue": "5", "pages": "809-823" }, { "id": "authors:pg7h2-wsy12", "collection": "authors", "collection_id": "pg7h2-wsy12", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210728-223917563", "type": "article", "title": "Brain-wide Cas9-mediated cleavage of a gene causing familial Alzheimer's disease alleviates amyloid-related pathologies in mice", "author": [ { "family_name": "Duan", "given_name": "Yangyang", "orcid": "0000-0002-0823-6574", "clpid": "Duan-Yangyang" }, { "family_name": "Ye", "given_name": "Tao", "orcid": "0000-0002-8075-7323", "clpid": "Ye-Tao" }, { "family_name": "Qu", "given_name": "Zhe", "clpid": "Qu-Zhe" }, { "family_name": "Chen", "given_name": "Yuewen", "clpid": "Chen-Yuewen" }, { "family_name": "Miranda", "given_name": "Abigail", "clpid": "Miranda-Abigail" }, { "family_name": "Zhou", "given_name": "Xiaopu", "clpid": "Zhou-Xiaopu" }, { "family_name": "Lok", "given_name": "Ka-Chun", "clpid": "Lok-Ka-Chun" }, { "family_name": "Chen", "given_name": "Yu", "clpid": "Chen-Yu" }, { "family_name": "Fu", "given_name": "Amy K. Y.", "clpid": "Fu-Amy-K-Y" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Ip", "given_name": "Nancy Y.", "orcid": "0000-0002-2763-8907", "clpid": "Ip-Nancy-Y" } ], "abstract": "The pathology of familial Alzheimer's disease, which is caused by dominant mutations in the gene that encodes amyloid-beta precursor protein (APP) and in those that encode presenilin 1 and presenilin 2, is characterized by extracellular amyloid plaques and intracellular neurofibrillary tangles in multiple brain regions. Here we show that the brain-wide selective disruption of a mutated APP allele in transgenic mouse models carrying the human APP Swedish mutation alleviates amyloid-beta-associated pathologies for at least six months after a single intrahippocampal administration of an adeno-associated virus that encodes both Cas9 and a single-guide RNA that targets the mutation. We also show that the deposition of amyloid-beta, as well as microgliosis, neurite dystrophy and the impairment of cognitive performance, can all be ameliorated when the CRISPR\u2013Cas9 construct is delivered intravenously via a modified adeno-associated virus that can cross the blood\u2013brain barrier. Brain-wide disease-modifying genome editing could represent a viable strategy for the treatment of familial Alzheimer's disease and other monogenic diseases that affect multiple brain regions.", "doi": "10.1038/s41551-021-00759-0", "issn": "2157-846X", "publisher": "Nature Publishing Group", "publication": "Nature Biomedical Engineering", "publication_date": "2022-02", "series_number": "2", "volume": "6", "issue": "2", "pages": "168-180" }, { "id": "authors:ejc84-8zb49", "collection": "authors", "collection_id": "ejc84-8zb49", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211210-240709000", "type": "article", "title": "AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset", "author": [ { "family_name": "Goertsen", "given_name": "David", "orcid": "0000-0001-7138-1697", "clpid": "Goertsen-David" }, { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-Nicholas-C" }, { "family_name": "Goeden", "given_name": "Nick", "clpid": "Goeden-Nicholas" }, { "family_name": "Chuapoco", "given_name": "Miguel R.", "orcid": "0000-0001-5397-996X", "clpid": "Chuapoco-Miguel-R" }, { "family_name": "Cummins", "given_name": "Alexander", "clpid": "Cummins-Alexander" }, { "family_name": "Chen", "given_name": "Yijing", "clpid": "Chen-Yijing" }, { "family_name": "Fan", "given_name": "Yingying", "clpid": "Fan-Yingying" }, { "family_name": "Zhang", "given_name": "Qiangge", "orcid": "0000-0001-7297-0717", "clpid": "Zhang-Qiangge" }, { "family_name": "Sharma", "given_name": "Jitendra", "clpid": "Sharma-Jitendra" }, { "family_name": "Duan", "given_name": "Yangyang", "orcid": "0000-0002-0823-6574", "clpid": "Duan-Yangyang" }, { "family_name": "Wang", "given_name": "Liping", "orcid": "0000-0001-6893-3809", "clpid": "Wang-Liping" }, { "family_name": "Feng", "given_name": "Guoping", "orcid": "0000-0002-8021-277X", "clpid": "Feng-Guoping" }, { "family_name": "Chen", "given_name": "Yu", "clpid": "Chen-Yu" }, { "family_name": "Ip", "given_name": "Nancy Y.", "orcid": "0000-0002-2763-8907", "clpid": "Ip-Nancy-Y" }, { "family_name": "Pickel", "given_name": "James", "orcid": "0000-0002-3617-3072", "clpid": "Pickel-James" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood\u2013brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.", "doi": "10.1038/s41593-021-00969-4", "issn": "1097-6256", "publisher": "Nature Publishing Group", "publication": "Nature Neuroscience", "publication_date": "2021-12-09", "series_number": "1", "volume": "25", "issue": "1", "pages": "106-115" }, { "id": "authors:zvjg3-dry41", "collection": "authors", "collection_id": "zvjg3-dry41", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210629-153450418", "type": "article", "title": "Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional Changes via Single-Cell RNA Sequencing", "author": [ { "family_name": "Brown", "given_name": "David", "orcid": "0000-0002-9757-1744", "clpid": "Brown-David" }, { "family_name": "Altermatt", "given_name": "Michael", "orcid": "0000-0003-2841-5374", "clpid": "Altermatt-Michael" }, { "family_name": "Dobreva", "given_name": "Tatyana", "orcid": "0000-0002-2625-8873", "clpid": "Dobreva-Tatyana" }, { "family_name": "Chen", "given_name": "Sisi", "orcid": "0000-0001-9448-9713", "clpid": "Chen-Sisi" }, { "family_name": "Wang", "given_name": "Alexander", "orcid": "0000-0001-7375-5445", "clpid": "Wang-Alexander" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at high resolution. Using this platform, we have both corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Transcriptomic analysis revealed that this neuronal bias is due mainly to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific transitory responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. The presented experimental and computational approaches for parallel characterization of AAV tropism will facilitate the advancement of safe and precise gene delivery vehicles, and showcase the power of understanding responses to gene therapies at the single-cell level.", "doi": "10.3389/fimmu.2021.730825", "pmcid": "PMC8574206", "issn": "1664-3224", "publisher": "Frontiers Media", "publication": "Frontiers in Immunology", "publication_date": "2021-10-21", "volume": "12", "pages": "Art. No. 730825" }, { "id": "authors:h1gp6-1c240", "collection": "authors", "collection_id": "h1gp6-1c240", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210930-210049351", "type": "article", "title": "Improved systemic AAV gene therapy with a neurotrophic capsid in Niemann\u2013Pick disease type C1 mice", "author": [ { "family_name": "Davidson", "given_name": "Cristin D.", "orcid": "0000-0002-5508-8113", "clpid": "Davidson-Cristin-D" }, { "family_name": "Gibson", "given_name": "Alana L.", "orcid": "0000-0003-2247-7064", "clpid": "Gibson-Alana-L" }, { "family_name": "Gu", "given_name": "Tansy", "clpid": "Gu-Tansy" }, { "family_name": "Baxter", "given_name": "Laura L.", "clpid": "Baxter-Laura-L" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-Benjamin-E" }, { "family_name": "Beadle", "given_name": "Keith", "orcid": "0000-0002-5695-6461", "clpid": "Beadle-Keith" }, { "family_name": "Incao", "given_name": "Arturo A.", "clpid": "Incao-Arturo-A" }, { "family_name": "Rodriguez-Gil", "given_name": "Jorge L.", "orcid": "0000-0002-1125-1281", "clpid": "Rodriguez-Gil-Jorge-L" }, { "family_name": "Fujiwara", "given_name": "Hideji", "clpid": "Fujiwara-Hideji" }, { "family_name": "Jiang", "given_name": "Xuntian", "clpid": "Jiang-Xuntian" }, { "family_name": "Chandler", "given_name": "Randy J.", "orcid": "0000-0002-6047-5230", "clpid": "Chandler-Randy-J" }, { "family_name": "Ory", "given_name": "Daniel S.", "clpid": "Ory-Daniel-S" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Venditti", "given_name": "Charles P.", "orcid": "0000-0001-6599-1253", "clpid": "Venditti-Charles-P" }, { "family_name": "Pavan", "given_name": "William J.", "orcid": "0000-0001-8281-5120", "clpid": "Pavan-William-J" } ], "abstract": "Niemann\u2013Pick C1 disease (NPC1) is a rare, fatal neurodegenerative disease caused by mutations in NPC1, which encodes the lysosomal cholesterol transport protein NPC1. Disease pathology involves lysosomal accumulation of cholesterol and lipids, leading to neurological and visceral complications. Targeting the central nervous system (CNS) from systemic circulation complicates treatment of neurological diseases with gene transfer techniques. Selected and engineered capsids, for example, adeno-associated virus (AAV)-PHP.B facilitate peripheral-to-CNS transfer and hence greater CNS transduction than parental predecessors. We report that systemic delivery to Npc1^(m1N/m1N) mice using an AAV-PHP.B vector ubiquitously expressing NPC1 led to greater disease amelioration than an otherwise identical AAV9 vector. In addition, viral copy number and biodistribution of GFP-expressing reporters showed that AAV-PHP.B achieved more efficient, albeit variable, CNS transduction than AAV9 in Npc1^(m1N/m1N) mice. This variability was associated with segregation of two alleles of the putative AAV-PHP.B receptor Ly6a in Npc1^(m1N/m1N) mice. Our data suggest that robust improvements in NPC1 disease phenotypes occur even with modest CNS transduction and that improved neurotrophic capsids have the potential for superior NPC1 AAV gene therapy vectors.", "doi": "10.26508/lsa.202101040", "pmcid": "PMC8380657", "issn": "2575-1077", "publisher": "Life Science Alliance, LLC", "publication": "Life Science Alliance", "publication_date": "2021-10", "series_number": "10", "volume": "4", "issue": "10", "pages": "Art. No. e202101040" }, { "id": "authors:6ftpg-4wj22", "collection": "authors", "collection_id": "6ftpg-4wj22", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211008-224633454", "type": "article", "title": "Light-guided sectioning for precise in situ localization and tissue interface analysis for brain-implanted optical fibers and GRIN lenses", "author": [ { "family_name": "Kahan", "given_name": "Anat", "orcid": "0000-0002-4799-3017", "clpid": "Kahan-Anat" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-Alon" }, { "family_name": "Jang", "given_name": "Min J.", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Robinson", "given_name": "J. Elliott", "orcid": "0000-0001-9417-3938", "clpid": "Robinson-J-Elliott" }, { "family_name": "Cho", "given_name": "Jounhong Ryan", "orcid": "0000-0001-9542-716X", "clpid": "Cho-Jounhong-Ryan" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Kassraian", "given_name": "Pegah", "orcid": "0000-0002-6562-7918", "clpid": "Kassraian-Fard-Pegah" }, { "family_name": "Wagenaar", "given_name": "Daniel A.", "orcid": "0000-0002-6222-761X", "clpid": "Wagenaar-D-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Optical implants to control and monitor neuronal activity in vivo have become foundational tools of neuroscience. Standard two-dimensional histology of the implant location, however, often suffers from distortion and loss during tissue processing. To address that, we developed a three-dimensional post hoc histology method called \"light-guided sectioning\" (LiGS), which preserves the tissue with its optical implant in place and allows staining and clearing of a volume up to 500 \u03bcm in depth. We demonstrate the use of LiGS to determine the precise location of an optical fiber relative to a deep brain target and to investigate the implant-tissue interface. We show accurate cell registration of ex vivo histology with single-cell, two-photon calcium imaging, obtained through gradient refractive index (GRIN) lenses, and identify subpopulations based on immunohistochemistry. LiGS provides spatial information in experimental paradigms that use optical fibers and GRIN lenses and could help increase reproducibility through identification of fiber-to-target localization and molecular profiling.", "doi": "10.1016/j.celrep.2021.109744", "pmcid": "PMC8552649", "issn": "2211-1247", "publisher": "Cell Press", "publication": "Cell Reports", "publication_date": "2021-09-28", "series_number": "13", "volume": "36", "issue": "13", "pages": "Art. No. 109744" }, { "id": "authors:k7txd-cgj35", "collection": "authors", "collection_id": "k7txd-cgj35", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200925-104734056", "type": "article", "title": "Human embryo polarization requires PLC signaling to mediate trophectoderm specification", "author": [ { "family_name": "Zhu", "given_name": "Meng", "orcid": "0000-0001-6157-8840", "clpid": "Zhu-Meng" }, { "family_name": "Shahbazi", "given_name": "Marta", "orcid": "0000-0002-1599-5747", "clpid": "Shahbazi-Marta-N" }, { "family_name": "Martin", "given_name": "Angel", "clpid": "Martin-Angel" }, { "family_name": "Zhang", "given_name": "Chuanxin", "orcid": "0000-0003-0814-9275", "clpid": "Zhang-Chuanxin" }, { "family_name": "Sozen", "given_name": "Berna", "orcid": "0000-0001-5834-5819", "clpid": "Sozen-Berna" }, { "family_name": "Borsos", "given_name": "Mate", "orcid": "0000-0002-2801-8910", "clpid": "Borsos-M\u00e1t\u00e9" }, { "family_name": "Mandelbaum", "given_name": "Rachel S.", "orcid": "0000-0001-8611-1462", "clpid": "Mandelbaum-Rachel-S" }, { "family_name": "Paulson", "given_name": "Richard J.", "clpid": "Paulson-Richard-J" }, { "family_name": "Mol\u00e8", "given_name": "Matteo A.", "clpid": "Mol\u00e8-Matteo-A" }, { "family_name": "Esbert", "given_name": "Marga", "orcid": "0000-0002-0002-2272", "clpid": "Esbert-Marga" }, { "family_name": "Titus", "given_name": "Shiny", "orcid": "0000-0002-8391-825X", "clpid": "Titus-Shiny" }, { "family_name": "Scott", "given_name": "Richard T.", "clpid": "Scott-Richard-T-W" }, { "family_name": "Campbell", "given_name": "Alison", "orcid": "0000-0002-4065-5672", "clpid": "Campbell-Alison" }, { "family_name": "Fishel", "given_name": "Simon", "clpid": "Fishel-Simon" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Zhao", "given_name": "Han", "clpid": "Zhao-Han" }, { "family_name": "Wu", "given_name": "Keliang", "clpid": "Wu-Keliang" }, { "family_name": "Chen", "given_name": "Zi-Jiang", "clpid": "Chen-Zi-Jiang" }, { "family_name": "Seli", "given_name": "Emre", "orcid": "0000-0001-7464-8203", "clpid": "Seli-Emre" }, { "family_name": "de los Santos", "given_name": "Maria J.", "clpid": "de-los-Santos-Maria-Jose" }, { "family_name": "Zernicka-Goetz", "given_name": "Magdalena", "orcid": "0000-0002-7004-2471", "clpid": "Zernicka-Goetz-M" } ], "abstract": "Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8\u201316 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the eight-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos.", "doi": "10.7554/eLife.65068", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2021-09-27", "volume": "10", "pages": "Art. No. e65068" }, { "id": "authors:gdjer-36e66", "collection": "authors", "collection_id": "gdjer-36e66", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210625-220915439", "type": "article", "title": "Specific and behaviorally consequential astrocyte G_q GPCR signaling attenuation in\u00a0vivo with i\u03b2ARK", "author": [ { "family_name": "Nagai", "given_name": "Jun", "clpid": "Nagai-Jun" }, { "family_name": "Bellafard", "given_name": "Arash", "clpid": "Bellafard-Arash" }, { "family_name": "Qu", "given_name": "Zhen", "orcid": "0000-0002-3766-9838", "clpid": "Qu-Zhen" }, { "family_name": "Yu", "given_name": "Xinzhu", "orcid": "0000-0003-1244-0110", "clpid": "Yu-Xinzhu" }, { "family_name": "Ollivier", "given_name": "Matthias", "clpid": "Ollivier-Matthias" }, { "family_name": "Gangwani", "given_name": "Mohitkumar R.", "clpid": "Gangwani-Mohitkumar-R" }, { "family_name": "Diaz-Castro", "given_name": "Blanca", "clpid": "Diaz-Castro-Blanca" }, { "family_name": "Coppola", "given_name": "Giovanni", "orcid": "0000-0003-2105-1061", "clpid": "Coppola-Giovanni" }, { "family_name": "Schumacher", "given_name": "Sarah M.", "clpid": "Schumacher-Sarah-M" }, { "family_name": "Golshani", "given_name": "Peyman", "clpid": "Golshani-Peyman" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Khakh", "given_name": "Baljit S.", "orcid": "0000-0002-0939-1218", "clpid": "Khakh-Baljit-S" } ], "abstract": "Astrocytes respond to neurotransmitters and neuromodulators using G-protein-coupled receptors (GPCRs) to mediate physiological responses. Despite their importance, there has been no method to genetically, specifically, and effectively attenuate astrocyte G_q GPCR pathways to explore consequences of this prevalent signaling mechanism in vivo. We report a 122-residue inhibitory peptide from \u03b2-adrenergic receptor kinase 1 (i\u03b2ARK; and inactive D110A control) to attenuate astrocyte G_q GPCR signaling. i\u03b2ARK significantly attenuated G_q GPCR Ca\u00b2\u207a signaling in brain slices and, in vivo, altered behavioral responses, spared other GPCR responses, and did not alter astrocyte spontaneous Ca\u00b2\u207a signals, morphology, electrophysiological properties, or gene expression in the striatum. Furthermore, brain-wide attenuation of astrocyte G_q GPCR signaling with i\u03b2ARK using PHP.eB adeno-associated viruses (AAVs), when combined with c-Fos mapping, suggested nuclei-specific contributions to behavioral adaptation and spatial memory. i\u03b2ARK extends the toolkit needed to explore functions of astrocyte G_q GPCR signaling within neural circuits in vivo.", "doi": "10.1016/j.neuron.2021.05.023", "pmcid": "PMC8418870", "issn": "0896-6273", "publisher": "Cell Press", "publication": "Neuron", "publication_date": "2021-07-21", "series_number": "14", "volume": "109", "issue": "14", "pages": "2256-2274" }, { "id": "authors:3mzpv-c6728", "collection": "authors", "collection_id": "3mzpv-c6728", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210706-202344110", "type": "article", "title": "Microbiota regulate social behaviour via stress response neurons in the brain", "author": [ { "family_name": "Wu", "given_name": "Wei-Li", "orcid": "0000-0003-2610-1881", "clpid": "Wu-Wei-Li" }, { "family_name": "Adame", "given_name": "Mark D.", "clpid": "Adame-Mark-D" }, { "family_name": "Liou", "given_name": "Chia-Wei", "orcid": "0000-0002-9003-4065", "clpid": "Liou-Chia-Wei" }, { "family_name": "Barlow", "given_name": "Jacob T.", "orcid": "0000-0002-1842-4835", "clpid": "Barlow-Jacob-T" }, { "family_name": "Lai", "given_name": "Tzu-Ting", "clpid": "Lai-Tzu-Ting" }, { "family_name": "Sharon", "given_name": "Gil", "orcid": "0000-0002-4605-9943", "clpid": "Sharon-Gil" }, { "family_name": "Schretter", "given_name": "Catherine E.", "orcid": "0000-0002-3957-6838", "clpid": "Schretter-Catherine-E" }, { "family_name": "Needham", "given_name": "Brittany D.", "orcid": "0000-0002-0280-1886", "clpid": "Needham-Brittany-D" }, { "family_name": "Wang", "given_name": "Madelyn I.", "orcid": "0000-0001-7576-1179", "clpid": "Wang-Madelyn-I" }, { "family_name": "Tang", "given_name": "Weiyi", "orcid": "0000-0002-1279-1001", "clpid": "Tang-Weiyi" }, { "family_name": "Ousey", "given_name": "James", "orcid": "0000-0003-4886-0053", "clpid": "Ousey-James" }, { "family_name": "Lin", "given_name": "Yuan-Yuan", "clpid": "Lin-Yuan-Yuan" }, { "family_name": "Yao", "given_name": "Tzu-Hsuan", "clpid": "Yao-Tzu-Hsuan" }, { "family_name": "Abdel-Haq", "given_name": "Reem", "orcid": "0000-0002-7418-5736", "clpid": "Abdel-Haq-Reem" }, { "family_name": "Beadle", "given_name": "Keith", "orcid": "0000-0002-5695-6461", "clpid": "Beadle-Keith" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "abstract": "Social interactions among animals mediate essential behaviours, including mating, nurturing, and defence. The gut microbiota contribute to social activity in mice, but the gut\u2013brain connections that regulate this complex behaviour and its underlying neural basis are unclear. Here we show that the microbiome modulates neuronal activity in specific brain regions of male mice to regulate canonical stress responses and social behaviours. Social deviation in germ-free and antibiotic-treated mice is associated with elevated levels of the stress hormone corticosterone, which is primarily produced by activation of the hypothalamus\u2013pituitary\u2013adrenal (HPA) axis. Adrenalectomy, antagonism of glucocorticoid receptors, or pharmacological inhibition of corticosterone synthesis effectively corrects social deficits following microbiome depletion. Genetic ablation of glucocorticoid receptors in specific brain regions or chemogenetic inactivation of neurons in the paraventricular nucleus of the hypothalamus that produce corticotrophin-releasing hormone (CRH) reverse social impairments in antibiotic-treated mice. Conversely, specific activation of CRH-expressing neurons in the paraventricular nucleus induces social deficits in mice with a normal microbiome. Via microbiome profiling and in vivo selection, we identify a bacterial species, Enterococcus faecalis, that promotes social activity and reduces corticosterone levels in mice following social stress. These studies suggest that specific gut bacteria can restrain the activation of the HPA axis, and show that the microbiome can affect social behaviours through discrete neuronal circuits that mediate stress responses in the brain.", "doi": "10.1038/s41586-021-03669-y", "pmcid": "PMC8346519", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2021-07-15", "series_number": "7867", "volume": "595", "issue": "7867", "pages": "409-414" }, { "id": "authors:xbner-74v22", "collection": "authors", "collection_id": "xbner-74v22", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210519-141318892", "type": "article", "title": "The SHREAD gene therapy platform for paracrine delivery improves tumor localization and intratumoral effects of a clinical antibody", "author": [ { "family_name": "Smith", "given_name": "Sheena N.", "orcid": "0000-0002-2162-7107", "clpid": "Smith-Sheena-N" }, { "family_name": "Schubert", "given_name": "Rajib", "orcid": "0000-0002-7071-0134", "clpid": "Schubert-Rajib" }, { "family_name": "Simic", "given_name": "Branko", "orcid": "0000-0002-7314-3913", "clpid": "Simic-Branko" }, { "family_name": "Br\u00fccher", "given_name": "Dominik", "orcid": "0000-0002-3160-5542", "clpid": "Br\u00fccher-Dominik" }, { "family_name": "Schmid", "given_name": "Markus", "orcid": "0000-0002-4533-371X", "clpid": "Schmid-Markus" }, { "family_name": "Kirk", "given_name": "Niels", "clpid": "Kirk-Niels" }, { "family_name": "Freitag", "given_name": "Patrick C.", "orcid": "0000-0001-8803-6048", "clpid": "Freitag-Patrick-C" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Pluckthun", "given_name": "Andreas", "orcid": "0000-0003-4191-5306", "clpid": "Pluckthun-Andreas" } ], "abstract": "The goal of cancer-drug delivery is to achieve high levels of therapeutics within tumors with minimal systemic exposure that could cause toxicity. Producing biologics directly in situ where they diffuse and act locally is an attractive alternative to direct administration of recombinant therapeutics, as secretion by the tumor itself provides high local concentrations that act in a paracrine fashion continuously over an extended duration (paracrine delivery). We have engineered a SHielded, REtargeted ADenovirus (SHREAD) gene therapy platform that targets specific cells based on chosen surface markers and converts them into biofactories secreting therapeutics. In a proof of concept, a clinically approved antibody is delivered to orthotopic tumors in a model system in which precise biodistribution can be determined using tissue clearing with passive CLARITY technique (PACT) with high-resolution three-dimensional imaging and feature quantification within the tumors made transparent. We demonstrate high levels of tumor cell\u2013specific transduction and significant and durable antibody production. PACT gives a localized quantification of the secreted therapeutic and allows us to directly observe enhanced pore formation in the tumor and destruction of the intact vasculature. In situ production of the antibody led to an 1,800-fold enhanced tumor-to-serum antibody concentration ratio compared to direct administration. Our detailed biochemical and microscopic analyses thus show that paracrine delivery with SHREAD could enable the use of highly potent therapeutic combinations, including those with systemic toxicity, to reach adequate therapeutic windows.", "doi": "10.1073/pnas.2017925118", "pmcid": "PMC8166199", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2021-05-25", "series_number": "21", "volume": "118", "issue": "21", "pages": "Art. No. e2017925118" }, { "id": "authors:zgj58-7kp67", "collection": "authors", "collection_id": "zgj58-7kp67", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210629-201655221", "type": "article", "title": "Use of high-content imaging to quantify transduction of AAV-PHP viruses in the brain following systemic delivery", "author": [ { "family_name": "Smith", "given_name": "Edward J.", "clpid": "Smith-Edward-J" }, { "family_name": "Farshim", "given_name": "Pamela P.", "orcid": "0000-0003-4816-6635", "clpid": "Farshim-Pamela-P" }, { "family_name": "Flomen", "given_name": "Rachel", "orcid": "0000-0002-6434-5804", "clpid": "Flomen-Rachel" }, { "family_name": "Jones", "given_name": "Samuel T.", "clpid": "Jones-Samuel-T" }, { "family_name": "McAteer", "given_name": "Sean J.", "clpid": "McAteer-Sean-J" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-Benjamin-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Bates", "given_name": "Gillian P.", "orcid": "0000-0002-4041-6305", "clpid": "Bates-Gillian-P" } ], "abstract": "The engineering of the AAV-PHP capsids was an important development for CNS research and the modulation of gene expression in the brain. They cross the blood brain barrier and transduce brain cells after intravenous systemic delivery, a property dependent on the genotype of Ly6a, the AAV-PHP capsid receptor. It is important to determine the transduction efficiency of a given viral preparation, as well as the comparative tropism for different brain cells; however, manual estimation of adeno-associated viral transduction efficiencies can be biased and time consuming. Therefore, we have used the Opera Phenix high-content screening system, equipped with the Harmony processing and analysis software, to reduce bias and develop an automated approach to determining transduction efficiency in the mouse brain. We used R Studio and 'gatepoints' to segment the data captured from coronal brain sections into brain regions of interest. C57BL/6J and CBA/Ca mice were injected with an AAV-PHP.B virus containing a green fluorescent protein reporter with a nuclear localization signal. Coronal sections at 600\u2009\u03bcm intervals throughout the entire brain were stained with Hoechst dye, combined with immunofluorescence to NeuN and green fluorescent protein to identify all cell nuclei, neurons and transduced cells, respectively. Automated data analysis was applied to give an estimate of neuronal percentages and transduction efficiencies throughout the entire brain as well as for the cortex, striatum and hippocampus. The data from each coronal section from a given mouse were highly comparable. The percentage of neurons in the C57BL/6J and CBA/Ca brains was approximately 40% and this was higher in the cortex than striatum and hippocampus. The systemic injection of AAV-PHP.B resulted in similar transduction rates across the entire brain for C57BL/6J mice. Approximately 10\u201315% of all cells were transduced, with neuronal transduction efficiencies ranging from 5% to 15%, estimates that were similar across brain regions, and were in contrast to the much more localized transduction efficiencies achieved through intracerebral injection. We confirmed that the delivery of the AAV-PHP.B viruses to the brain from the vasculature resulted in widespread transduction. Our methodology allows the rapid comparison of transduction rates between brain regions producing comparable data to more time-consuming approaches. The methodology developed here can be applied to the automated quantification of any parameter of interest that can be captured as a fluorescent signal.", "doi": "10.1093/braincomms/fcab105", "pmcid": "PMC8200048", "issn": "2632-1297", "publisher": "Oxford University Press", "publication": "Brain Communications", "publication_date": "2021-05-17", "series_number": "2", "volume": "3", "issue": "2", "pages": "Art. No. fcab105" }, { "id": "authors:k5yvf-a4q19", "collection": "authors", "collection_id": "k5yvf-a4q19", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200421-092814882", "type": "article", "title": "Enhancer viruses for combinatorial cell-subclass-specific labeling", "author": [ { "family_name": "Graybuck", "given_name": "Lucas T.", "orcid": "0000-0002-8814-6818", "clpid": "Graybuck-Lucas-T" }, { "family_name": "Daigle", "given_name": "Tanya L.", "orcid": "0000-0001-9700-8452" }, { "family_name": "Sede\u00f1o-Cort\u00e9s", "given_name": "Adriana E.", "orcid": "0000-0001-7959-8970" }, { "family_name": "Walker", "given_name": "Miranda", "orcid": "0000-0002-3767-1591" }, { "family_name": "Kalmbach", "given_name": "Brian", "orcid": "0000-0003-3136-8097" }, { "family_name": "Lenz", "given_name": "Garreck H.", "orcid": "0000-0002-3233-0763" }, { "family_name": "Morin", "given_name": "Elyse", "orcid": "0000-0002-7310-1561" }, { "family_name": "Nguyen", "given_name": "Thuc Nghi", "orcid": "0000-0002-6466-5883" }, { "family_name": "Garren", "given_name": "Emma" }, { "family_name": "Bendrick", "given_name": "Jacqueline L.", "orcid": "0000-0002-2694-9914" }, { "family_name": "Kim", "given_name": "Tae Kyung", "orcid": "0000-0001-9646-5969" }, { "family_name": "Zhou", "given_name": "Thomas" }, { "family_name": "Mortrud", "given_name": "Marty", "orcid": "0000-0002-4570-9887" }, { "family_name": "Yao", "given_name": "Shenqin", "orcid": "0000-0003-2992-4752" }, { "family_name": "Sieverts", "given_name": "La'Akea", "orcid": "0000-0002-1385-086X" }, { "family_name": "Larsen", "given_name": "Rachael", "orcid": "0000-0003-0178-003X" }, { "family_name": "Gore", "given_name": "Bryan B.", "orcid": "0000-0003-1721-4235" }, { "family_name": "Szelenyi", "given_name": "Eric R.", "orcid": "0000-0003-2636-0912" }, { "family_name": "Trader", "given_name": "Cameron", "orcid": "0000-0001-8214-1541" }, { "family_name": "Balaram", "given_name": "Pooja" }, { "family_name": "van Velthoven", "given_name": "Cindy T. J.", "orcid": "0000-0001-5120-4546" }, { "family_name": "Chiang", "given_name": "Megan", "orcid": "0000-0002-7306-3016" }, { "family_name": "Mich", "given_name": "John K.", "orcid": "0000-0002-1626-1139" }, { "family_name": "Dee", "given_name": "Nick", "orcid": "0000-0002-2831-9254" }, { "family_name": "Goldy", "given_name": "Jeff", "orcid": "0000-0001-5140-6922" }, { "family_name": "Cetin", "given_name": "Ali H.", "orcid": "0000-0003-1510-0517" }, { "family_name": "Smith", "given_name": "Kimberly", "orcid": "0000-0002-3142-1970" }, { "family_name": "Way", "given_name": "Sharon W.", "orcid": "0000-0002-9598-2240" }, { "family_name": "Esposito", "given_name": "Luke", "orcid": "0000-0002-3565-9126" }, { "family_name": "Yao", "given_name": "Zizhen", "orcid": "0000-0002-9361-5607" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Sunkin", "given_name": "Susan M.", "orcid": "0000-0001-9893-3834" }, { "family_name": "Lein", "given_name": "Ed", "orcid": "0000-0001-9012-6552" }, { "family_name": "Levi", "given_name": "Boaz P.", "orcid": "0000-0002-8346-872X" }, { "family_name": "Ting", "given_name": "Jonathan T.", "orcid": "0000-0001-8266-0392" }, { "family_name": "Zeng", "given_name": "Hongkui", "orcid": "0000-0002-0326-5878" }, { "family_name": "Tasic", "given_name": "Bosiljka", "orcid": "0000-0002-6861-4506" } ], "abstract": "Rapid cell type identification by new genomic single-cell analysis methods has not been met with efficient experimental access to these cell types. To facilitate access to specific neural populations in mouse cortex, we collected chromatin accessibility data from individual cells and identified enhancers specific for cell subclasses and types. When cloned into recombinant adeno-associated viruses (AAVs) and delivered to the brain, these enhancers drive transgene expression in specific cortical cell subclasses. We extensively characterized several enhancer AAVs to show that they label different projection neuron subclasses as well as a homologous neuron subclass in human cortical slices. We also show how coupling enhancer viruses expressing recombinases to a newly generated transgenic mouse, Ai213, enables strong labeling of three different neuronal classes/subclasses in the brain of a single transgenic animal. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.", "doi": "10.1016/j.neuron.2021.03.011", "pmcid": "PMC8610077", "issn": "0896-6273", "publisher": "Cell Press", "publication": "Neuron", "publication_date": "2021-05-05", "series_number": "9", "volume": "109", "issue": "9", "pages": "1449-1464" }, { "id": "authors:pf45e-w5a27", "collection": "authors", "collection_id": "pf45e-w5a27", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210527-160526973", "type": "article", "title": "Simultaneous Detection of AAV Genome, Transcript, and Protein Localization in Intact Cells and Tissues at High Resolution", "author": [ { "family_name": "Borsos", "given_name": "M\u00e1t\u00e9", "clpid": "Borsos-M\u00e1t\u00e9" }, { "family_name": "Chen", "given_name": "Xinhong", "clpid": "Chen-Xinhong" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Recent years have seen significant progress in AAV capsid engineering for gene delivery with increased efficiency and desired cell-type specificity to match the needs of pre-clinical research and gene therapy. The cellular uptake of AAVs, however, can stop short of the ultimate goal of cargo protein production - due to AAV silencing, insufficient nuclear transport, inefficient uncoating, failed second-strand synthesis, or other still to be discovered mechanisms. Defining the relationship between AAV genome uptake, transcription and cargo protein synthesis efficiencies in different cell types and tissues can help bypass key bottlenecks in gene delivery and guide effective AAV engineering. We adapted a recently published in-situ-transcription-based signal amplification method, the \"Zombie technique\" (1), to detect AAV genomes in a variety of fixed cells and tissues. Zombie involves producing 20 to 380 base-pair long barcode transcripts by exogenously-supplied T7 phage polymerase, which are then visualized at high resolution in intact cells through fluorescent in situ hybridization (FISH) by hybridization chain reaction (HCR). We demonstrate that Zombie enables simultaneous detection of AAV genomes and AAV transcripts by FISH and cargo proteins by immunofluorescence. Consequently, we can determine the rates of AAV genome nuclear uptake, cargo transcription and cargo translation at different time points post-infection. Combining the Zombie method with cell-type-specific markers (protein immunolabelling or mRNA FISH) can give insights into subcellular AAV processing in complex tissues and help pinpoint to limiting factors in the overall transduction process. Interestingly, we find that after direct brain injection of AAV6, although cargo genomes do enter the nuclei of microglia, these genomes do not result in functional cargo protein production in this cell type. Furthermore, we analyze the pattern of AAV genome nuclear uptake, transcription and translation in mice after systemic delivery of AAV9 and engineered AAV capsids (AAV.PHP.eB, AAV.CAP-B10) (2, 3) across brain, liver and spleen. In conclusion, the Zombie technique adapted to AAV vectors enables simultaneous cargo genome, mRNA and protein visualization and thus can help differentiate cellular and subcellular processing steps of engineered gene delivery vectors, facilitating their refinement for basic and translational research.", "doi": "10.1016/j.ymthe.2021.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2021-04-27", "series_number": "4, S1", "volume": "29", "issue": "4, S1", "pages": "142-143" }, { "id": "authors:tf90z-d3h15", "collection": "authors", "collection_id": "tf90z-d3h15", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210527-161220137", "type": "article", "title": "Deep Tropism Profiling of Barcoded AAV Capsid and Cargo Pools in Intact Tissue Using High-Throughput Ultrasensitive Sequential FISH", "author": [ { "family_name": "Jang", "given_name": "Min Jee", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Coughlin", "given_name": "Gerard M.", "clpid": "Coughlin-Gerard-M" }, { "family_name": "Zhang", "given_name": "Yameng", "clpid": "Zhang-Yameng" }, { "family_name": "Koulena", "given_name": "Noushin", "orcid": "0000-0002-9419-5712", "clpid": "Koulena-Noushin" }, { "family_name": "Schindler", "given_name": "Simone", "clpid": "Schindler-Simone" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Genetic access to specific cell types through minimally invasive routes\nis of particular interest in basic research and clinical applications.\nExtensive efforts have been made in engineering gene delivery\nvectors, such as recombinant adeno-associated viruses (rAAVs),\nand gene regulatory elements to achieve this goal. Despite many\ninteresting candidates, revealed for example from directed evolution\nvia M-CREATE (Sripriya Ravindra Kumar et al., Nature Methods,\n2020), histology-based characterization presents a bottleneck due to\nthe limited number of variants and/or cell types that can be investigated\nat once. To address this, we have developed ultrasensitive sequential\nFISH (useqFISH) for multiplexed detection of both endogenous and\nbarcoded transgene transcripts in intact tissue with single-molecule\nresolution. By combining two amplification strategies (rolling circle\namplification, RCA, and hybridization chain reaction, HCR), we\nachieved a 2.7- or 6.7-fold increased signal-to-background ratio of\nuseqFISH in comparison to one with RCA or HCR only amplification,\nrespectively. UseqFISH allowed us to detect endogenous genes with\na single probe pair (20-nucleotide (nt) for each) and, in transfected\ncell cultures, to distinguish capsid variants with genomes differing by\nonly 7-mer peptide modification. We further improved useqFISH by\nestablishing an automated single-molecule imaging and microfluidic\nsolution exchange system and an analytical pipeline for 3D imaging\ndata. To demonstrate the applicability of useqFISH for in vivo AAV\nprofiling, we employed this method to further characterize a pool of\n6 AAV capsid variants that we found to be highly efficient for brainwide\nand/or cell-type biased transduction in the mouse brain following\nsystemic delivery. We designed unique nucleic acid barcodes (160-nt)\nin the 3'UTR of each viral genome and retro-orbitally injected the\npooled AAVs into 2 C57BL6/J mice at a dose of 5e10 viral genomes\n(vg) per variant (total 3e11 vg/mouse). For transcript detection, 11\ncanonical cell-type markers (e.g., Slc17a7, Gad1, Pvalb, SST, VIP, etc)\nwere used together with probes against the viral genome barcodes, to\ncharacterize the cell-type tropisms of each variant. Next, we designed\na pool of 103 barcoded AAV genomes carrying 4 tandem repeats of a\nunique miRNA target site. We packaged these genomes into AAV-PHP.\neB and delivered to 3 C57BL6/J mice at a dose of 1e10 vg/variant (total\n~1e12 vg/mouse). Using useqFISH, we were able to assess the ability of\neach miRNA target site to dampen transgene expression in different cell\ntypes, thereby revealing useful intersectional strategies to refine celltype-\nspecific transgene expression with capsid/cargo combinations.\nThese results demonstrate that useqFISH allows for high-throughput\ncharacterization of pooled genetic variants of viral capsids and gene\nregulatory elements in intact tissue and thus enables comprehensive\nprofiling of genetic toolkits for precise access to targets of interest.", "doi": "10.1016/j.ymthe.2021.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2021-04-27", "series_number": "4, S1", "volume": "29", "issue": "4, S1", "pages": "142" }, { "id": "authors:hfhnb-zj890", "collection": "authors", "collection_id": "hfhnb-zj890", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210428-094505098", "type": "article", "title": "Cholinergic neurons constitutively engage the ISR for dopamine modulation and skill learning in mice", "author": [ { "family_name": "Helseth", "given_name": "Ashley R.", "orcid": "0000-0002-7383-823X", "clpid": "Helseth-Ashley-R" }, { "family_name": "Hernandez-Martinez", "given_name": "Ricardo", "orcid": "0000-0002-9376-8363", "clpid": "Hernandez-Martinez-Ricardo" }, { "family_name": "Hall", "given_name": "Victoria L.", "orcid": "0000-0001-8882-9443", "clpid": "Hall-Victoria-L" }, { "family_name": "Oliver", "given_name": "Matthew L.", "orcid": "0000-0002-8474-4589", "clpid": "Oliver-Matthew-L" }, { "family_name": "Turner", "given_name": "Brandon D.", "orcid": "0000-0002-2887-1719", "clpid": "Turner-Brandon-D" }, { "family_name": "Caffall", "given_name": "Zachary F.", "orcid": "0000-0003-4601-6799", "clpid": "Caffall-Zachary-F" }, { "family_name": "Rittiner", "given_name": "Joseph E.", "orcid": "0000-0003-3592-6500", "clpid": "Rittiner-Joseph-E" }, { "family_name": "Shipman", "given_name": "Miranda K.", "orcid": "0000-0002-4418-8717", "clpid": "Shipman-Miranda-K" }, { "family_name": "King", "given_name": "Connor S.", "orcid": "0000-0002-3740-9817", "clpid": "King-Connor-S" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Gerfen", "given_name": "Charles", "orcid": "0000-0001-9008-4882", "clpid": "Gerfen-Charles" }, { "family_name": "Costa-Mattioli", "given_name": "Mauro", "orcid": "0000-0002-9809-4732", "clpid": "Costa-Mattioli-Mauro" }, { "family_name": "Calakos", "given_name": "Nicole", "orcid": "0000-0002-9918-3294", "clpid": "Calakos-Nicole" } ], "abstract": "Introduction: The integrated stress response (ISR) is a highly conserved biochemical pathway that, upon activation, markedly shifts which proteins are synthesized. Its roles in proteostasis, synaptic plasticity, learning, and memory have made the pathway an attractive therapeutic target for systemic and brain diseases. Preclinical studies showing the capacity of small-molecule ISR inhibitors to enhance some forms of learning and memory have further highlighted its translational potential. Despite strong and accumulating evidence for the ISR as a potent modifier of plasticity, learning, and memory in diverse behavioral paradigms, the cellular sites of action and time course of ISR engagement are less well understood. \n\nRationale: To better understand ISR roles in the brain, we developed a two-color reporter of ISR activation state, SPOTlight, and delivered it through a viral vector for brainwide imaging with cellular resolution. SPOTlight was designed to differentially translate green or red fluorescent proteins from a single transcript based on ISR activation state. We first established that reporter readouts corresponded to known manipulations of the ISR using immunohistochemical analyses. SPOTlight signal indicating high ISR activation at steady state in striatal cholinergic interneurons (CINs) was validated using immunohistochemical analyses. To understand the factors driving ISR activation in CINs, we inhibited CIN activity chemogenetically and assessed the ISR state. Selective pharmacological and genetic manipulations were combined with electrophysiological CIN recordings to establish the mechanisms by which ISR state influences CIN physiology. Fluorescent dopamine reporter imaging was used to examine the circuit-level effects of CIN ISR state on evoked striatal dopamine transients in acute brain slices. Finally, task training assays were used to measure the behavioral effects of CIN ISR activity. \n\nResults: Pharmacological ISR activators and inhibitors delivered in vivo differentially modified levels of SPOTlight-encoded fluorescent proteins and corresponded to immunohistochemical markers of ISR activation in the mouse brain. As expected, in the normal mature mouse brain, SPOTlight revealed only sparse cells with evidence of ISR activation. Unexpectedly, we also found a class of neurons that showed population-wide ISR activation: tonically firing striatal CINs. Chemogenetic inhibition of CIN firing reduced ISR activation, indicating an activity-dependent component. CINs also appeared to be distinctive in ISR engagement; a survey of SPOTlight in other cell types with tonic or high-firing properties did not show similarly high and population-wide ISR engagement. In CINs, manipulations inhibiting the ISR inverted the normal type 2 dopamine receptor (D2R) response from slowing to increasing CIN firing through a mechanism that reduced small-conductance calcium-activated potassium channel currents. Cell-autonomous ISR inhibition in CINs also inverted D2R modulation of evoked striatal dopamine and altered skill learning by increasing the velocity of movement in two learned tasks. \n\nConclusion: Our study defines a distinct role for the ISR in brain, neuromodulation, which expands our understanding of how the ISR influences learning and memory. We show that steady-state ISR activation in striatal cholinergic interneurons determines their response to dopaminergic modulation, shapes circuit-level dopamine release, and regulates learned skill performance. In this context, ISR activation is activity dependent and influences CIN cellular excitability. As ISR-inhibiting drugs move toward the clinical setting, our results highlight an unappreciated mechanism for their effects on learned behaviors. Our results also prompt further examination of the sites of ISR action in various forms of synaptic plasticity given the importance of cholinergic and dopaminergic neuromodulation in this process. Finally, SPOTlight provides a tool with which to explore when and where the ISR is activated across diverse contexts, including developmental, learning-related, and diseased states.", "doi": "10.1126/science.abe1931", "pmcid": "PMC8457366", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2021-04-23", "series_number": "6540", "volume": "372", "issue": "6540", "pages": "Art. No. eabe1931" }, { "id": "authors:twtz6-z9n14", "collection": "authors", "collection_id": "twtz6-z9n14", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200421-093728277", "type": "article", "title": "Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex", "author": [ { "family_name": "Mich", "given_name": "John K.", "clpid": "Mich-John-K" }, { "family_name": "Graybuck", "given_name": "Lucas T." }, { "family_name": "Hess", "given_name": "Erik E." }, { "family_name": "Mahoney", "given_name": "Joseph T." }, { "family_name": "Kojimo", "given_name": "Yoshiko" }, { "family_name": "Ding", "given_name": "Yi" }, { "family_name": "Somasundaram", "given_name": "Saroja" }, { "family_name": "Miller", "given_name": "Jeremy A." }, { "family_name": "Kalmbach", "given_name": "Brian E.", "orcid": "0000-0003-3136-8097" }, { "family_name": "Radaelli", "given_name": "Cristina" }, { "family_name": "Gore", "given_name": "Bryan B." }, { "family_name": "Weed", "given_name": "Natalie" }, { "family_name": "Omstead", "given_name": "Victoria" }, { "family_name": "Bishaw", "given_name": "Yemeserach" }, { "family_name": "Shapovalova", "given_name": "Nadiya V." }, { "family_name": "Martinez", "given_name": "Refugio A." }, { "family_name": "Fong", "given_name": "Olivia" }, { "family_name": "Yao", "given_name": "Shenqin" }, { "family_name": "Mortrud", "given_name": "Marty" }, { "family_name": "Chong", "given_name": "Peter" }, { "family_name": "Loftus", "given_name": "Luke" }, { "family_name": "Bertagnolli", "given_name": "Darren" }, { "family_name": "Goldy", "given_name": "Jeff" }, { "family_name": "Casper", "given_name": "Tamara" }, { "family_name": "Dee", "given_name": "Nick" }, { "family_name": "Opitz-Araya", "given_name": "Ximena" }, { "family_name": "Cetin", "given_name": "Ali" }, { "family_name": "Smith", "given_name": "Kimberly A." }, { "family_name": "Gwinn", "given_name": "Ryder P." }, { "family_name": "Cobbs", "given_name": "Charles" }, { "family_name": "Ko", "given_name": "Andrew L." }, { "family_name": "Ojemann", "given_name": "Jeffrey G." }, { "family_name": "Keene", "given_name": "C. Dirk" }, { "family_name": "Silbergeld", "given_name": "Daniel L." }, { "family_name": "Sunkin", "given_name": "Susan M." }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Horwitz", "given_name": "Gregory D." }, { "family_name": "Zeng", "given_name": "Hongkui" }, { "family_name": "Tasic", "given_name": "Bosiljka" }, { "family_name": "Lein", "given_name": "Ed S." }, { "family_name": "Ting", "given_name": "Jonathan T." }, { "family_name": "Levi", "given_name": "Boaz P." } ], "abstract": "Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.", "doi": "10.1016/j.celrep.2021.108754", "issn": "2211-1247", "publisher": "Elsevier", "publication": "Cell Reports", "publication_date": "2021-03-30", "series_number": "13", "volume": "34", "issue": "13", "pages": "Art. No. 108754" }, { "id": "authors:hk6b4-y9189", "collection": "authors", "collection_id": "hk6b4-y9189", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200728-094010838", "type": "article", "title": "Dorsal Raphe Dopamine Neurons Signal Motivational Salience Dependent on Internal State, Expectation, and Behavioral Context", "author": [ { "family_name": "Cho", "given_name": "Jounhong Ryan", "orcid": "0000-0001-9542-716X", "clpid": "Cho-Jounhong-Ryan" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Kahan", "given_name": "Anat", "orcid": "0000-0002-4799-3017", "clpid": "Kahan-Anat" }, { "family_name": "Robinson", "given_name": "J. Elliott", "orcid": "0000-0001-9417-3938", "clpid": "Robinson-J-Elliott" }, { "family_name": "Wagenaar", "given_name": "Daniel A.", "orcid": "0000-0002-6222-761X", "clpid": "Wagenaar-D-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "The ability to recognize motivationally salient events and respond to them adaptively is critical for survival. Here we tested whether dopamine (DA) neurons in the dorsal raphe nucleus (DRN) contribute to this process. Population recordings of DRN^(DA) neurons during associative learning tasks showed that their activity dynamically tracks salience, developing excitation to both reward- and punishment-paired cues. The DRNDA response to reward-predicting cues was diminished after satiety, suggesting modulation by internal states. DRN^(DA) activity was also greater for unexpected outcomes than for expected outcomes. Two-photon imaging of DRN^(DA) neurons demonstrated that the majority of individual neurons developed activation to reward predicting cues but not to punishment-predicting cues, which was surprising and qualitatively distinct from the population results. Head-fixation during fear learning abolished the neural response to aversive cues, indicating modulation by behavioral context. Overall, these results suggest that DRN^(DA) neurons encode motivational salience, dependent on internal and external factors.", "doi": "10.1523/JNEUROSCI.2690-20.2021", "pmcid": "PMC8018733", "issn": "0270-6474", "publisher": "Society for Neuroscience", "publication": "Journal of Neuroscience", "publication_date": "2021-03-24", "series_number": "12", "volume": "41", "issue": "12", "pages": "2645-2655" }, { "id": "authors:hjhgm-hc812", "collection": "authors", "collection_id": "hjhgm-hc812", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201217-143607354", "type": "article", "title": "Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning", "author": [ { "family_name": "Unger", "given_name": "Elizabeth K.", "orcid": "0000-0002-8235-7534", "clpid": "Unger-Elizabeth-K" }, { "family_name": "Keller", "given_name": "Jacob P.", "orcid": "0000-0001-7487-4104" }, { "family_name": "Altermatt", "given_name": "Michael", "orcid": "0000-0003-2841-5374", "clpid": "Altermatt-Michael" }, { "family_name": "Liang", "given_name": "Ruqiang", "orcid": "0000-0002-3075-4554" }, { "family_name": "Matsui", "given_name": "Aya", "orcid": "0000-0003-4437-8278" }, { "family_name": "Dong", "given_name": "Chunyang", "orcid": "0000-0002-4820-4454" }, { "family_name": "Hon", "given_name": "Olivia J.", "orcid": "0000-0003-1086-1421" }, { "family_name": "Yao", "given_name": "Zizhen", "orcid": "0000-0002-9361-5607" }, { "family_name": "Sun", "given_name": "Junqing" }, { "family_name": "Banala", "given_name": "Sambashiva", "orcid": "0000-0002-9463-7664" }, { "family_name": "Flanigan", "given_name": "Meghan E.", "orcid": "0000-0002-3185-7459" }, { "family_name": "Jaffe", "given_name": "David A.", "orcid": "0000-0003-4773-6982" }, { "family_name": "Hartanto", "given_name": "Samantha", "orcid": "0000-0001-8513-5294" }, { "family_name": "Carlen", "given_name": "Jane", "orcid": "0000-0002-2538-6670" }, { "family_name": "Mizuno", "given_name": "Grace O.", "orcid": "0000-0003-4786-3084" }, { "family_name": "Borden", "given_name": "Phillip M.", "orcid": "0000-0003-1653-7067" }, { "family_name": "Shivange", "given_name": "Amol V.", "orcid": "0000-0002-4169-2969", "clpid": "Shivange-Amol-V" }, { "family_name": "Cameron", "given_name": "Lindsay P.", "orcid": "0000-0002-8420-7898" }, { "family_name": "Sinning", "given_name": "Steffen", "orcid": "0000-0001-6971-6929" }, { "family_name": "Underhill", "given_name": "Suzanne M." }, { "family_name": "Olson", "given_name": "David E.", "orcid": "0000-0002-4517-0543" }, { "family_name": "Amara", "given_name": "Susan G.", "orcid": "0000-0001-8914-1106" }, { "family_name": "Temple Lang", "given_name": "Duncan", "orcid": "0000-0003-0159-1546" }, { "family_name": "Rudnick", "given_name": "Gary", "orcid": "0000-0002-7622-4110" }, { "family_name": "Marvin", "given_name": "Jonathan S.", "orcid": "0000-0003-2294-4515" }, { "family_name": "Lavis", "given_name": "Luke D.", "orcid": "0000-0002-0789-6343" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Alvarez", "given_name": "Veronica A.", "orcid": "0000-0003-2611-8675" }, { "family_name": "Fisher", "given_name": "Andrew J.", "orcid": "0000-0003-3488-6594" }, { "family_name": "Prescher", "given_name": "Jennifer A.", "orcid": "0000-0002-9250-4702" }, { "family_name": "Kash", "given_name": "Thomas L.", "orcid": "0000-0002-4747-4495" }, { "family_name": "Yarov-Yarovoy", "given_name": "Vladimir", "orcid": "0000-0002-2325-4834" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Looger", "given_name": "Loren L.", "orcid": "0000-0002-7531-1757" }, { "family_name": "Tian", "given_name": "Lin", "orcid": "0000-0001-7012-6926" } ], "abstract": "Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.", "doi": "10.1016/j.cell.2020.11.040", "pmcid": "PMC8025677", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "2020-12-23", "series_number": "7", "volume": "183", "issue": "7", "pages": "1986-2002" }, { "id": "authors:0pen5-78s90", "collection": "authors", "collection_id": "0pen5-78s90", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200817-140210217", "type": "article", "title": "Negative feedback control of neuronal activity by microglia", "author": [ { "family_name": "Badimon", "given_name": "Ana", "clpid": "Badimon-Ana" }, { "family_name": "Strasburger", "given_name": "Hayley J." }, { "family_name": "Ayata", "given_name": "Pinar" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Nair", "given_name": "Aditya", "clpid": "Nair-Aditya" }, { "family_name": "Ikegami", "given_name": "Ako" }, { "family_name": "Hwang", "given_name": "Philip" }, { "family_name": "Chan", "given_name": "Andrew T." }, { "family_name": "Graves", "given_name": "Steven M." }, { "family_name": "Uweru", "given_name": "Jospeh O." }, { "family_name": "Ledderose", "given_name": "Carola" }, { "family_name": "Gunes Kutlu", "given_name": "Munir" }, { "family_name": "Wheeler", "given_name": "Michael A." }, { "family_name": "Kahan", "given_name": "Anat", "clpid": "Kahan-Anat" }, { "family_name": "Ishikawa", "given_name": "Masago" }, { "family_name": "Wang", "given_name": "Ying-Chih" }, { "family_name": "Loh", "given_name": "Yong-Hwee E." }, { "family_name": "Jiang", "given_name": "Jean X." }, { "family_name": "Sumeier", "given_name": "D. James" }, { "family_name": "Robson", "given_name": "Simon C." }, { "family_name": "Junger", "given_name": "Wolfgang G." }, { "family_name": "Sebra", "given_name": "Robert" }, { "family_name": "Calipari", "given_name": "Erin S." }, { "family_name": "Kenny", "given_name": "Paul J." }, { "family_name": "Eyo", "given_name": "Ukpong B." }, { "family_name": "Colonna", "given_name": "Marco" }, { "family_name": "Qunitana", "given_name": "Francisco J." }, { "family_name": "Wake", "given_name": "Hiroaki" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Schaefer", "given_name": "Anne" } ], "abstract": "Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A1R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.", "doi": "10.1038/s41586-020-2777-8", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2020-10-15", "series_number": "7829", "volume": "586", "issue": "7829", "pages": "417-423" }, { "id": "authors:m1wxm-btm54", "collection": "authors", "collection_id": "m1wxm-btm54", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200810-121624161", "type": "article", "title": "Expanding the brain researcher's toolkit", "author": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Despite the wealth and quality of basic neuroscience research, there is still little we can do to treat or prevent most brain disorders. Industry efforts, meanwhile, have shied away from this field, particularly after a series of major drug candidates for the treatment of Alzheimer's disease failed to meet expectations (1).", "doi": "10.1126/science.abd2660", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2020-08-07", "series_number": "6504", "volume": "369", "issue": "6504", "pages": "637" }, { "id": "authors:zrpnd-mj732", "collection": "authors", "collection_id": "zrpnd-mj732", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200121-141523675", "type": "article", "title": "Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types", "author": [ { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-Sripriya" }, { "family_name": "Miles", "given_name": "Timothy F.", "orcid": "0000-0001-6591-3271", "clpid": "Miles-T-F" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Brown", "given_name": "David", "orcid": "0000-0002-9757-1744", "clpid": "Brown-David" }, { "family_name": "Dobreva", "given_name": "Tatyana", "orcid": "0000-0002-2625-8873", "clpid": "Dobreva-Tatyana" }, { "family_name": "Huang", "given_name": "Qin", "clpid": "Huang-Qin" }, { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Luo", "given_name": "Yicheng", "orcid": "0000-0003-3704-2389", "clpid": "Luo-Yicheng" }, { "family_name": "Einarsson", "given_name": "Petur H.", "clpid": "Einarsson-Petur-H" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-Alon" }, { "family_name": "Jang", "given_name": "Min J.", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-Benjamin-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood\u2013brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications.", "doi": "10.1038/s41592-020-0799-7", "pmcid": "PMC7219404", "issn": "1548-7091", "publisher": "Nature Publishing Group", "publication": "Nature Methods", "publication_date": "2020-05", "series_number": "5", "volume": "17", "issue": "5", "pages": "541-550" }, { "id": "authors:jev5y-8ky19", "collection": "authors", "collection_id": "jev5y-8ky19", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200504-132509459", "type": "article", "title": "Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation", "author": [ { "family_name": "Seo", "given_name": "Jai Woong", "orcid": "0000-0002-2732-7498", "clpid": "Seo-Jai-Woong" }, { "family_name": "Ingham", "given_name": "Elizabeth S.", "clpid": "Ingham-E-S" }, { "family_name": "Mahakian", "given_name": "Lisa", "clpid": "Mahakian-L" }, { "family_name": "Tumbale", "given_name": "Spencer", "clpid": "Tumbale-S" }, { "family_name": "Wu", "given_name": "Bo", "clpid": "Wu-Bo" }, { "family_name": "Aghevlian", "given_name": "Sadaf", "clpid": "Aghevlian-S" }, { "family_name": "Shams", "given_name": "Shahin", "orcid": "0000-0001-9122-0364", "clpid": "Shams-S" }, { "family_name": "Baikoghli", "given_name": "Mo", "clpid": "Baikoghli-M" }, { "family_name": "Jain", "given_name": "Poorva", "clpid": "Jain-P" }, { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Goeden", "given_name": "Nick", "clpid": "Goeden-N" }, { "family_name": "Dobreva", "given_name": "Tatyana", "orcid": "0000-0002-2625-8873", "clpid": "Dobreva-T" }, { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-N-C" }, { "family_name": "Chavez", "given_name": "Michael", "clpid": "Chavez-M" }, { "family_name": "Singhal", "given_name": "Kratika", "clpid": "Singhal-K" }, { "family_name": "Leib", "given_name": "Ryan", "orcid": "0000-0002-8129-1916", "clpid": "Leib-R" }, { "family_name": "James", "given_name": "Michelle L.", "clpid": "James-M-L" }, { "family_name": "Segal", "given_name": "David J.", "orcid": "0000-0001-8962-3105", "clpid": "Segal-D-J" }, { "family_name": "Cheng", "given_name": "R. Holland", "orcid": "0000-0002-2068-7271", "clpid": "Cheng-R-Holland" }, { "family_name": "Silva", "given_name": "Eduardo A.", "orcid": "0000-0003-3173-7622", "clpid": "Silva-E-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Ferrara", "given_name": "Katherine W.", "orcid": "0000-0002-4976-9107", "clpid": "Ferrara-K-W" } ], "abstract": "Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t_(1/2)\u2009=\u200912.7\u2009h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design.", "doi": "10.1038/s41467-020-15818-4", "pmcid": "PMC7193641", "issn": "2041-1723", "publisher": "Nature Publishing Group", "publication": "Nature Communications", "publication_date": "2020-04-30", "volume": "11", "pages": "Art. No. 2102" }, { "id": "authors:zk45d-25a12", "collection": "authors", "collection_id": "zk45d-25a12", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-102237108", "type": "article", "title": "A Computational and Experimental Platform for Detecting Full Transcriptome Cell Type Tropism of Lowly Expressed Barcoded and Pooled AAV Variants via Single-Cell RNA Sequencing", "author": [ { "family_name": "Brown", "given_name": "David", "clpid": "Brown-D" }, { "family_name": "Altermatt", "given_name": "Michael", "clpid": "Altermatt-M" }, { "family_name": "Dobreva", "given_name": "Tatyana", "orcid": "0000-0002-2625-8873", "clpid": "Dobreva-T" }, { "family_name": "Park", "given_name": "Jong H.", "clpid": "Park-Jong-H" }, { "family_name": "Ravindra-Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-S" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Coughlin", "given_name": "Gerard M.", "clpid": "Coughlin-G-M" }, { "family_name": "Pool", "given_name": "Allan-Hermann", "orcid": "0000-0002-0811-9861", "clpid": "Pool-A-H" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Despite being one of the primary gene therapy delivery vehicles, adeno-associated viruses (AAVs) are limited in their specificity towards certain cell types implicated in disease. Recombinant AAVs (rAAVs) are addressing these limitations through both capsid engineering and gene regulatory approaches that alter viral tropism or viral expression patterns. Current rAAV targeting, selection, screening, and characterization methods are typically based on single- or few-molecule read-outs, such as promoter and enhancer-driven constructs, mouse lines expressing Cre recombinase under a cell type-specific promoter, or cell type-specific antibodies for imaging. Such methods harbor challenges for parallelizing rAAV characterization, or extending characterization and engineering to complex or previously unknown cell types. The recent advent of single-cell RNA sequencing (scRNAseq) has revealed a rich diversity of cell types and states, many of which are not associated with canonical cell type markers, and can even be defined by multi-gene programs. To aid in the engineering of rAAVs aimed at such complex cell states and aid in the discovery of novel tropisms, we have developed a scRNA-seq AAV screening method, whereby we inspect full transcriptomes of cells transduced with pools of AAV vectors in a single animal. To generate pools of variants that can be differentiated in sequencing, we package variants with either unique transgenes, or the same transgene with unique barcodes incorporated in the polyA region. We then co-inject mice with these pools of variants, wait for expression, and harvest tissue slices for downstream cell dissociation and single-cell sequencing using the Chromium 10X Single Cell Kit. In order to accommodate the low expression rates of virally delivered cargo and the loss of the region of mRNA upstream of the polyA capture site that identifies the capsid variant, we amplify the viral transcripts from the full cDNA library with primers near the differentiating region of the cargo. To characterize variants, we developed a customized computational pipeline that addresses the unique challenges of these datasets: (1) to discern the variant that delivered each transgene read, we demultiplex the amplified viral transgene reads based on their differentiating sequences; (2) to reduce the effects of PCR amplification noise, we convert variant transgene reads into probabilistic estimates of the number of transcripts per cell; and (3) to calculate cell type biases, we automatically identify a cell type hierarchy and compare the distribution of viral transcripts by cell type to a null model of empty droplets. Thus far, our platform has corroborated several expected virus tropism findings from imaging (e.g. for brain vasculature or neuronal preference). To apply this barcoding strategy to even larger pools without individually cloning and producing each variant, and link arbitrary mutations in the capsid genome to the barcode, we have further developed a plasmid that contains both the expressed transgene and the capsid gene, but inverted in orientation, with their 3' ends adjacent. With these barcoding strategies and computational methods, we enable fast identification and characterization of rAAV variant pools with precise disease-relevant tropisms, with the ultimate goal of aiding the gene therapy field in developing precision delivery vehicles.", "doi": "10.1016/j.ymthe.2020.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2020-04-28", "series_number": "4", "volume": "28", "issue": "4", "pages": "80-81" }, { "id": "authors:jtkjw-hfq07", "collection": "authors", "collection_id": "jtkjw-hfq07", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-083821035", "type": "article", "title": "Evolution and Investigation of Engineered AAV Capsids Exhibiting Enhanced Transduction of the Central Nervous System with or without Murine Strain Specificity", "author": [ { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-S" }, { "family_name": "Miles", "given_name": "Timothy F.", "orcid": "0000-0001-6591-3271", "clpid": "Miles-T-F" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Brown", "given_name": "David", "clpid": "Brown-D" }, { "family_name": "Dobreva", "given_name": "Tatyana", "orcid": "0000-0002-2625-8873", "clpid": "Dobreva-T" }, { "family_name": "Huang", "given_name": "Qin", "clpid": "Huang-Qin" }, { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Luo", "given_name": "Yicheng", "orcid": "0000-0003-3704-2389", "clpid": "Luo-Yicheng" }, { "family_name": "Einarsson", "given_name": "Petur", "clpid": "Einarsson-P-H" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-A" }, { "family_name": "Jang", "given_name": "Min Jee", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Recombinant adeno-associated viral (rAAV) capsids are increasingly used as gene delivery vectors in science and in therapeutics. However, there is room for further improvement on the efficiency and specificity to transduce the central nervous system (CNS) via non-invasive systemic delivery. We have shown in the past that marked improvement is possible using a Cre recombination-based AAV targeted evolution (CREATE) platform to identify AAV-PHP.B and AAV-PHP.eB capsids, which broadly transduce the CNS (Deverman et al, 2016; Chan et al, 2017). While CREATE was successful in identifying efficient CNS vectors in two rounds of evolution, the method was limited by its ability to identify only a few top candidates from the selection. To truly utilize the potential of a large selection design (such as a library of ~1.28 billion theoretical size using 7-mer-NNK mutagenesis strategy) across multiple different selection targets (such as endothelial cells, neurons and astrocytes), we developed Multiplexed-CREATE (M-CREATE). \n\nThis method involves: (1) application of a positive selection pressure for on-target delivery and post-hoc negative selection against off-target delivery using next generation sequencing, (2) an unbiased selection design, and (3) a novel data analysis platform. M-CREATE identified distinct families of variants based on shared amino acid motifs. These include a family of PHP.B-like variants that appear to be dominant across in vivo selections for CNS transduction in Cre-transgenic adult mice. While some variants from this family showed similar tropism to PHP.B as expected, two variants (PHP.Vs) were distinct by exhibiting biased transduction to the vascular cells forming the blood-brain barrier (BBB). In addition, the new analysis platform allowed us to further mine the selection that yielded PHP.eB from its PHP.B parent (Chan et al, 2017) and therefore uncover PHP.N, that differs by 3 amino acids from PHP.B and shows greater specificity in transducing neurons over other CNS cell-types. This further demonstrates that small sequence variations within a family can yield different tropisms. \n\nRecent reports on the non-permissibility of PHP.B or PHP.eB in BALB/cJ (Hordeaux et al, 2018; Matsuzaki et al, 2019), and the subsequent identification of LY6A as the receptor underlying the improved BBB entry (Hordeaux et al, 2019; Huang et al, 2019; Batista et al, 2019) has motivated us to investigate the non-dominant families with distinct amino acid signatures in our 7-mer NNK library. We identified several non-dominant families (PHP.Cs), exhibiting better transduction of the CNS compared to parent AAV9 while also being efficient in crossing the BBB across mouse strains, including BALB/cJ. This suggests that variants with different amino acid motifs from the PHP.C-like families may have different mechanisms of BBB transmission, thereby making them promising candidates for further study toward translation of AAV vectors across strains and species.", "doi": "10.1016/j.ymthe.2020.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2020-04-28", "series_number": "4", "volume": "28", "issue": "4", "pages": "11-12" }, { "id": "authors:eyhgz-80z17", "collection": "authors", "collection_id": "eyhgz-80z17", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-073642424", "type": "article", "title": "Targeting the Rodent Peripheral Nervous System Efficiently and with Greater Specificity through Intravenous Delivery of AAV Capsids Evolved by Multiplexed-CREATE", "author": [ { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-S" }, { "family_name": "Wolfe", "given_name": "Damien", "clpid": "Wolfe-D" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "The peripheral nervous system (PNS) is critical in regulating end-organ function and feedback signal transmission to the central nervous system (CNS). The sensory and enteric nervous systems (SNS and ENS), key components of the PNS, are of increasing interest to both scientific and clinical communities. With the proper gene delivery vectors, SNS gene therapy could be applied to neurological disorders such as neuropathic pain whereas ENS gene therapy could address the gastrointestinal symptoms, including dysregulated digestion and transit, common to many disorders including Parkinson's. To achieve precise genetic manipulation within the PNS however, more efficient and targeted gene delivery vehicles are needed. Adeno-associated viruses (AAVs) have become the vector of choice for gene delivery in vivo but non-invasive systemic administration of natural serotypes targets the PNS with mixed efficiency and specificity. Attempts to circumvent these limitations by direct administration of AAV are often either surgically challenging (e.g. for dorsal root ganglia (DRG)) or have incomplete coverage (e.g. for ENS). In prior work we have shown that modification of the AAV9 capsid by directed evolution generates AAV variants that target the CNS or selected PNS regions by systemic delivery (Chan et al, 2017). To further explore the potential of targeting PNS with engineered-capsid, we utilized the advanced selection platform Multiplexed-CREATE (Kumar et al, Nature Methods, 2020), that builds upon our prior method CREATE (Deverman et al, Nature Biotechnology, 2016). M-CREATE is a high-confidence AAV selection platform that uses next generation sequencing (NGS) and Cre-transgenic mouse lines (e.g. neuronspecific) to perform positive selection in vivo on PNS areas of interest (including DRG, small and large intestine), and a post-hoc negative selection against off-targets (including liver). After 2 rounds of in vivo M-CREATE selection in C57BL/6J adult mice, we recovered several variants that were positively enriched in the PNS areas of interest compared to their parent capsid AAV9. Preliminary NGS analysis revealed two novel AAV variants: AAV-PNS1, with the greatest relative enrichment between the PNS and off-target tissues, and AAV-PNS2 with the greatest overall enrichment in the PNS compared to AAV9. In vivo validation in C57BL/6J adult mice by intravenous delivery of the variants packaging nuclear-localized EGFP under a strong ubiquitous CAG promoter (ssAAV-CAG-NLS-EGFP) showed enhanced EGFP expression in neurons of the nodose ganglia and DRG. Consistent with NGS enrichment data, AAV-PNS1 showed greater specificity to the nodose ganglia and DRG by exhibiting lower overall transduction in peripheral organs including liver (an otherwise strong AAV9 target), while AAV-PNS2 showed higher overall efficiency in targeting both the ENS and SNS. The preliminary outcomes described here demonstrate the potential of M-CREATE to identify designer AAVs with greater efficiency and specificity towards the PNS in adult mice and thereby enabling basic and pre-clinical research.", "doi": "10.1016/j.ymthe.2020.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2020-04-28", "series_number": "4", "volume": "28", "issue": "4", "pages": "252-253" }, { "id": "authors:n1hwc-rqm20", "collection": "authors", "collection_id": "n1hwc-rqm20", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-074008042", "type": "article", "title": "Method for High-Throughput, In Situ Characterization of AAV Variant Pools in Intact Tissue Using Ultrasensitive Sequential FISH", "author": [ { "family_name": "Jang", "given_name": "Min Jee", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Coughlin", "given_name": "Gerard M.", "clpid": "Coughlin-G-M" }, { "family_name": "Zhang", "given_name": "Yameng", "clpid": "Zhang-Yameng" }, { "family_name": "Koulena", "given_name": "Noushin", "orcid": "0000-0002-9419-5712", "clpid": "Koulena-N" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Extensive efforts have been made to engineer adeno-associated viruses (AAVs) with desirable characteristics, such as enhanced transduction efficiency and tissue- or cell-type specific tropisms. In-vivo selection, followed by next-generation sequencing (NGS)-based screening, has enabled us to uncover novel viral capsid variants, such as the AAV-PHP series (Deverman et al., Nat Biotech, 2016; Chan et al., Nat Neurosci, 2017; Kumar et al., Nat Methods, 2020). Despite successful library-based selections, the characterization of viral tropisms is slow and labor-intensive and is thus limited to only a handful of variants. To overcome this bottleneck and allow for high-throughput screening, we introduce an imaging-based approach that detects viral transcripts in intact tissue by using ultrasensitive, sequential fluorescence in situ hybridization (FISH). We first developed a new FISH method to enable detection of relatively low abundance viral transcripts compared to endogenous genes in tissue. Compared to two signal amplification methods, rolling-circle amplification (RCA) and hybridization chain reaction (HCR), our method resulted in a 2.7- or 6.7-fold higher signal-to-background ratio, respectively, with the same number of probes. The high sensitivity of our method also allowed us to detect RNA transcripts with 1 probe and distinguish capsid variants packaging an identical viral genome with a short mutated region (7 amino acids, equivalent to 21 base pairs) transduced in HEK293T cells. We also developed an efficient two-step probe stripping method to enable multiple rounds of labeling (up to 8), which increases the number of targets that can be characterized in the same tissue beyond the spectral limit (e.g., 4 colors x 8 rounds = 32 variants). The high sensitivity and ability for sequential labeling allowed us to examine the cell-type tropism of capsid variants and/or gene regulatory elements in intact tissue. For this purpose, we generated AAV pools, comprising a combination of novel AAV-PHP.B-like capsids and cell-type specific promoters, that package the same coding sequence with a unique barcode in the 3'UTR. The pool was injected into one animal at a low dose (~1e10 for each), and after 3-4 weeks of injection, the transcripts of each variant were detected with a custom probe set targeting the unique barcodes. As a proof-of-concept, we were able to characterize the cell-type tropism of 6 variants in one tissue within 4 hours. Further refinement of barcode designs (e.g., temporal barcoding or in situ sequencing) and single-molecule imaging will allow us to either reduce the screening time or increase the number of variants that can be characterized to hundreds. These approaches enable high-throughput characterization of virally delivered transgenes in intact tissue, thus complementing the active field of viral vector engineering with scalable tropism identification or validation. Moreover, visualizing the distribution of many variants while preserving spatial context will offer insights into AAV biology, which can include entry mechanisms as well as cell- and tissue-type associated expression.", "doi": "10.1016/j.ymthe.2020.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2020-04-28", "series_number": "4", "volume": "28", "issue": "4", "pages": "78-79" }, { "id": "authors:rc28g-k9d42", "collection": "authors", "collection_id": "rc28g-k9d42", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-134728909", "type": "article", "title": "Building a Toolbox of Compact Enhancers for Cell Type-Specific Gene Expression from AAVs in the Primate Brain", "author": [ { "family_name": "Mich", "given_name": "John K.", "clpid": "Mich-J-K" }, { "family_name": "Gore", "given_name": "Bryan B." }, { "family_name": "Martinez", "given_name": "Refugio A." }, { "family_name": "Graybuck", "given_name": "Lucas T." }, { "family_name": "Mahoney", "given_name": "Joseph T." }, { "family_name": "Miller", "given_name": "Jeremy A." }, { "family_name": "Yao", "given_name": "Shenqin" }, { "family_name": "Bertagnolli", "given_name": "Darren" }, { "family_name": "Goldy", "given_name": "Jeff" }, { "family_name": "Hodge", "given_name": "Rebecca D." }, { "family_name": "Bakken", "given_name": "Trygve E." }, { "family_name": "Yao", "given_name": "Zizhen" }, { "family_name": "Dee", "given_name": "Nick" }, { "family_name": "Smith", "given_name": "Kimberly A." }, { "family_name": "Gwinn", "given_name": "Ryder P." }, { "family_name": "Cobbs", "given_name": "Charles" }, { "family_name": "Ko", "given_name": "Andrew L." }, { "family_name": "Ojemann", "given_name": "Jeffrey G." }, { "family_name": "Keene", "given_name": "C. Dirk" }, { "family_name": "Silbergeld", "given_name": "Daniel L." }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Zeng", "given_name": "Hongkui" }, { "family_name": "Tasic", "given_name": "Bosiljka" }, { "family_name": "Lein", "given_name": "Ed S." }, { "family_name": "Ting", "given_name": "Jonathan T." }, { "family_name": "Levi", "given_name": "Boaz P." } ], "abstract": "AAV gene therapy has tremendous potential to transform the treatment of brain diseases. Tissue and cell class-selective transgene expression could improve the safety and efficacy of AAV-based therapeutics for brain circuit-related disorders compared to ubiquitous transgene expression. Unfortunately, there are few known compact genetic regulatory elements that limit transgene expression to specific cell populations. We undertook a systematic screen to find enhancer elements capable of targeting AAV expression to all major neocortical cell populations. Our strategy leverages multi-modal transcriptomic and epigenetic analyses of brain cells from mouse and human, followed by brain-wide in vivo testing in mouse and ex vivo testing in NHP and human. These studies generated a diverse collection of enhancer-AAV vectors that specifically target neocortical neuronal and glial cell populations across species. Furthermore, we show endogenous enhancers can be rationally optimized to increase expression levels for preclinical lead optimization studies. These studies yield a growing toolbox of enhancer elements for cell type-specific targeting in the human brain that will enable more precise gene therapy.", "doi": "10.1016/j.ymthe.2020.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2020-04-28", "series_number": "4", "volume": "28", "issue": "4", "pages": "70" }, { "id": "authors:wbsg5-7hw28", "collection": "authors", "collection_id": "wbsg5-7hw28", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-072416648", "type": "article", "title": "Structure-Guided Rational Design of Adeno-Associated Viral Capsids with Expanded Sizes", "author": [ { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "The application of recombinant Adeno-Associated Virus (rAAV) vectors as gene delivery vehicles has been limited by the modest packaging capacity (5.2kb) of rAAV. Th is limitation is imposed by the capsid size of 25nm diameter, which is determined by the capsid's T=1 icosahedral geometry. Current methods for rAAV delivery of oversized cargo, typically involving co-delivery of dual vectors, suffer from challenges such as low efficiency in co-infection and reassembly of full-length products, indefinite ratios of the two vectors in individual cells, and necessity of re-design and re-validation for every new cargo. \n\nInspired by the size polymorphism observed in natural viruses, we hypothesized that the T=1 icosahedral geometry of AAV capsids could be changed so that each capsid is built from more than 60 subunits. \n\nHere we present two rational design strategies that lead to eXtra Large AAV capsids (XL-AAVs). Th ese strategies involve modifying the intersubunit interactions and the assembly pathway of AAV capsids. Capsids engineered through both strategies form heterogeneous, 35nm-70nm spherical particles (Figure 1). Th e XL-AAV capsids can still package rAAV genomes, and the same protein design principles can be applied across multiple serotypes we tested (including AAV9, AAV2, AAV5, and AAVDJ). Ongoing work seeks to determine whether the XL-AAV capsids can fully protect and deliver conventionally oversized rAAV genomes. These design principles and the emerging XL-AAV capsids represent the first steps towards creating rAAV vectors with larger clone capacities, opening a path towards the delivery of disease-related genes and genetically-encoded tools with long coding sequences in single rAAV vectors.", "doi": "10.1016/j.ymthe.2020.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2020-04-28", "series_number": "4", "volume": "28", "issue": "4", "pages": "226-227" }, { "id": "authors:rrtwx-57q24", "collection": "authors", "collection_id": "rrtwx-57q24", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-075354300", "type": "article", "title": "In-Depth Parallel Profiling of Tissue and Cell-Type Tropism of AAV Variants by Single-Cell RNA Sequencing", "author": [ { "family_name": "Altermatt", "given_name": "Michael", "clpid": "Altermatt-M" }, { "family_name": "Brown", "given_name": "David", "clpid": "Brown-D" }, { "family_name": "Dobreva", "given_name": "Tatyana", "orcid": "0000-0002-2625-8873", "clpid": "Dobreva-T" }, { "family_name": "Jang", "given_name": "Min Jee", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Coughlin", "given_name": "Gerard M.", "clpid": "Coughlin-G-M" }, { "family_name": "Pool", "given_name": "Allan-Hermann", "orcid": "0000-0002-0811-9861", "clpid": "Pool-A-H" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Adeno-associated viruses (AAVs) are popular gene delivery vehicles and there is a continuing high demand for AAV variants with improved transduction efficiency and specificity. Directed evolution and/or rational design have been used extensively to engineer the capsid of naturally occurring AAVs in order to better customize their properties for research and clinical work. While these engineering approaches are scalable and have generated useful variants, the subsequent transduction profiling of these variants remains either low throughput or lacks resolution across the many relevant cell and tissue types. Single-cell RNA sequencing (scRNA-seq) via droplet-based methods allows in-depth profiling of gene expression of several thousand individual cells. We established a tissue processing and data analysis pipeline that leverages the capabilities of scRNA-seq to achieve simultaneous characterization of AAV variants across multiplexed tissue cell types. To verify our approach, we retro-orbitally co-injected C57Bl/6 mice with PHP.eB (Chan et al., Nat. Neurosci., 2017) and a neuron-biased PHP.eB-evolved variant (Flytzanis*, Goeden* et al., ASGCT, 2019), each packaging a construct expressing different fluorophores. \n\nAfter two weeks of expression we harvested the brain and used one hemisphere for characterization by traditional immunohistochemistry and one hemisphere for characterization by scRNA-seq. Single-cell libraries were prepared with the Chromium Single Cell Kit by 10x Genomics and analyzed with multiplexed Illumina sequencing. As a proof of concept, we compared the two characterization methods by analyzing the infection rate of neurons (NeuN), astrocytes (S100b) or oligodendrocytes (Olig2). For immunohistochemistry, a cell was classified as infected based on expression of fluorophores while in scRNA-seq transduced cells were identified based on the presence of defining viral transcripts. Louvain community detection method (Blondel et al., J. Stat. Mech., 2008) followed by analysis of significantly differentially expressed genes was used to identify cell types in the scRNA-seq data set. Given the differences in RNA and protein abundance and detection thresholds between imaging and sequencing, the two characterization methods detect different absolute numbers of infection rates; however, the cell type transduction biases are consistent among the three different cell types we tested. After verifying our method, we further explored the data set beyond major cell types and discovered previously unnoticed sub-cell type enrichments in, for example, cortical inhibitory neurons. These findings are being confirmed by mapping mRNA expression using in situ hybridization chain reaction. Besides sub-cell type tropism characterization, we are analyzing the transcriptome of infected and non-infected cells in search of mechanistic insights into AAV transduction that could facilitate rational design of recombinant AAVs with disease-relevant cell-type specificity. Our approach will aid the gene therapy field to both characterize more thoroughly existing recombinant AAVs and guide engineering of novel AAV variants.", "doi": "10.1016/j.ymthe.2020.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2020-04-28", "series_number": "4", "volume": "28", "issue": "4", "pages": "399-400" }, { "id": "authors:6t92d-n0y91", "collection": "authors", "collection_id": "6t92d-n0y91", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-154158513", "type": "article", "title": "Transduction Profiles of Engineered Adeno-Associated Viral Capsids in Mouse and Marmoset", "author": [ { "family_name": "Goertsen", "given_name": "David", "clpid": "Goertsen-D" }, { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-N-C" }, { "family_name": "Goeden", "given_name": "Nick", "clpid": "Goeden-N" }, { "family_name": "Cummins", "given_name": "Alexander", "clpid": "Cummins-A" }, { "family_name": "Pickel", "given_name": "James", "clpid": "Pickel-J" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Adeno-associated viruses (AAVs) remain promising vectors for gene therapy due to stable expression in vivo and a strong clinical safety record. Unfortunately, naturally occurring AAV serotypes are inefficient transducers of many gene therapy relevant tissues, requiring high viral doses to achieve therapeutic efficacy for many disease indications. Fortunately, AAVs are amenable to engineering efforts to improve tissue tropism and specificity. Past efforts have produced AAV variants with improved transduction capabilities for clinically relevant cell populations, including crossing the blood-brain-barrier (BBB) in mice (AAV-PHP.eB) [1], but the efficacy of those variants has not translated across all strains and species. \n\nWith the aim of enhancing viral tropism for refractory targets, libraries of AAV9 were selected for novel characteristics using Multiplexed Cre-recombination-based AAV targeted evolution (M-CREATE) [2]. \n\nViral genomes from capsids that transduced tissues of interest across rodent Cre-lines are selectively amplified and recovered through the M-CREATE method, allowing simultaneous positive and negative selection of AAV variants. Systemic administration of AAV libraries through intravenous injection permits non-invasive transduction of tissues where direct administration is difficult, enabling subsequent variant selection within gene therapy relevant cell populations. \n\nWe are presenting data on three novel engineered capsids: variant AAV-CAP.A4, which was identified for improved transduction in mouse lung tissue, and variants AAV-CAP.B10 and AAV-CAP.B22 that, when administered systemically, can cross the blood\u2014brain barrier and efficiently transduce neurons in adult mice and marmosets. \n\nAAV-CAP.A4 was compared against serotypes AAV5 and AAV9 for lung tissue transduction after systemic injections of 1e11 viral capsids/animal. AAV-CAP.A4 displays a 17-fold higher total lung transduction over AAV9 and a 45-fold improvement over AAV5. In Alveolar Type II pneumocytes, the improvement in transduction over AAV9 and AAV5 reaches 29-fold and 100-fold, respectively. This enhancement in AAV-CAP.A4 transduction is specific to the lung, while the liver and other tissues targeted by AAV9 have similar transduction profiles. \n\nA panel of novel AAV9 variants was identified for similar transduction across the BBB as the strongest current neurotropic AAV, PHP.eB [1]. Promising variants were chosen for pooled screening via systemic delivery in adult marmosets, from which AAV-CAP.B10 and AAV-CAP.B22 were chosen for independent screening. Both variants display improved transduction across the brain relative to AAV9. In the marmoset cortex, AAV-CAP.B10 and AAV-CAP. \n\nB22 transduce neurons ~15 fold and ~17 fold higher than AAV9 respectively. These novel variants enable robust, non-invasive gene delivery to the adult marmoset brain following IV administration. \n\nThis work demonstrates that, through the M-CREATE method, novel AAV variants can be developed with sought-after transduction profiles in clinically relevant cell populations. We have also shown that relative improvements to the transduction profile obtained through in vivo selection in mice can be translated to non-human primates.", "doi": "10.1016/j.ymthe.2020.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2020-04-28", "series_number": "4", "volume": "28", "issue": "4", "pages": "269-270" }, { "id": "authors:e5ak1-qrv31", "collection": "authors", "collection_id": "e5ak1-qrv31", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-073231089", "type": "article", "title": "Cloud-based Software for NGS Data Management and Analysis for Directed Evolution of Peptide-Based Delivery Vectors", "author": [ { "family_name": "Padia", "given_name": "Umesh", "clpid": "Padia-U" }, { "family_name": "Brown", "given_name": "David", "clpid": "Brown-D" }, { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" }, { "family_name": "Kumar", "given_name": "Sripriya R.", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-S" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Adeno-associated viruses (AAVs) are widely used gene delivery vectors due to their ability to transduce dividing and non-dividing cells, their long-term persistence, and low immunogenicity. However, natural AAV serotypes have a limited set of tropisms. Directed evolution has been used to engineer recombinant AAVs to target specific cell types and tissues, leveraging next generation sequencing data. The deluge of data from these deep sequencing experiments has brought about data management and analysis challenges, for which there are no current commercially available solutions. Furthermore, classical approaches to analyzing data from directed evolution heavily involves manual inspection, and often overlooks patterns present in the larger datasets. To address these challenges, we developed robust cloud-based software that provides central management for next generation sequencing data, extracts variants, performs structural modeling, and can be extended to incorporate machine learning models to make predictions for variants with specific properties. The software is composed of a set of interconnected discrete components: a modern web user interface implemented in JavaScript with React, a relational database, a distributed task queue, task workers, and a Django-based API. This architecture allows computationally intensive tasks such as alignments, structural modeling, and machine learning to scale from a single machine to hundreds of machines, with minimal configuration. The software automatically imports and manages sequencing data from several different commercial and in-house sequencing providers. When the data is imported, sequence quality metrics are automatically generated and presented to the user. Variants are extracted by performing pairwise alignments between the natural serotype and the sequencing reads. The variants are further encoded into embeddings, grouped into families, and are analyzed for prevalent sequence motifs. We use the Rosetta software libraries to perform comparative modeling simulations on selected variants. Finally, we are developing and have extension support for Pytorch-based machine learning models to generate novel variants with desirable properties as well as to select candidate variants for additional rounds of optimization and characterization. This software represents a general tool for simple, scalable, and centralized analyses of next generation sequencing data for protein engineering by directed evolution, and could be generalized for all projects with large-scale deep sequencing datasets in the future.", "doi": "10.1016/j.ymthe.2020.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2020-04-28", "series_number": "4", "volume": "28", "issue": "4", "pages": "434-435" }, { "id": "authors:27n9s-n4j49", "collection": "authors", "collection_id": "27n9s-n4j49", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200220-151529138", "type": "article", "title": "LRP1 is a master regulator of tau uptake and spread", "author": [ { "family_name": "Rauch", "given_name": "Jennifer N.", "clpid": "Rauch-J-N" }, { "family_name": "Luna", "given_name": "Gabriel", "clpid": "Luna-G" }, { "family_name": "Guzman", "given_name": "Elmer", "clpid": "Guzman-E" }, { "family_name": "Audouard", "given_name": "Morgane", "clpid": "Audouard-M" }, { "family_name": "Challis", "given_name": "Collin", "orcid": "0000-0003-4716-6086", "clpid": "Challis-Collin" }, { "family_name": "Sibih", "given_name": "Youssef E.", "clpid": "Sibih-Y-E" }, { "family_name": "Leshuk", "given_name": "Carolina", "clpid": "Leshuk-C" }, { "family_name": "Hernandez", "given_name": "Israel", "clpid": "Hernandez-I" }, { "family_name": "Wegmann", "given_name": "Susanne", "clpid": "Wegmann-S" }, { "family_name": "Hyman", "given_name": "Bradley T.", "clpid": "Hyman-B-T" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Kampmann", "given_name": "Martin", "orcid": "0000-0002-3819-7019", "clpid": "Kampmann-M" }, { "family_name": "Kosik", "given_name": "Kenneth S.", "clpid": "Kosik-K-S" } ], "abstract": "The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau has a central role in the pathogenesis of several forms of dementia known as tauopathies\u2014including Alzheimer's disease, frontotemporal dementia and chronic traumatic encephalopathy. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity. This observation and complementary experimental studies have suggested that tau can spread in a prion-like manner, by passing to naive cells in which it templates misfolding and aggregation. However, although the propagation of tau has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein receptor-related protein 1 (LRP1) controls the endocytosis of tau and its subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and in induced pluripotent stem cell-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule-binding repeat region of tau. Furthermore, downregulation of LRP1 in an in vivo mouse model of tau spread was found to effectively reduce the propagation of tau between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain, and therefore a potential target for the treatment of diseases that involve tau spread and aggregation.", "doi": "10.1038/s41586-020-2156-5", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2020-04-16", "series_number": "7803", "volume": "580", "issue": "7803", "pages": "381-385" }, { "id": "authors:56ap2-xb768", "collection": "authors", "collection_id": "56ap2-xb768", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190220-102621411", "type": "article", "title": "RecV recombinase system for in vivo targeted optogenomic modifications of single cells or cell populations", "author": [ { "family_name": "Yao", "given_name": "Shenqin", "clpid": "Yao-Shenqin" }, { "family_name": "Yuan", "given_name": "Peng", "clpid": "Yuan-Peng" }, { "family_name": "Ouellette", "given_name": "Ben", "clpid": "Ouellette-B" }, { "family_name": "Zhou", "given_name": "Thomas", "clpid": "Zhou-Thomas" }, { "family_name": "Mortrud", "given_name": "Marty", "clpid": "Mortrud-M" }, { "family_name": "Balaram", "given_name": "Pooja", "clpid": "Balaram-P" }, { "family_name": "Chatterjee", "given_name": "Soumya", "orcid": "0000-0002-2878-1502", "clpid": "Chatterjee-S" }, { "family_name": "Wang", "given_name": "Yun", "clpid": "Wang-Yun" }, { "family_name": "Daigle", "given_name": "Tanya L.", "clpid": "Daigle-T-L" }, { "family_name": "Tasic", "given_name": "Bosiljka", "clpid": "Tasic-B" }, { "family_name": "Kuang", "given_name": "Xiuli", "clpid": "Kuang-Xiuli" }, { "family_name": "Gong", "given_name": "Hui", "clpid": "Gong-Hui" }, { "family_name": "Luo", "given_name": "Qingming", "clpid": "Luo-Qingming" }, { "family_name": "Zeng", "given_name": "Shaoqun", "clpid": "Zeng-Shaoqun" }, { "family_name": "Curtright", "given_name": "Andrew", "clpid": "Curtright-A" }, { "family_name": "Dhaka", "given_name": "Ajay", "clpid": "Dhaka-A" }, { "family_name": "Kahan", "given_name": "Anat", "clpid": "Kahan-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Chrapkiewicz", "given_name": "Rados\u0142aw", "clpid": "Chrapkiewicz-R" }, { "family_name": "Schnitzer", "given_name": "Mark", "clpid": "Schnitzer-M-J" }, { "family_name": "Zeng", "given_name": "Hongkui", "clpid": "Zeng-Hongkui" }, { "family_name": "Cetin", "given_name": "Ali", "clpid": "Cetin-A-H" } ], "abstract": "Brain circuits comprise vast numbers of interconnected neurons with diverse molecular, anatomical and physiological properties. To allow targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo. They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.", "doi": "10.1038/s41592-020-0774-3", "pmcid": "PMC7135964", "issn": "1548-7091", "publisher": "Nature Publishing Group", "publication": "Nature Methods", "publication_date": "2020-04", "series_number": "4", "volume": "17", "issue": "4", "pages": "422-429" }, { "id": "authors:2ncza-sht42", "collection": "authors", "collection_id": "2ncza-sht42", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200218-134259315", "type": "article", "title": "A gut bacterial amyloid promotes \u03b1-synuclein aggregation and motor impairment in mice", "author": [ { "family_name": "Sampson", "given_name": "Timothy R.", "orcid": "0000-0002-2486-8766", "clpid": "Sampson-Timothy-R" }, { "family_name": "Challis", "given_name": "Collin", "orcid": "0000-0003-4716-6086", "clpid": "Challis-Collin" }, { "family_name": "Jain", "given_name": "Neha", "clpid": "Jain-Neha" }, { "family_name": "Moiseyenko", "given_name": "Anastasiya", "clpid": "Moiseyenko-Anastasiya" }, { "family_name": "Ladinsky", "given_name": "Mark S.", "orcid": "0000-0002-1036-3513", "clpid": "Ladinsky-Mark-S" }, { "family_name": "Shastri", "given_name": "Gauri G.", "clpid": "Shastri-Gauri-G" }, { "family_name": "Thron", "given_name": "Taren", "clpid": "Thron-Taren" }, { "family_name": "Needham", "given_name": "Brittany D.", "orcid": "0000-0002-0280-1886", "clpid": "Needham-Brittany-D" }, { "family_name": "Horvath", "given_name": "Istvan", "clpid": "Horvath-Istvan-T" }, { "family_name": "Debelius", "given_name": "Justine W.", "orcid": "0000-0002-8605-3546", "clpid": "Debelius-Justine-W" }, { "family_name": "Janssen", "given_name": "Stefan", "orcid": "0000-0003-0955-0589", "clpid": "Janssen-Stefan" }, { "family_name": "Knight", "given_name": "Rob", "orcid": "0000-0002-0975-9019", "clpid": "Knight-Rob" }, { "family_name": "Wittung-Stafshede", "given_name": "Pernilla", "orcid": "0000-0003-1058-1964", "clpid": "Wittung-Stafshede-P" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Chapman", "given_name": "Matthew", "clpid": "Chapman-Matthew" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "abstract": "Amyloids are a class of protein with unique self-aggregation properties, and their aberrant accumulation can lead to cellular dysfunctions associated with neurodegenerative diseases. While genetic and environmental factors can influence amyloid formation, molecular triggers and/or facilitators are not well defined. Growing evidence suggests that non-identical amyloid proteins may accelerate reciprocal amyloid aggregation in a prion-like fashion. While humans encode ~30 amyloidogenic proteins, the gut microbiome also produces functional amyloids. For example, curli are cell surface amyloid proteins abundantly expressed by certain gut bacteria. In mice overexpressing the human amyloid \u03b1-synuclein (\u03b1Syn), we reveal that colonization with curli-producing Escherichia coli promotes \u03b1Syn pathology in the gut and the brain. Curli expression is required for E. coli to exacerbate \u03b1Syn-induced behavioral deficits, including intestinal and motor impairments. Purified curli subunits accelerate \u03b1Syn aggregation in biochemical assays, while oral treatment of mice with a gut-restricted amyloid inhibitor prevents curli-mediated acceleration of pathology and behavioral abnormalities. We propose that exposure to microbial amyloids in the gastrointestinal tract can accelerate \u03b1Syn aggregation and disease in the gut and the brain.", "doi": "10.7554/elife.53111", "pmcid": "PMC7012599", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2020-02-11", "volume": "9", "pages": "Art. No. e53111" }, { "id": "authors:4y22j-7j002", "collection": "authors", "collection_id": "4y22j-7j002", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200106-082556970", "type": "article", "title": "Tissue clearing and its applications in\u00a0neuroscience", "author": [ { "family_name": "Ueda", "given_name": "Hiroki R.", "orcid": "0000-0001-8342-9176", "clpid": "Ueda-Hiroki-R" }, { "family_name": "Ert\u00fcrk", "given_name": "Ali", "orcid": "0000-0001-5163-5100", "clpid": "Ert\u00fcrk-Ali" }, { "family_name": "Chung", "given_name": "Kwanghun", "orcid": "0000-0002-8167-3340", "clpid": "Chung-Kwanghun" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Ch\u00e9dotal", "given_name": "Alain", "orcid": "0000-0001-7577-3794", "clpid": "Ch\u00e9dotal-Alain" }, { "family_name": "Tomancak", "given_name": "Pavel", "clpid": "Tomancak-Pavel" }, { "family_name": "Keller", "given_name": "Philipp J.", "orcid": "0000-0003-2896-4920", "clpid": "Keller-Philipp-J" } ], "abstract": "State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.", "doi": "10.1038/s41583-019-0250-1", "pmcid": "PMC8121164", "issn": "1471-003X", "publisher": "Nature Publishing Group", "publication": "Nature Reviews. Neuroscience", "publication_date": "2020-02", "series_number": "2", "volume": "21", "issue": "2", "pages": "61-79" }, { "id": "authors:de2e1-ej682", "collection": "authors", "collection_id": "de2e1-ej682", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191203-132507070", "type": "article", "title": "Low- and high-thermogenic brown adipocyte subpopulations coexist in murine adipose tissue", "author": [ { "family_name": "Song", "given_name": "Anying", "clpid": "Song-Anying" }, { "family_name": "Dai", "given_name": "Wenting", "clpid": "Dai-Wenting" }, { "family_name": "Jang", "given_name": "Min Jee", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Medrano", "given_name": "Leonard", "orcid": "0000-0002-0782-5473", "clpid": "Medrano-L" }, { "family_name": "Li", "given_name": "Zhuo", "clpid": "Li-Zhuo" }, { "family_name": "Zhao", "given_name": "Hu", "clpid": "Zhao-Hu" }, { "family_name": "Shao", "given_name": "Mengle", "orcid": "0000-0002-5488-9904", "clpid": "Shao-Mengle" }, { "family_name": "Tan", "given_name": "Jiayi", "clpid": "Tan-Jiayi" }, { "family_name": "Li", "given_name": "Aimin", "clpid": "Li-Aimin" }, { "family_name": "Ning", "given_name": "Tinglu", "clpid": "Ning-Tinglu" }, { "family_name": "Miller", "given_name": "Marcia M.", "clpid": "Miller-M-M" }, { "family_name": "Armstrong", "given_name": "Brian", "clpid": "Armstrong-B" }, { "family_name": "Huss", "given_name": "Janice M.", "clpid": "Huss-J-M" }, { "family_name": "Zhu", "given_name": "Yi", "clpid": "Zhu-Yi" }, { "family_name": "Liu", "given_name": "Yong", "clpid": "Liu-Yong" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Wu", "given_name": "Xiwei", "clpid": "Wu-Xiwei" }, { "family_name": "Jiang", "given_name": "Lei", "clpid": "Jiang-Lei" }, { "family_name": "Scherer", "given_name": "Philipp E.", "orcid": "0000-0003-0680-3392", "clpid": "Scherer-P-E" }, { "family_name": "Wang", "given_name": "Qiong A.", "orcid": "0000-0003-2224-4287", "clpid": "Wang-Qiong-A" } ], "abstract": "Brown adipose tissue (BAT), as the main site of adaptive thermogenesis, exerts beneficial metabolic effects on obesity and insulin resistance. BAT has been previously assumed to contain a homogeneous population of brown adipocytes. Utilizing multiple mouse models capable of genetically labeling different cellular populations, as well as single-cell RNA sequencing and 3D tissue profiling, we discovered a new brown adipocyte subpopulation with low thermogenic activity coexisting with the classical high-thermogenic brown adipocytes within the BAT. Compared with the high-thermogenic brown adipocytes, these low-thermogenic brown adipocytes had substantially lower Ucp1 and Adipoq expression, larger lipid droplets, and lower mitochondrial content. Functional analyses showed that, unlike the high-thermogenic brown adipocytes, the low-thermogenic brown adipocytes have markedly lower basal mitochondrial respiration, and they are specialized in fatty acid uptake. Upon changes in environmental temperature, the 2 brown adipocyte subpopulations underwent dynamic interconversions. Cold exposure converted low-thermogenic brown adipocytes into high-thermogenic cells. A thermoneutral environment had the opposite effect. The recruitment of high-thermogenic brown adipocytes by cold stimulation is not affected by high fat diet feeding, but it does substantially decline with age. Our results revealed a high degree of functional heterogeneity of brown adipocytes.", "doi": "10.1172/jci129167", "pmcid": "PMC6934193", "issn": "0021-9738", "publisher": "American Society for Clinical Investigation", "publication": "Journal of Clinical Investigation", "publication_date": "2020-01", "series_number": "1", "volume": "130", "issue": "1", "pages": "247-257" }, { "id": "authors:8t3hd-cp839", "collection": "authors", "collection_id": "8t3hd-cp839", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190801-132221928", "type": "article", "title": "Imaging neuromodulators with high spatiotemporal resolution using genetically encoded indicators", "author": [ { "family_name": "Patriarchi", "given_name": "Tommaso", "orcid": "0000-0001-9351-3734", "clpid": "Patriarchi-T" }, { "family_name": "Cho", "given_name": "Jounhong Ryan", "orcid": "0000-0001-9542-716X", "clpid": "Cho-Jounhong-Ryan" }, { "family_name": "Merten", "given_name": "Katharina", "orcid": "0000-0002-0197-0186", "clpid": "Merten-K" }, { "family_name": "Marley", "given_name": "Aaron", "clpid": "Marley-A" }, { "family_name": "Broussard", "given_name": "Gerard Joey", "orcid": "0000-0003-1636-2615", "clpid": "Broussard-G-J" }, { "family_name": "Liang", "given_name": "Ruqiang", "orcid": "0000-0002-3075-4554", "clpid": "Liang-Ruqiang" }, { "family_name": "Williams", "given_name": "John", "clpid": "Williams-J-T" }, { "family_name": "Nimmerjahn", "given_name": "Axel", "orcid": "0000-0002-0875-7855", "clpid": "Nimmerjahn-A" }, { "family_name": "von Zastrow", "given_name": "Mark", "orcid": "0000-0003-1375-6926", "clpid": "von-Zastrow-M" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Tian", "given_name": "Lin", "orcid": "0000-0001-7012-6926", "clpid": "Tian-Lin" } ], "abstract": "Multiple aspects of neural activity, from neuronal firing to neuromodulator release and signaling, underlie brain function and ultimately shape animal behavior. The recently developed and constantly growing toolbox of genetically encoded sensors for neural activity, including calcium, voltage, neurotransmitter and neuromodulator sensors, allows precise measurement of these signaling events with high spatial and temporal resolution. Here, we describe the engineering, characterization and application of our recently developed dLight1, a suite of genetically encoded dopamine (DA) sensors based on human inert DA receptors. dLight1 offers high molecular specificity, requisite affinity and kinetics and great sensitivity for measuring DA release in vivo. The detailed workflow described in this protocol can be used to systematically characterize and validate dLight1 in increasingly intact biological systems, from cultured cells to acute brain slices to behaving mice. For tool developers, we focus on characterizing five distinct properties of dLight1: dynamic range, affinity, molecular specificity, kinetics and interaction with endogenous signaling; for end users, we provide comprehensive step-by-step instructions for how to leverage fiber photometry and two-photon imaging to measure dLight1 transients in vivo. The instructions provided in this protocol are designed to help laboratory personnel with a broad range of experience (at the graduate or post-graduate level) to develop and utilize novel neuromodulator sensors in vivo, by using dLight1 as a benchmark.", "doi": "10.1038/s41596-019-0239-2", "issn": "1754-2189", "publisher": "Nature Publishing Group", "publication": "Nature Protocols", "publication_date": "2019-12", "series_number": "12", "volume": "14", "issue": "12", "pages": "3471-3505" }, { "id": "authors:mce30-bne28", "collection": "authors", "collection_id": "mce30-bne28", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191216-134424777", "type": "article", "title": "Paracrine delivery of therapeutic biologics for cancer", "author": [ { "family_name": "Smith", "given_name": "S. N.", "clpid": "Smith-S-N" }, { "family_name": "Schubert", "given_name": "R.", "clpid": "Schubert-R" }, { "family_name": "Simic", "given_name": "B.", "clpid": "Simic-B" }, { "family_name": "Br\u00fccher", "given_name": "D.", "clpid": "Br\u00fccher-D" }, { "family_name": "Schmid", "given_name": "M.", "clpid": "Schmid-M" }, { "family_name": "Gradinaru", "given_name": "V.", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Pl\u00fcckthun", "given_name": "A.", "clpid": "Pl\u00fcckthun-A" } ], "abstract": "A fundamental goal of cancer drug delivery is to achieve sufficient levels within the tumour without leading to high systemic concentrations that might cause off-target toxicities. In situ production of protein-based therapeutics by tumour cells provides an attractive alternative to treatment with repeated high bolus injections, as secretion by the tumour itself could provide high local\nconcentrations that act in a paracrine fashion over an extended duration. For this purpose, we have developed a non-oncolytic adenoviral delivery system that allows for targeting of Ad5 to discrete cell types by redirecting viral tropism to cell surface biomarkers through the use of interchangeable adapters. Furthermore, we recently described the engineering of a protein-based 'shield' that is coated on the Ad5 capsid, which, together\nwith the retargeting adapters, allows for improved tumour specificity\nand prevention of viral clearance. To test this delivery\nstrategy in vivo, SCID-beige mice bearing orthotopic BT474\nxenografts were treated with three doses of either a cancerspecific,\nnon-replicative Ad5 that encodes a secreted anti-HER2\nantibody, trastuzumab, in its genome, or with the protein therapeutic\nitself (Herceptin\u00ae). We have employed state-of-the-art\nwhole tumour clearing and imaging with confocal microscopy at\nhigh spatial resolution in 3D to assess biodistribution, and large\nvolumetric imaging has revealed that the secreted therapeutic\ndiffuses significantly throughout the tumour leading to a therapeutic\neffect and delayed tumour outgrowth. Moreover, the systemic\nconcentration of antibody is significantly reduced with viral\ndelivery, suggesting that paracrine delivery may be a promising\nstrategy for delivery of biologics with narrow therapeutic indices.", "doi": "10.1089/hum.2019.29095.abstracts", "issn": "1043-0342", "publisher": "Mary Ann Liebert", "publication": "Human Gene Therapy", "publication_date": "2019-11-01", "series_number": "11", "volume": "30", "issue": "11", "pages": "A3" }, { "id": "authors:jh7xg-zzf80", "collection": "authors", "collection_id": "jh7xg-zzf80", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190304-085432637", "type": "article", "title": "Machine learning-guided channelrhodopsin engineering enables minimally-invasive optogenetics", "author": [ { "family_name": "Bedbrook", "given_name": "Claire N.", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-Claire-N" }, { "family_name": "Yang", "given_name": "Kevin K.", "orcid": "0000-0001-9045-6826", "clpid": "Yang-Kevin-K" }, { "family_name": "Robinson", "given_name": "J. Elliott", "orcid": "0000-0001-9417-3938", "clpid": "Robinson-J-Elliott" }, { "family_name": "Mackey", "given_name": "Elisha D.", "clpid": "Mackey-Elisha-D-W" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Arnold", "given_name": "Frances H.", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "abstract": "We engineered light-gated channelrhodopsins (ChRs) whose current strength and light sensitivity enable minimally invasive neuronal circuit interrogation. Current ChR tools applied to the mammalian brain require intracranial surgery for transgene delivery and implantation of fiber-optic cables to produce light-dependent activation of a small volume of tissue. To facilitate expansive optogenetics without the need for invasive implants, our engineering approach leverages the substantial literature of ChR variants to train statistical models for the design of high-performance ChRs. With Gaussian process models trained on a limited experimental set of 102 functionally characterized ChRs, we designed high-photocurrent ChRs with high light sensitivity. Three of these, ChRger1\u20133, enable optogenetic activation of the nervous system via systemic transgene delivery. ChRger2 enables light-induced neuronal excitation without fiber-optic implantation; that is, this opsin enables transcranial optogenetics.", "doi": "10.1038/s41592-019-0583-8", "pmcid": "PMC6858556", "issn": "1548-7091", "publisher": "Nature Publishing Group", "publication": "Nature Methods", "publication_date": "2019-11", "series_number": "11", "volume": "16", "issue": "11", "pages": "1176-1184" }, { "id": "authors:e3khm-sn905", "collection": "authors", "collection_id": "e3khm-sn905", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190926-135813898", "type": "article", "title": "Proceedings of the Sixth Deep Brain Stimulation Think Tank Modulation of Brain Networks and Application of Advanced Neuroimaging, Neurophysiology, and Optogenetics", "author": [ { "family_name": "Ramirez-Zamora", "given_name": "Adolfo", "clpid": "Ramirez-Zamora-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "The annual deep brain stimulation (DBS) Think Tank aims to create an opportunity for a multidisciplinary discussion in the field of neuromodulation to examine developments, opportunities and challenges in the field. The proceedings of the Sixth Annual Think Tank recapitulate progress in applications of neurotechnology, neurophysiology, and emerging techniques for the treatment of a range of psychiatric and neurological conditions including Parkinson's disease, essential tremor, Tourette syndrome, epilepsy, cognitive disorders, and addiction. Each section of this overview provides insight about the understanding of neuromodulation for specific disease and discusses current challenges and future directions. This year's report addresses key issues in implementing advanced neurophysiological techniques, evolving use of novel modulation techniques to deliver DBS, ans improved neuroimaging techniques. The proceedings also offer insights into the new era of brain network neuromodulation and connectomic DBS to define and target dysfunctional brain networks. The proceedings also focused on innovations in applications and understanding of adaptive DBS (closed-loop systems), the use and applications of optogenetics in the field of neurostimulation and the need to develop databases for DBS indications. Finally, updates on neuroethical, legal, social, and policy issues relevant to DBS research are discussed.", "doi": "10.3389/fnins.2019.00936", "pmcid": "PMC6751331", "issn": "1662-453X", "publisher": "Frontiers", "publication": "Frontiers in Neuroscience", "publication_date": "2019-09-12", "volume": "13", "pages": "Art. No. 936" }, { "id": "authors:ggwey-18p67", "collection": "authors", "collection_id": "ggwey-18p67", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191017-140230343", "type": "article", "title": "Structural Studies of Engineered Adeno-Associated Virus Capsids that Cross Blood-Brain Barrier Efficiently", "author": [ { "family_name": "Ding", "given_name": "Xiaozhe", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-S" }, { "family_name": "Malyutin", "given_name": "Andrey", "clpid": "Malyutin-A-G" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "[no abstract]", "doi": "10.1002/pro.3710", "issn": "0961-8368", "publisher": "Wiley", "publication": "Protein Science", "publication_date": "2019-09", "series_number": "S1", "volume": "28", "issue": "S1", "pages": "116-117" }, { "id": "authors:tvp5y-12b43", "collection": "authors", "collection_id": "tvp5y-12b43", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190624-113552525", "type": "article", "title": "The Serotonergic Raphe Promote Sleep in Zebrafish and Mice", "author": [ { "family_name": "Oikonomou", "given_name": "Grigorios", "orcid": "0000-0001-6797-7375", "clpid": "Oikonomou-Grigorios" }, { "family_name": "Altermatt", "given_name": "Michael", "orcid": "0000-0003-2841-5374", "clpid": "Altermatt-Michael" }, { "family_name": "Zhang", "given_name": "Rong-wei", "orcid": "0000-0003-1145-8589", "clpid": "Zhang-Rong-wei" }, { "family_name": "Coughlin", "given_name": "Gerard M.", "clpid": "Coughlin-Gerard-M" }, { "family_name": "Montz", "given_name": "Christin", "clpid": "Montz-Christin" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Prober", "given_name": "David A.", "orcid": "0000-0002-7371-4675", "clpid": "Prober-D-A" } ], "abstract": "The role of serotonin (5-HT) in sleep is controversial: early studies suggested a sleep-promoting role, but eventually the paradigm shifted toward a wake-promoting function for the serotonergic raphe. Here, we provide evidence from zebrafish and mice that the raphe are critical for the initiation and maintenance of sleep. In zebrafish, genetic ablation of 5-HT production by the raphe reduces sleep, sleep depth, and the homeostatic response to sleep deprivation. Pharmacological inhibition or ablation of the raphe reduces sleep, while optogenetic stimulation increases sleep. Similarly, in mice, ablation of the raphe increases wakefulness and impairs the homeostatic response to sleep deprivation, whereas tonic optogenetic stimulation at a rate similar to baseline activity induces sleep. Interestingly, burst optogenetic stimulation induces wakefulness in accordance with previously described burst activity of the raphe during arousing stimuli. These results indicate that the serotonergic system promotes sleep in both diurnal zebrafish and nocturnal rodents.", "doi": "10.1016/j.neuron.2019.05.038", "pmcid": "PMC6706304", "issn": "0896-6273", "publisher": "Cell Press", "publication": "Neuron", "publication_date": "2019-08-21", "series_number": "4", "volume": "103", "issue": "4", "pages": "686-701" }, { "id": "authors:bmacn-7y974", "collection": "authors", "collection_id": "bmacn-7y974", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20181107-092306925", "type": "article", "title": "Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies", "author": [ { "family_name": "Rajendran", "given_name": "Pradeep S.", "orcid": "0000-0001-9790-0408", "clpid": "Rajendran-Pradeep-S" }, { "family_name": "Challis", "given_name": "Rosemary C.", "orcid": "0000-0003-3086-6553", "clpid": "Challis-Rosemary-C" }, { "family_name": "Fowlkes", "given_name": "Charless C.", "orcid": "0000-0002-2990-1780", "clpid": "Fowlkes-Charless-C" }, { "family_name": "Hanna", "given_name": "Peter", "clpid": "Hanna-Peter" }, { "family_name": "Tompkins", "given_name": "John D.", "clpid": "Tompkins-John-D" }, { "family_name": "Jordan", "given_name": "Maria C.", "clpid": "Jordan-Maria-C" }, { "family_name": "Hiyari", "given_name": "Sarah", "clpid": "Hiyari-Sarah" }, { "family_name": "Gabris-Weber", "given_name": "Beth A.", "clpid": "Gabris-Weber-Beth-A" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-Alon" }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-Benjamin-E" }, { "family_name": "Munzberg", "given_name": "Heike", "clpid": "Munzberg-Heike" }, { "family_name": "Ardell", "given_name": "Jeffrey L.", "clpid": "Ardell-Jeffrey-L" }, { "family_name": "Salama", "given_name": "Guy", "orcid": "0000-0002-5306-8178", "clpid": "Salama-Guy" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Shivkumar", "given_name": "Kalyanam", "orcid": "0000-0002-4121-1766", "clpid": "Shivkumar-Kalyanam" } ], "abstract": "Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.", "doi": "10.1038/s41467-019-09770-1", "pmcid": "PMC6486614", "issn": "2041-1723", "publisher": "Nature Publishing Group", "publication": "Nature Communications", "publication_date": "2019-04-26", "volume": "10", "pages": "Art. No. 1944" }, { "id": "authors:4wh9a-76m48", "collection": "authors", "collection_id": "4wh9a-76m48", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190502-130505856", "type": "article", "title": "Engineered AAVs for Enhanced Transduction of Submucosal Cells within the Lung Following Intravenous Delivery", "author": [ { "family_name": "Goeden", "given_name": "Nick", "clpid": "Goeden-N" }, { "family_name": "Flytzanis", "given_name": "Nicholas", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-N-C" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Lung diseases are one of the leading causes of morbidity and\nmortality worldwide, highlighting the need for vectors capable of\nefficiently transducing cells within the lung. Recent developments\nin gene delivery have demonstrated that the use of gene therapies\nto treat disease is a realistic and achievable goal. Adeno-associated\nviruses (AAVs) have quickly become the vector of choice in both\nlaboratory and clinical settings due to their strong safety record and\nstable expression in vivo, especially when compared to other viruses.\nHowever, the naturally occurring AAVs tend to have severely limited\ntropisms in lung tissues, as expression following intranasal delivery\nis generally inefficient and restricted to the airway epithelium. One\nsolution to this obstacle is to utilize a systemically injected AAV in\nconjunction with inhalant based delivery to cover a broad population\nof cells throughout the lung.", "doi": "10.1016/j.ymthe.2019.04.004", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2019-04-22", "series_number": "4", "volume": "27", "issue": "4", "pages": "Art. No. 101" }, { "id": "authors:ebpm3-mcm94", "collection": "authors", "collection_id": "ebpm3-mcm94", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190503-152305499", "type": "article", "title": "Multiplexed-CREATE Selection Yields AAV Vectors Targeting Different Cell Types of the Central Nervous System Following Systemic Delivery", "author": [ { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-S" }, { "family_name": "Chen", "given_name": "Xinhong", "clpid": "Chen-Xinhong" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Brown", "given_name": "David", "clpid": "Brown-D" }, { "family_name": "Dobreva", "given_name": "Tatyana", "orcid": "0000-0002-2625-8873", "clpid": "Dobreva-T" }, { "family_name": "Huang", "given_name": "Qin", "clpid": "Huang-Qin" }, { "family_name": "Ding", "given_name": "Xiaozhe", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Luo", "given_name": "Yicheng", "orcid": "0000-0003-3704-2389", "clpid": "Luo-Yicheng" }, { "family_name": "Einarsson", "given_name": "Petur H.", "clpid": "Einarsson-P-H" }, { "family_name": "Goeden", "given_name": "Nick", "clpid": "Goeden-N" }, { "family_name": "Flytzanis", "given_name": "Nicholas", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-N-C" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Recombinant adeno-associated viral (rAAV) capsids are widely accepted as safe gene delivery vehicles in research laboratories and in gene therapy clinical trials and there is potential to further improve their usage by evolving the surface of the capsids to enhance their affinity to specific cell-types or tissues after intravenous (IV) delivery. To this end, we built upon our previous Cre recombination-based AAV targeted evolution (CREATE) method (Deverman et al, Nat. Biotech., 2016; Chan et al., Nat. Neurosci., 2017) to develop Multiplexed-CREATE (M-CREATE). M-CREATE facilitates both positive and negative selections and minimizes the propagation of biases from successive rounds of selection via synthetic library generation. In addition to increasing the confidence in the selections, M-CREATE enables a detailed characterization of the selection process, improving our understanding of selection progression and outcome.", "doi": "10.1016/j.ymthe.2019.04.004", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2019-04-22", "series_number": "4", "volume": "27", "issue": "4", "pages": "Art. No. 99" }, { "id": "authors:568ya-eb855", "collection": "authors", "collection_id": "568ya-eb855", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190502-130921368", "type": "article", "title": "Engineered AAVS for CNS Transduction and Peripheral Organ De-Targeting across Species after Systemic Delivery", "author": [ { "family_name": "Flytzanis", "given_name": "Nicholas", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-N-C" }, { "family_name": "Goeden", "given_name": "Nick", "clpid": "Goeden-N" }, { "family_name": "Pickel", "given_name": "James", "clpid": "Pickel-J" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "In recent years, we have witnessed the development and clinical use of gene therapies at an accelerated pace. The option to introduce, silence, or edit genes greatly increases the therapeutic avenues for a variety of diseases, with adeno-associated viruses (AAVs) being the vehicle of\nchoice due to their low immunogenicity, stable expression, and strong\nclinical safety record. While the route of delivery has traditionally been\nlocalized, systemic administration via the blood is an option in cases\nwhere direct injection is impractical or widespread cell populations are\naffected. However, the naturally occurring AAV serotypes have evolved\nto broadly infect tissue, an undesirable characteristic for targeting\ntherapeutics to distinct cells.", "doi": "10.1016/j.ymthe.2019.04.004", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2019-04-22", "series_number": "4", "volume": "27", "issue": "4", "pages": "Art. No. 102" }, { "id": "authors:qewst-m1m10", "collection": "authors", "collection_id": "qewst-m1m10", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190409-100816489", "type": "article", "title": "TRIM9-Mediated Resolution of Neuroinflammation Confers Neuroprotection upon Ischemic Stroke in Mice", "author": [ { "family_name": "Zeng", "given_name": "Jianxiong", "clpid": "Zeng-Jianxiong" }, { "family_name": "Wang", "given_name": "Yaoming", "clpid": "Wang-Yaoming" }, { "family_name": "Luo", "given_name": "Zhifei", "clpid": "Luo-Zhifei" }, { "family_name": "Chang", "given_name": "Lin-Chun", "clpid": "Chang-Lin-Chun" }, { "family_name": "Yoo", "given_name": "Ji Seung", "clpid": "Yoo-Ji-Seung" }, { "family_name": "Yan", "given_name": "Huan", "clpid": "Yan-Huan" }, { "family_name": "Choi", "given_name": "Younho", "clpid": "Choi-Younho" }, { "family_name": "Xie", "given_name": "Xiaochun", "clpid": "Xie-Xiaochun" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-Benjamin-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Gupton", "given_name": "Stephanie L.", "clpid": "Gupton-Stephanie-L" }, { "family_name": "Zlokovic", "given_name": "Berislav V.", "orcid": "0000-0002-6802-8232", "clpid": "Zlokovic-Berislav-V" }, { "family_name": "Zhao", "given_name": "Zhen", "clpid": "Zhao-Zhen" }, { "family_name": "Jung", "given_name": "Jae U.", "clpid": "Jung-Jae-U" } ], "abstract": "Excessive and unresolved neuroinflammation is a key component of the pathological cascade in brain injuries such as ischemic stroke. Here, we report that TRIM9, a brain-specific tripartite motif (TRIM) protein, was highly expressed in the peri-infarct areas shortly after ischemic insults in mice, but expression was decreased in aged mice, which are known to have increased neuroinflammation after stroke. Mechanistically, TRIM9 sequestered \u03b2-transducin repeat-containing protein (\u03b2-TrCP) from the Skp-Cullin-F-box ubiquitin ligase complex, blocking I\u03baB\u03b1 degradation and thereby dampening nuclear factor \u03baB (NF-\u03baB)-dependent proinflammatory mediator production and immune cell infiltration to limit neuroinflammation. Consequently, Trim9-deficient mice were highly vulnerable to ischemia, manifesting uncontrolled neuroinflammation and exacerbated neuropathological outcomes. Systemic administration of a recombinant TRIM9 adeno-associated virus that drove brain-wide TRIM9 expression effectively resolved neuroinflammation and alleviated neuronal death, especially in aged mice. These findings reveal that TRIM9 is essential for resolving NF-\u03baB-dependent neuroinflammation to promote recovery and repair after brain injury and may represent an attractive therapeutic target.", "doi": "10.1016/j.celrep.2018.12.055", "pmcid": "PMC6485958", "issn": "2211-1247", "publisher": "Cell Press", "publication": "Cell Reports", "publication_date": "2019-04-09", "series_number": "2", "volume": "27", "issue": "2", "pages": "549-560" }, { "id": "authors:7yjfx-pw756", "collection": "authors", "collection_id": "7yjfx-pw756", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180816-125507808", "type": "article", "title": "Systemic AAV vectors for widespread and targeted gene delivery in rodents", "author": [ { "family_name": "Challis", "given_name": "Rosemary C.", "orcid": "0000-0003-3086-6553", "clpid": "Challis-R-C" }, { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-S" }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Challis", "given_name": "Collin", "orcid": "0000-0003-4716-6086", "clpid": "Challis-Collin" }, { "family_name": "Beadle", "given_name": "Keith", "clpid": "Beadle-K" }, { "family_name": "Jang", "given_name": "Min J.", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Kim", "given_name": "Hyun Min", "clpid": "Kim-Hyun-Min" }, { "family_name": "Rajendran", "given_name": "Pradeep S.", "orcid": "0000-0001-9790-0408", "clpid": "Rajendran-P-S" }, { "family_name": "Tompkins", "given_name": "John D.", "clpid": "Tompkins-J-D" }, { "family_name": "Shivkumar", "given_name": "Kalyanam", "orcid": "0000-0002-4121-1766", "clpid": "Shivkumar-K" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.", "doi": "10.1038/s41596-018-0097-3", "issn": "1754-2189", "publisher": "Nature Publishing Group", "publication": "Nature Protocols", "publication_date": "2019-02", "series_number": "2", "volume": "14", "issue": "2", "pages": "379-414" }, { "id": "authors:k4pg5-fsn12", "collection": "authors", "collection_id": "k4pg5-fsn12", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20181105-084438080", "type": "article", "title": "Whole-Brain Analysis of Cells and Circuits by Tissue Clearing and Light-Sheet Microscopy", "author": [ { "family_name": "Mano", "given_name": "Tomoyuki", "clpid": "Mano-Tomoyuki" }, { "family_name": "Albanese", "given_name": "Alexandre", "orcid": "0000-0001-7093-574X", "clpid": "Albanese-Alexandre" }, { "family_name": "Dodt", "given_name": "Hans-Ulrich", "orcid": "0000-0002-9784-9689", "clpid": "Dodt-Hans-Ulrich" }, { "family_name": "Erturk", "given_name": "Ali", "orcid": "0000-0001-5163-5100", "clpid": "Erturk-v" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Treweek", "given_name": "Jennifer B.", "clpid": "Treweek-Jennifer-B" }, { "family_name": "Miyawaki", "given_name": "Atsushi", "clpid": "Miyawaki-Atsushi" }, { "family_name": "Chung", "given_name": "Kwanghun", "orcid": "0000-0002-8167-3340", "clpid": "Chung-Kwanghun" }, { "family_name": "Ueda", "given_name": "Hiroki R.", "orcid": "0000-0001-8342-9176", "clpid": "Ueda-Hiroki-R" } ], "abstract": "In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for high-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous Mini-Symposium presented at the Society for Neuroscience's 2018 annual meeting.", "doi": "10.1523/jneurosci.1677-18.2018", "pmcid": "PMC6706004", "issn": "0270-6474", "publisher": "Society for Neuroscience", "publication": "Journal of Neuroscience", "publication_date": "2018-10-31", "series_number": "44", "volume": "38", "issue": "44", "pages": "9330-9337" }, { "id": "authors:3ngwx-46089", "collection": "authors", "collection_id": "3ngwx-46089", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180717-145659939", "type": "article", "title": "Two-photon microscopy with a double-wavelength metasurface objective lens", "author": [ { "family_name": "Arbabi", "given_name": "Ehsan", "orcid": "0000-0002-5328-3863", "clpid": "Arbabi-E" }, { "family_name": "Li", "given_name": "Jiaqi", "orcid": "0000-0003-2021-2310", "clpid": "Li-Jiaqi" }, { "family_name": "Hutchins", "given_name": "Romanus J.", "clpid": "Hutchins-R-J" }, { "family_name": "Kamali", "given_name": "Seyedeh Mahsa", "orcid": "0000-0002-6968-811X", "clpid": "Kamali-S-M" }, { "family_name": "Arbabi", "given_name": "Amir", "orcid": "0000-0001-8831-7552", "clpid": "Arbabi-A" }, { "family_name": "Horie", "given_name": "Yu", "orcid": "0000-0001-7083-1270", "clpid": "Horie-Yu" }, { "family_name": "Van Dorpe", "given_name": "Pol", "clpid": "Van-Dorpe-P" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Wagenaar", "given_name": "Daniel A.", "orcid": "0000-0002-6222-761X", "clpid": "Wagenaar-D-A" }, { "family_name": "Faraon", "given_name": "Andrei", "orcid": "0000-0002-8141-391X", "clpid": "Faraon-A" } ], "abstract": "Two-photon microscopy is a key imaging technique in life sciences due to its superior deep-tissue imaging capabilities. Light-weight and compact two-photon microscopes are of great interest because of their applications for in vivo deep brain imaging. Recently, dielectric metasurfaces have enabled a new category of small and lightweight optical elements, including objective lenses. Here we experimentally demonstrate two-photon microscopy using a double-wavelength metasurface lens. It is specifically designed to focus 820 and 605 nm light, corresponding to the excitation and emission wavelengths of the measured fluorophors, to the same focal distance. The captured two-photon images are qualitatively comparable to the ones taken by a conventional objective lens. Our metasurface lens can enable ultracompact two-photon microscopes with similar performance compared to current systems that are usually based on graded-index-lenses. In addition, further development of tunable metasurface lenses will enable fast axial scanning for volumetric imaging.", "doi": "10.1021/acs.nanolett.8b01737", "issn": "1530-6984", "publisher": "American Chemical Society", "publication": "Nano Letters", "publication_date": "2018-08-08", "series_number": "8", "volume": "18", "issue": "8", "pages": "4943-4948" }, { "id": "authors:k41np-yb952", "collection": "authors", "collection_id": "k41np-yb952", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180507-085656725", "type": "article", "title": "Viral Strategies for Targeting the Central and Peripheral Nervous Systems", "author": [ { "family_name": "Bedbrook", "given_name": "Claire N.", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-C-N" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Recombinant viruses allow for targeted transgene expression in specific cell populations throughout the nervous system. The adeno-associated virus (AAV) is among the most commonly used viruses for neuroscience research. Recombinant AAVs (rAAVs) are highly versatile and can package most cargo composed of desired genes within the capsid's \u223c5-kb carrying capacity. Numerous regulatory elements and intersectional strategies have been validated in rAAVs to enable cell type\u2013specific expression. rAAVs can be delivered to specific neuronal populations or globally throughout the animal. The AAV capsids have natural cell type or tissue tropism and trafficking that can be modified for increased specificity. Here, we describe recently engineered AAV capsids and associated cargo that have extended the utility of AAVs in targeting molecularly defined neurons throughout the nervous system, which will further facilitate neuronal circuit interrogation and discovery.", "doi": "10.1146/annurev-neuro-080317-062048", "issn": "0147-006X", "publisher": "Annual Reviews", "publication": "Annual Review of Neuroscience", "publication_date": "2018-07", "volume": "41", "pages": "323-348" }, { "id": "authors:dqwzv-z0c80", "collection": "authors", "collection_id": "dqwzv-z0c80", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180601-103720713", "type": "article", "title": "Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors", "author": [ { "family_name": "Patriarchi", "given_name": "Tommaso", "orcid": "0000-0001-9351-3734", "clpid": "Patriarchi-Tommaso" }, { "family_name": "Cho", "given_name": "Jounhong Ryan", "orcid": "0000-0001-9542-716X", "clpid": "Cho-Jounhong-Ryan" }, { "family_name": "Merten", "given_name": "Katharina", "orcid": "0000-0002-0197-0186", "clpid": "Merten-Katharina" }, { "family_name": "Howe", "given_name": "Mark W.", "clpid": "Howe-Mark-W" }, { "family_name": "Marley", "given_name": "Aaron", "clpid": "Marley-Aaron" }, { "family_name": "Xiong", "given_name": "Wei-Hong", "clpid": "Xiong-Wei-Hong" }, { "family_name": "Folk", "given_name": "Robert W.", "clpid": "Folk-Robert-W" }, { "family_name": "Broussard", "given_name": "Gerard Joey", "orcid": "0000-0003-1636-2615", "clpid": "Broussard-Gerard-Joey" }, { "family_name": "Liang", "given_name": "Ruqiang", "orcid": "0000-0002-3075-4554", "clpid": "Liang-Ruqiang" }, { "family_name": "Jang", "given_name": "Min Jee", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Zhong", "given_name": "Haining", "clpid": "Zhong-Haining" }, { "family_name": "Dombeck", "given_name": "Daniel", "clpid": "Dombeck-Daniel" }, { "family_name": "von Zastrow", "given_name": "Mark", "orcid": "0000-0003-1375-6926", "clpid": "von-Zastrow-Mark" }, { "family_name": "Nimmerjahn", "given_name": "Axel", "orcid": "0000-0002-0875-7855", "clpid": "Nimmerjahn-Axel" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Williams", "given_name": "John T.", "clpid": "Williams-John-T" }, { "family_name": "Tian", "given_name": "Lin", "orcid": "0000-0001-7012-6926", "clpid": "Tian-Lin" } ], "abstract": "Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires measuring spatiotemporally precise neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.", "doi": "10.1126/science.aat4422", "pmcid": "PMC6287765", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2018-06-29", "series_number": "6396", "volume": "360", "issue": "6396", "pages": "Art. No. eaat4422" }, { "id": "authors:xgk8s-ec475", "collection": "authors", "collection_id": "xgk8s-ec475", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180518-105323206", "type": "article", "title": "The Neuropeptide Tac2 Controls a Distributed Brain State Induced by Chronic Social Isolation Stress", "author": [ { "family_name": "Zelikowsky", "given_name": "Moriel", "clpid": "Zelikowski-Moriel" }, { "family_name": "Hui", "given_name": "May", "orcid": "0000-0002-6231-7383", "clpid": "Hui-May" }, { "family_name": "Karigo", "given_name": "Tomomi", "clpid": "Karigo-Tomomi" }, { "family_name": "Choe", "given_name": "Andrea", "clpid": "Choe-Andrea" }, { "family_name": "Yang", "given_name": "Bin", "clpid": "Yang-Bin" }, { "family_name": "Blanco", "given_name": "Mario R.", "orcid": "0000-0002-9852-2231", "clpid": "Blanco-Mario-R" }, { "family_name": "Beadle", "given_name": "Keith", "orcid": "0000-0002-5695-6461", "clpid": "Beadle-Keith" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-Benjamin-E" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" } ], "abstract": "Chronic social isolation causes severe psychological effects in humans, but their neural bases remain poorly understood. 2 weeks (but not 24 hr) of social isolation stress (SIS) caused multiple behavioral changes in mice and induced brain-wide upregulation of the neuropeptide tachykinin 2 (Tac2)/neurokinin B (NkB). Systemic administration of an Nk3R antagonist prevented virtually all of the behavioral effects of chronic SIS. Conversely, enhancing NkB expression and release phenocopied SIS in group-housed mice, promoting aggression and converting stimulus-locked defensive behaviors to persistent responses. Multiplexed analysis of Tac2/NkB function in multiple brain areas revealed dissociable, region-specific requirements for both the peptide and its receptor in different SIS-induced behavioral changes. Thus, Tac2 coordinates a pleiotropic brain state caused by SIS via a distributed mode of action. These data reveal the profound effects of prolonged social isolation on brain chemistry and function and suggest potential new therapeutic applications for Nk3R antagonists.", "doi": "10.1016/j.cell.2018.03.037", "pmcid": "PMC5967263", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "2018-05-17", "series_number": "5", "volume": "173", "issue": "5", "pages": "1265-1279" }, { "id": "authors:dpmpe-csf66", "collection": "authors", "collection_id": "dpmpe-csf66", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180706-154255761", "type": "article", "title": "Enhanced Efficacy of Gene Therapy Treatment for Niemann-Pick C1 Disease Using a Novel Serotype, AAV-PHP.B", "author": [ { "family_name": "Gu", "given_name": "Tansy", "clpid": "Gu-Tansy" }, { "family_name": "Davidson", "given_name": "Cristin", "clpid": "Davidson-C-D" }, { "family_name": "Gibson", "given_name": "Alana", "clpid": "Gibson-A-L" }, { "family_name": "Chandler", "given_name": "Randy", "clpid": "Chandler-R-J" }, { "family_name": "Beadle", "given_name": "Keith", "clpid": "Beadle-K" }, { "family_name": "Deverman", "given_name": "Ben", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Ory", "given_name": "Daniel S.", "clpid": "Ory-D-S" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Pavan", "given_name": "William J.", "clpid": "Pavan-W-J" }, { "family_name": "Venditti", "given_name": "Charles P.", "clpid": "Venditti-C-P" } ], "abstract": "Accessing the central nervous system (CNS) continues to present a challenge when developing therapies for the treatment of neurological diseases. Overcoming the barrier of gene transfer to brains of animals and patients from systemic circulation has been difficult. Recent advances using Cre recombination-based adeno-associated virus (AAV) targeted evolution (CREATE) has yielded a promising new serotype, AAV-PHP.B, with greater transduction than AAV9 in the adult mouse CNS after systemic delivery. Here we show systemic delivery of a therapeutic AAV-PHP.B vector outperforms the naturally occurring AAV9 in treatment of a murine model of a rare lysosomal storage disorder, Niemann-Pick C1 (NPC1) disease. Approximately 95% of patients have a mutation in NPC1 which results in either absence or a significant reduction in functional NPC1, a lysosomal transmembrane protein involved in cholesterol transport. NPC1 pathology involves lysosomal accumulation of unesterified cholesterol and other lipids. Patients typically present with neurological symptoms and visceral complications including hepatosplenomegaly. Disease progression in the null mouse model of NPC1 (Npc1-/-) is characterized by weight loss, ataxia, and early death. Results: We previously reported that systemic delivery of an AAV9 vector expressing the human NPC1 gene under transcriptional control of a ubiquitous promoter (EF1a) improved lifespan and ameliorated disease phenotype of Npc1-/- mice. Using a similar study design, we find that an otherwise identical AAV-PHP.B vector improved lifespan in Npc1-/- mice more effectively than an AAV9 vector.", "doi": "10.1016/j.ymthe.2018.05.001", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2018-05", "series_number": "5", "volume": "26", "issue": "5", "pages": "404" }, { "id": "authors:1849v-gb697", "collection": "authors", "collection_id": "1849v-gb697", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180529-083813817", "type": "article", "title": "Hydrogel-Tissue Chemistry: Principles and Applications", "author": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Treweek", "given_name": "Jennifer", "clpid": "Treweek-Jennifer-B" }, { "family_name": "Overton", "given_name": "Kristin", "clpid": "Overton-Kristin" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-Karl" } ], "abstract": "Over the past five years, a rapidly developing experimental approach has enabled high-resolution and high-content information retrieval from intact multicellular animal (metazoan) systems. New chemical and physical forms are created in the hydrogel-tissue chemistry process, and the retention and retrieval of crucial phenotypic information regarding constituent cells and molecules (and their joint interrelationships) are thereby enabled. For example, rich data sets defining both single-cell-resolution gene expression and single-cell-resolution activity during behavior can now be collected while still preserving information on three-dimensional positioning and/or brain-wide wiring of those very same neurons\u2014even within vertebrate brains. This new approach and its variants, as applied to neuroscience, are beginning to illuminate the fundamental cellular and chemical representations of sensation, cognition, and action. More generally, reimagining metazoans as metareactants\u2014or positionally defined three-dimensional graphs of constituent chemicals made available for ongoing functionalization, transformation, and readout\u2014is stimulating innovation across biology and medicine.", "doi": "10.1146/annurev-biophys-070317-032905", "pmcid": "PMC6359929", "issn": "1936-122X", "publisher": "Annual Reviews", "publication": "Annual Review of Biophysics", "publication_date": "2018-05", "volume": "47", "pages": "355-376" }, { "id": "authors:vhrr1-zfn16", "collection": "authors", "collection_id": "vhrr1-zfn16", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180706-151207096", "type": "article", "title": "Engineering Cell Type Specific Delivery Vectors for Noninvasive Modulation of Brain Circuits and Behaviors", "author": [ { "family_name": "Flytzanis", "given_name": "Nicholas", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-N-C" }, { "family_name": "Goeden", "given_name": "Nicholas", "clpid": "Goeden-N" }, { "family_name": "Cho", "given_name": "Jounhong Ryan", "orcid": "0000-0001-9542-716X", "clpid": "Cho-Jounhong-Ryan" }, { "family_name": "Kahan", "given_name": "Anat", "clpid": "Kahan-A" }, { "family_name": "Luongo", "given_name": "Francisco", "orcid": "0000-0002-6648-682X", "clpid": "Luongo-F" }, { "family_name": "Tsao", "given_name": "Doris Y.", "orcid": "0000-0003-1083-1919", "clpid": "Tsao-D-Y" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Within neuroscience the modulation and monitoring of specific circuits is achieved using a combination of transgenic animals and direct injection of viral vectors. To provide an alternative to invasive brain injection and increase transduction coverage and efficiency, we previously engineered adeno-associated viruses (AAVs) that efficiently cross the BBB in adult mice (Deverman et al, 2016; Chan et al, 2017). When delivering genes systemically, however, it is currently not possible to achieve a high degree of specificity for defined neuronal cell types in specific brain regions without the use of transgenic approaches. Here, we attempt to address this limitation by selectively engineering capsids with regional and cell-type specificity using CREATE, the Cre recombination-dependent AAV targeted evolution (Deverman et al, 2016) methodology that recovers capsids that transduce predefined Cre+ target cell populations.", "doi": "10.1016/j.ymthe.2018.05.001", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2018-05", "series_number": "5", "volume": "26", "issue": "5", "pages": "304" }, { "id": "authors:eswzw-tcp82", "collection": "authors", "collection_id": "eswzw-tcp82", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180706-160530988", "type": "article", "title": "Developing AAV Vectors for More Efficient and Selective Gene Expression in Specific Cell Types of the Nervous System Following Systemic Delivery", "author": [ { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-S" }, { "family_name": "Chan", "given_name": "Ken", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Jang", "given_name": "Min J.", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Huang", "given_name": "Qin", "clpid": "Huang-Qin" }, { "family_name": "Brown", "given_name": "David", "clpid": "Brown-D" }, { "family_name": "Dobreva", "given_name": "Tatyana", "orcid": "0000-0002-2625-8873", "clpid": "Dobreva-T" }, { "family_name": "Kim", "given_name": "Hyun M.", "clpid": "Kim-Hyun-Min" }, { "family_name": "Luo", "given_name": "Yicheng", "orcid": "0000-0003-3704-2389", "clpid": "Luo-Yicheng" }, { "family_name": "Hurt", "given_name": "Robert C.", "orcid": "0000-0002-4347-6901", "clpid": "Hurt-R-C" }, { "family_name": "Chen", "given_name": "Xinhong", "clpid": "Chen-Xinhong" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Recombinant adeno-associated viruses (rAAVs) are commonly used as gene delivery vehicles in biomedical research and have shown great potential for gene therapy. Systemic administration of AAVs can be used to achieve broad vector distribution. However, key challenges remain with systemic administration of AAVs as their lack of organ and/or cell-type specificity may cofound experiments and cause off-target effects. We and others have evolved AAVs to direct their tropism towards specific organs, such as the brain, after systemic delivery. We previously described CREATE, a Cre-based selection method, and used it to develop AAVs that efficiently transduce the central nervous system (CNS) in adult rodents, namely AAV-PHP.B (Deverman et al, Nat. Biotech., 2016), a further enhanced variant, AAV-PHP.eB (Chan et al., Nat. Neurosci., 2017), and AAV-PHP.S (Chan et al., Nat. Neurosci., 2017), a variant that can efficiently transduce the peripheral nervous system (PNS) and several visceral organs.", "doi": "10.1016/j.ymthe.2018.05.001", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2018-05", "series_number": "5", "volume": "26", "issue": "5", "pages": "321" }, { "id": "authors:gvqes-kwv35", "collection": "authors", "collection_id": "gvqes-kwv35", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180301-095437009", "type": "article", "title": "Gene therapy for the treatment of Niemann-Pick disease type C1: Comparison of AAV9 to a novel serotype, AAV-PHP.B", "author": [ { "family_name": "Davidson", "given_name": "Cristin", "clpid": "Davidson-C-D" }, { "family_name": "Gibson", "given_name": "Alana", "clpid": "Gibson-A-L" }, { "family_name": "Gu", "given_name": "Tansy", "clpid": "Gu-Tansy" }, { "family_name": "Chandler", "given_name": "Randy", "clpid": "Chandler-R-J" }, { "family_name": "Deverman", "given_name": "Benjamin", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Venditti", "given_name": "Charles", "clpid": "Venditti-C-P" }, { "family_name": "Pavan", "given_name": "William", "clpid": "Pavan-W-J" } ], "abstract": "[no abstract]", "doi": "10.1016/j.ymgme.2017.12.075", "issn": "1096-7192", "publisher": "Elsevier", "publication": "Molecular Genetics and Metabolism", "publication_date": "2018-02", "series_number": "2", "volume": "123", "issue": "2", "pages": "S36-S37" }, { "id": "authors:j25jc-5t646", "collection": "authors", "collection_id": "j25jc-5t646", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170918-075519928", "type": "article", "title": "Dopaminergic dysfunction in neurodevelopmental disorders: recent advances and synergistic technologies to aid basic research", "author": [ { "family_name": "Robinson", "given_name": "J. Elliott", "orcid": "0000-0001-9417-3938", "clpid": "Robinson-J-Elliott" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Neurodevelopmental disorders (NDDs) represent a diverse group of syndromes characterized by abnormal development of the central nervous system and whose symptomatology includes cognitive, emotional, sensory, and motor impairments. The identification of causative genetic defects has allowed for creation of transgenic NDD mouse models that have revealed pathophysiological mechanisms of disease phenotypes in a neural circuit- and cell type-specific manner. Mouse models of several syndromes, including Rett syndrome, Fragile X syndrome, Angelman syndrome, Neurofibromatosis type 1, etc., exhibit abnormalities in the structure and function of dopaminergic circuitry, which regulates motivation, motor behavior, sociability, attention, and executive function. Recent advances in technologies for functional circuit mapping, including tissue clearing, viral vector-based tracing methods, and optical readouts of neural activity, have refined our knowledge of dopaminergic circuits in unperturbed states, yet these tools have not been widely applied to NDD research. Here, we will review recent findings exploring dopaminergic function in NDD models and discuss the promise of new tools to probe NDD pathophysiology in these circuits.", "doi": "10.1016/j.conb.2017.08.003", "pmcid": "PMC5825239", "issn": "0959-4388", "publisher": "Elsevier", "publication": "Current Opinion in Neurobiology", "publication_date": "2018-02", "volume": "48", "pages": "17-29" }, { "id": "authors:p04pt-mv098", "collection": "authors", "collection_id": "p04pt-mv098", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171107-132336726", "type": "article", "title": "Deep tissue optical focusing and optogenetic modulation with time-reversed ultrasonically encoded light", "author": [ { "family_name": "Ruan", "given_name": "Haowen", "orcid": "0000-0002-4917-4509", "clpid": "Ruan-Haowen" }, { "family_name": "Brake", "given_name": "Joshua", "orcid": "0000-0002-5113-6886", "clpid": "Brake-Joshua-H" }, { "family_name": "Robinson", "given_name": "J. Elliott", "orcid": "0000-0001-9417-3938", "clpid": "Robinson-J-Elliott" }, { "family_name": "Liu", "given_name": "Yan", "orcid": "0000-0002-5837-4908", "clpid": "Liu-Yan" }, { "family_name": "Jang", "given_name": "Mooseok", "orcid": "0000-0003-1977-9539", "clpid": "Jang-Mooseok" }, { "family_name": "Xiao", "given_name": "Cheng", "orcid": "0000-0001-9649-7450", "clpid": "Xiao-Cheng" }, { "family_name": "Zhou", "given_name": "Chunyi", "clpid": "Zhou-Chunyi" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" } ], "abstract": "Noninvasive light focusing deep inside living biological tissue has long been a goal in biomedical optics. However, the optical scattering of biological tissue prevents conventional optical systems from tightly focusing visible light beyond several hundred micrometers. The recently developed wavefront shaping technique time-reversed ultrasonically encoded (TRUE) focusing enables noninvasive light delivery to targeted locations beyond the optical diffusion limit. However, until now, TRUE focusing has only been demonstrated inside nonliving tissue samples. We present the first example of TRUE focusing in 2-mm-thick living brain tissue and demonstrate its application for optogenetic modulation of neural activity in 800-\u03bcm-thick acute mouse brain slices at a wavelength of 532 nm. We found that TRUE focusing enabled precise control of neuron firing and increased the spatial resolution of neuronal excitation fourfold when compared to conventional lens focusing. This work is an important step in the application of TRUE focusing for practical biomedical uses.", "doi": "10.1126/sciadv.aao5520", "pmcid": "PMC5722648", "issn": "2375-2548", "publisher": "American Association for the Advancement of Science", "publication": "Science Advances", "publication_date": "2017-12", "series_number": "12", "volume": "3", "issue": "12", "pages": "Art. No. eaao5520" }, { "id": "authors:cn3ba-htx34", "collection": "authors", "collection_id": "cn3ba-htx34", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180108-144650592", "type": "article", "title": "Bone CLARITY reveals that anti-sclerostin antibody penetrates the lacunar canalicular system in mice under physiological loading conditions", "author": [ { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-A" }, { "family_name": "Chan", "given_name": "Ken", "clpid": "Chan-Ken" }, { "family_name": "Boyce", "given_name": "Rogely", "clpid": "Boyce-R" }, { "family_name": "Salimi-Moosavi", "given_name": "Hossein", "clpid": "Salimi-Moosavi-H" }, { "family_name": "McBride", "given_name": "Helen J.", "clpid": "McBride-H-J" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Anti-sclerostin antibody (Scl-Ab) neutralizes sclerostin activity, a secreted protein from osteocytes, resulting in WNT mediated increases in bone formation and decreases in bone resorption; this dual effect on bone results in rapid increases in bone mass and strength.", "doi": "10.1002/jbmr.3363", "issn": "0884-0431", "publisher": "Wiley", "publication": "Journal of Bone and Mineral Research", "publication_date": "2017-12", "series_number": "S1", "volume": "32", "issue": "S1", "pages": "S293" }, { "id": "authors:6tav9-rp997", "collection": "authors", "collection_id": "6tav9-rp997", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171127-142458372", "type": "article", "title": "Overriding sleep", "author": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "The need for sleep is recognized across the animal kingdom: Short-term sleep deprivation affects proper functioning, and extended sleep deprivation is fatal. However, animals can override the physiological drive to sleep when necessary or advantageous. Some can choose to sleep for shorter periods to avoid predators. Others, like the great frigate-bird, can sleep minimally and with half the brain during migratory flight. Still others, like the male pectoral sandpiper, can skip sleep altogether for weeks to maximize mating.", "doi": "10.1126/science.aap9535", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2017-10-27", "series_number": "6362", "volume": "358", "issue": "6362", "pages": "457-457" }, { "id": "authors:ptv4z-1sb38", "collection": "authors", "collection_id": "ptv4z-1sb38", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171030-140148566", "type": "article", "title": "Machine learning to design integral membrane channelrhodopsins for efficient eukaryotic expression and plasma membrane localization", "author": [ { "family_name": "Bedbrook", "given_name": "Claire N.", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-C-N" }, { "family_name": "Yang", "given_name": "Kevin K.", "orcid": "0000-0001-9045-6826", "clpid": "Yang-Kevin-K" }, { "family_name": "Rice", "given_name": "Austin J.", "clpid": "Rice-A-J" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Arnold", "given_name": "Frances H.", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "abstract": "There is growing interest in studying and engineering integral membrane proteins (MPs) that play key roles in sensing and regulating cellular response to diverse external signals. A MP must be expressed, correctly inserted and folded in a lipid bilayer, and trafficked to the proper cellular location in order to function. The sequence and structural determinants of these processes are complex and highly constrained. Here we describe a predictive, machine-learning approach that captures this complexity to facilitate successful MP engineering and design. Machine learning on carefully-chosen training sequences made by structure-guided SCHEMA recombination has enabled us to accurately predict the rare sequences in a diverse library of channelrhodopsins (ChRs) that express and localize to the plasma membrane of mammalian cells. These light-gated channel proteins of microbial origin are of interest for neuroscience applications, where expression and localization to the plasma membrane is a prerequisite for function. We trained Gaussian process (GP) classification and regression models with expression and localization data from 218 ChR chimeras chosen from a 118,098-variant library designed by SCHEMA recombination of three parent ChRs. We use these GP models to identify ChRs that express and localize well and show that our models can elucidate sequence and structure elements important for these processes. We also used the predictive models to convert a naturally occurring ChR incapable of mammalian localization into one that localizes well.", "doi": "10.1371/journal.pcbi.1005786", "pmcid": "PMC5695628", "issn": "1553-7358", "publisher": "Public Library of Science", "publication": "PLOS Computational Biology", "publication_date": "2017-10-23", "series_number": "10", "volume": "13", "issue": "10", "pages": "Art. No. e1005786" }, { "id": "authors:psp7j-nmv35", "collection": "authors", "collection_id": "psp7j-nmv35", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170921-075913502", "type": "article", "title": "The Jellyfish Cassiopea Exhibits a Sleep-like State", "author": [ { "family_name": "Nath", "given_name": "Ravi D.", "clpid": "Nath-Ravi-D" }, { "family_name": "Bedbrook", "given_name": "Claire N.", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-Claire-N" }, { "family_name": "Abrams", "given_name": "Michael J.", "orcid": "0000-0003-1864-1706", "clpid": "Abrams-Michael-J" }, { "family_name": "Basinger", "given_name": "Ty", "clpid": "Basinger-Ty" }, { "family_name": "Bois", "given_name": "Justin S.", "orcid": "0000-0001-7137-8746", "clpid": "Bois-J-S" }, { "family_name": "Prober", "given_name": "David A.", "orcid": "0000-0002-7371-4675", "clpid": "Prober-D-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Goentoro", "given_name": "Lea", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" } ], "abstract": "Do all animals sleep? Sleep has been observed in many vertebrates, and there is a growing body of evidence for sleep-like states in arthropods and nematodes. Here we show that sleep is also present in Cnidaria, an earlier-branching metazoan lineage. Cnidaria and Ctenophora are the first metazoan phyla to evolve tissue-level organization and differentiated cell types, such as neurons and muscle. In Cnidaria, neurons are organized into a non-centralized radially symmetric nerve net that nevertheless shares fundamental properties with the vertebrate nervous system: action potentials, synaptic transmission, neuropeptides, and neurotransmitters . It was reported that cnidarian soft corals and box jellyfish exhibit periods of quiescence, a pre-requisite for sleep-like states, prompting us to ask whether sleep is present in Cnidaria. Within Cnidaria, the upside-down jellyfish Cassiopea spp. displays a quantifiable pulsing behavior, allowing us to perform long-term behavioral tracking. Monitoring of Cassiopea pulsing activity for consecutive days and nights revealed behavioral quiescence at night that is rapidly reversible, as well as a delayed response to stimulation in the quiescent state. When deprived of nighttime quiescence, Cassiopea exhibited decreased activity and reduced responsiveness to a sensory stimulus during the subsequent day, consistent with homeostatic regulation of the quiescent state. Together, these results indicate that Cassiopea has a sleep-like state, supporting the hypothesis that sleep arose early in the metazoan lineage, prior to the emergence of a centralized nervous system.", "doi": "10.1016/j.cub.2017.08.014", "pmcid": "PMC5653286", "issn": "0960-9822", "publisher": "Cell Press", "publication": "Current Biology", "publication_date": "2017-10-09", "series_number": "19", "volume": "27", "issue": "19", "pages": "2984-2990" }, { "id": "authors:nr034-pht93", "collection": "authors", "collection_id": "nr034-pht93", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170926-130537624", "type": "article", "title": "Q&A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology?", "author": [ { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-Alon" }, { "family_name": "Jang", "given_name": "Min J.", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Challis", "given_name": "Collin", "orcid": "0000-0003-4716-6086", "clpid": "Challis-Collin" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Mammalian organs comprise a variety of cells that interact with each other and have distinct biological roles. Access to evaluate and perturb intact biological systems at the cellular and molecular levels is essential to fully understand their functioning in normal and diseased conditions, yet technical limitations have constrained most research to small pieces of tissue. Tissue clearing and optogenetics can help overcome this hurdle: tissue clearing affords optical interrogation of whole organs at the molecular level, and optogenetics enables the scalable control and measurement of cellular activity with light. In this Q&A, we delineate recent advances and practical challenges associated with these two techniques when applied body-wide.", "doi": "10.1186/s12915-017-0421-3", "pmcid": "PMC5613628", "issn": "1741-7007", "publisher": "BioMed Central", "publication": "BMC Biology", "publication_date": "2017-09-25", "volume": "15", "pages": "Art. No. 87" }, { "id": "authors:kehhk-szz26", "collection": "authors", "collection_id": "kehhk-szz26", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170626-152125765", "type": "article", "title": "Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems", "author": [ { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Jang", "given_name": "Min J.", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Yoo", "given_name": "Bryan B.", "orcid": "0000-0003-1450-2696", "clpid": "Yoo-Bryan-B" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-Alon" }, { "family_name": "Ravi", "given_name": "Namita", "clpid": "Ravi-Namita" }, { "family_name": "Wu", "given_name": "Wei-Li", "orcid": "0000-0003-2610-1881", "clpid": "Wu-Wei-Li" }, { "family_name": "S\u00e1nchez-Guardado", "given_name": "Luis", "orcid": "0000-0001-5598-8608", "clpid": "S\u00e1nchez-Guardado-Luis" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-C" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-Benjamin-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1 \u00d7 1011 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 \u00d7 1012 vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust cotransduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell-type-specific promoters and enhancers, these AAVs enable efficient and targetable genetic modification of cells throughout the nervous system of transgenic and non-transgenic animals.", "doi": "10.1038/nn.4593", "pmcid": "PMC5529245", "issn": "1097-6256", "publisher": "Springer Nature", "publication": "Nature Neuroscience", "publication_date": "2017-08", "series_number": "8", "volume": "20", "issue": "8", "pages": "1172-1179" }, { "id": "authors:9tv57-7fw75", "collection": "authors", "collection_id": "9tv57-7fw75", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170609-094413808", "type": "article", "title": "Dorsal Raphe Dopamine Neurons Modulate Arousal and Promote Wakefulness by Salient Stimuli", "author": [ { "family_name": "Cho", "given_name": "Jounhong Ryan", "orcid": "0000-0001-9542-716X", "clpid": "Cho-Jounhong-Ryan" }, { "family_name": "Treweek", "given_name": "Jennifer B.", "clpid": "Treweek-J-B" }, { "family_name": "Robinson", "given_name": "J. Elliott", "orcid": "0000-0001-9417-3938", "clpid": "Robinson-J-E" }, { "family_name": "Xiao", "given_name": "Cheng", "orcid": "0000-0001-9649-7450", "clpid": "Xiao-Cheng" }, { "family_name": "Bremner", "given_name": "Lindsay R.", "clpid": "Bremner-L-R" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Ventral midbrain dopamine (DA) is unambiguously involved in motivation and behavioral arousal, yet the contributions of other DA populations to these processes are poorly understood. Here, we demonstrate that the dorsal raphe nucleus DA neurons are critical modulators of behavioral arousal and sleep-wake patterning. Using simultaneous fiber photometry and polysomnography, we observed time-delineated dorsal raphe nucleus dopaminergic (DRNDA) activity upon exposure to arousal-evoking salient cues, irrespective of their hedonic valence. We also observed broader fluctuations of DRNDA activity across sleep-wake cycles with highest activity during wakefulness. Both endogenous DRNDA activity and optogenetically driven DRNDA activity were associated with waking from sleep, with DA signal strength predictive of wake duration. Conversely, chemogenetic inhibition opposed wakefulness and promoted NREM sleep, even in the face of salient stimuli. Therefore, the DRNDA population is a critical contributor to wake-promoting pathways and is capable of modulating sleep-wake states according to the outside environment, wherein the perception of salient stimuli prompts vigilance and arousal.", "doi": "10.1016/j.neuron.2017.05.020", "issn": "0896-6273", "publisher": "Elsevier", "publication": "Neuron", "publication_date": "2017-06-21", "series_number": "6", "volume": "94", "issue": "6", "pages": "1205-1219" }, { "id": "authors:m8ah5-thb30", "collection": "authors", "collection_id": "m8ah5-thb30", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170522-103736207", "type": "article", "title": "Global Representations of Goal-Directed Behavior in Distinct Cell Types of Mouse Neocortex", "author": [ { "family_name": "Allen", "given_name": "William E.", "clpid": "Allen-William-E" }, { "family_name": "Kauvar", "given_name": "Isaac V.", "clpid": "Kauvar-Isaac-V" }, { "family_name": "Chen", "given_name": "Michael Z.", "clpid": "Chen-Michael-Z" }, { "family_name": "Richman", "given_name": "Ethan B.", "clpid": "Richman-Ethan-B" }, { "family_name": "Yang", "given_name": "Samuel J.", "clpid": "Yang-Samuel-J" }, { "family_name": "Chan", "given_name": "Ken", "clpid": "Chan-Ken" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-Benjamin-E" }, { "family_name": "Luo", "given_name": "Liqun", "clpid": "Luo-Liqun" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-Karl" } ], "abstract": "The successful planning and execution of adaptive behaviors in mammals may require long-range coordination of neural networks throughout cerebral cortex. The neuronal implementation of signals that could orchestrate cortex-wide activity remains unclear. Here, we develop and apply methods for cortex-wide Ca^(2+) imaging in mice performing decision-making behavior and identify a global cortical representation of task engagement encoded in the activity dynamics of both single cells and superficial neuropil distributed across the majority of dorsal cortex. The activity of multiple molecularly defined cell types was found to reflect this representation with type-specific dynamics. Focal optogenetic inhibition tiled across cortex revealed a crucial role for frontal cortex in triggering this cortex-wide phenomenon; local inhibition of this region blocked both the cortex-wide response to task-initiating cues and the voluntary behavior. These findings reveal cell-type-specific processes in cortex for globally representing goal-directed behavior and identify a major cortical node that gates the global broadcast of task-related information.", "doi": "10.1016/j.neuron.2017.04.017", "pmcid": "PMC5723385", "issn": "0896-6273", "publisher": "Cell Press", "publication": "Neuron", "publication_date": "2017-05-17", "series_number": "4", "volume": "94", "issue": "4", "pages": "891-907" }, { "id": "authors:k9dq3-nnt81", "collection": "authors", "collection_id": "k9dq3-nnt81", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170601-080728015", "type": "article", "title": "Adeno-Associated Viral Gene Therapy Using PHP.B:NPC1 Ameliorates Disease Phenotype in Mouse Model of Niemann-Pick C1 Disease", "author": [ { "family_name": "Gibson", "given_name": "Alana L.", "clpid": "Gibson-A-L" }, { "family_name": "Davidson", "given_name": "Cristin D.", "clpid": "Davidson-C-D" }, { "family_name": "Chandler", "given_name": "Randy J.", "clpid": "Chandler-R-J" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Incao", "given_name": "Arturo A.", "clpid": "Incao-A-A" }, { "family_name": "Hubbard", "given_name": "Brandon T.", "clpid": "Hubbard-B-T" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Pavan", "given_name": "William J.", "clpid": "Pavan-W-J" }, { "family_name": "Venditti", "given_name": "Charles P.", "clpid": "Venditti-C-P" } ], "abstract": "[no abstract]", "doi": "10.1016/j.ymthe.2017.04.025", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2017-05", "series_number": "5", "volume": "25", "issue": "5", "pages": "Art. No. 225" }, { "id": "authors:j7rz1-8mn46", "collection": "authors", "collection_id": "j7rz1-8mn46", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20161221-135116035", "type": "article", "title": "Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow", "author": [ { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-A" }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Dobreva", "given_name": "Tatyana", "orcid": "0000-0002-2625-8873", "clpid": "Dobreva-T" }, { "family_name": "Brown", "given_name": "David", "clpid": "Brown-D" }, { "family_name": "Balani", "given_name": "Deepak H.", "clpid": "Balani-D-H" }, { "family_name": "Boyce", "given_name": "Rogely", "clpid": "Boyce-R" }, { "family_name": "Kronenberg", "given_name": "Henry M.", "clpid": "Kronenberg-H-M" }, { "family_name": "McBride", "given_name": "Helen J.", "clpid": "McBride-H-J" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Bone tissue harbors unique and essential physiological processes, such as hematopoiesis, bone growth, and bone remodeling. To enable visualization of these processes at the cellular level in an intact environment, we developed \"Bone CLARITY,\" a bone tissue clearing method. We used Bone CLARITY and a custom-built light-sheet fluorescence microscope to detect the endogenous fluorescence of Sox9-tdTomato+ osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice. To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semiautomatically detects individual Sox9-tdTomato+ cells in their native three-dimensional environment. Our computational method counted all labeled osteoprogenitor cells without relying on sampling techniques and displayed increased precision when compared with traditional stereology techniques for estimating the total number of these rare cells. We demonstrate the value of the clearing-imaging pipeline by quantifying changes in the population of Sox9-tdTomato\u2013labeled osteoprogenitor cells after sclerostin antibody treatment. Bone tissue clearing is able to provide fast and comprehensive visualization of biological processes in intact bone tissue.", "doi": "10.1126/scitranslmed.aah6518", "issn": "1946-6234", "publisher": "American Association for the Advancement of Science", "publication": "Science Translational Medicine", "publication_date": "2017-04-26", "series_number": "387", "volume": "9", "issue": "387", "pages": "Art. No. eaah6518" }, { "id": "authors:239ws-b2z58", "collection": "authors", "collection_id": "239ws-b2z58", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170310-130542741", "type": "article", "title": "Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins", "author": [ { "family_name": "Bedbrook", "given_name": "Claire N.", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-Claire-N" }, { "family_name": "Rice", "given_name": "Austin J.", "clpid": "Rice-Austin-J" }, { "family_name": "Yang", "given_name": "Kevin K.", "orcid": "0000-0001-9045-6826", "clpid": "Yang-Kevin-K" }, { "family_name": "Ding", "given_name": "Xiaozhe", "orcid": "0000-0002-0267-0791", "clpid": "Ding-Xiaozhe-Z" }, { "family_name": "Chen", "given_name": "Siyuan", "clpid": "Chen-Siyuan" }, { "family_name": "LeProust", "given_name": "Emily M.", "clpid": "Le-Proust-Emily-M" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Arnold", "given_name": "Frances H.", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "abstract": "Integral membrane proteins (MPs) are key engineering targets due to their critical roles in regulating cell function. In engineering MPs, it can be extremely challenging to retain membrane localization capability while changing other desired properties. We have used structure-guided SCHEMA recombination to create a large set of functionally diverse chimeras from three sequence-diverse channelrhodopsins (ChRs). We chose 218 ChR chimeras from two SCHEMA libraries and assayed them for expression and plasma membrane localization in human embryonic kidney cells. The majority of the chimeras express, with 89% of the tested chimeras outperforming the lowest-expressing parent; 12% of the tested chimeras express at even higher levels than any of the parents. A significant fraction (23%) also localize to the membrane better than the lowest-performing parent ChR. Most (93%) of these well-localizing chimeras are also functional light-gated channels. Many chimeras have stronger light-activated inward currents than the three parents, and some have unique off-kinetics and spectral properties relative to the parents. An effective method for generating protein sequence and functional diversity, SCHEMA recombination can be used to gain insights into sequence\u2013function relationships in MPs.", "doi": "10.1073/pnas.1700269114", "pmcid": "PMC5380088", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2017-03-28", "series_number": "13", "volume": "114", "issue": "13", "pages": "E2624-E2633" }, { "id": "authors:eyt49-dzw75", "collection": "authors", "collection_id": "eyt49-dzw75", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170307-082231020", "type": "article", "title": "Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore", "author": [ { "family_name": "Herwig", "given_name": "Lukas", "clpid": "Herwig-Lukas" }, { "family_name": "Rice", "given_name": "Austin J.", "clpid": "Rice-Austin-J" }, { "family_name": "Bedbrook", "given_name": "Claire N.", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-Claire-N" }, { "family_name": "Zhang", "given_name": "Ruijie K.", "orcid": "0000-0002-7251-5527", "clpid": "Zhang-Ruijie-K" }, { "family_name": "Lignell", "given_name": "Antti", "orcid": "0000-0001-7664-5583", "clpid": "Lignell-Antti" }, { "family_name": "Cahn", "given_name": "Jackson K. B.", "clpid": "Cahn-Jackson-K-B" }, { "family_name": "Renata", "given_name": "Hans", "orcid": "0000-0003-2468-2328", "clpid": "Renata-Hans" }, { "family_name": "Dodani", "given_name": "Sheel C.", "clpid": "Dodani-Sheel-C" }, { "family_name": "Cho", "given_name": "Inha", "orcid": "0000-0002-7564-5378", "clpid": "Cho-Inha" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Arnold", "given_name": "Frances H.", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "abstract": "By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore, we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems.", "doi": "10.1016/j.chembiol.2017.02.008", "pmcid": "PMC5357175", "issn": "2451-9456", "publisher": "Cell Press", "publication": "Cell Chemical Biology", "publication_date": "2017-03-16", "series_number": "3", "volume": "24", "issue": "3", "pages": "415-425" }, { "id": "authors:xwekr-he073", "collection": "authors", "collection_id": "xwekr-he073", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20161115-114016210", "type": "article", "title": "Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson's Disease", "author": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Sampson", "given_name": "Timothy R.", "orcid": "0000-0002-2486-8766", "clpid": "Sampson-Timothy-R" }, { "family_name": "Debelius", "given_name": "Justine W.", "orcid": "0000-0002-8605-3546", "clpid": "Debelius-Justine-W" }, { "family_name": "Thron", "given_name": "Taren", "orcid": "0000-0001-9577-2617", "clpid": "Thron-Taren-M" }, { "family_name": "Janssen", "given_name": "Stefan", "orcid": "0000-0003-0955-0589", "clpid": "Janssen-Stefan" }, { "family_name": "Shastri", "given_name": "Gauri G.", "clpid": "Shastri-Gauri-G" }, { "family_name": "Ilhan", "given_name": "Esra", "clpid": "Ilhan-Esra" }, { "family_name": "Challis", "given_name": "Collin", "orcid": "0000-0003-4716-6086", "clpid": "Challis-Collin" }, { "family_name": "Schretter", "given_name": "Catherine E.", "orcid": "0000-0002-3957-6838", "clpid": "Schretter-Catherine-E" }, { "family_name": "Rocha", "given_name": "Sandra", "clpid": "Rocha-Sandra" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Chesselet", "given_name": "Marie-Francoise", "clpid": "Chesselet-Marie-Francoise" }, { "family_name": "Keshavarzian", "given_name": "Ali", "clpid": "Keshavarzian-Ali" }, { "family_name": "Shannon", "given_name": "Kathleen M.", "clpid": "Shannon-Kathleen-M" }, { "family_name": "Krajmalnik-Brown", "given_name": "Rosa", "orcid": "0000-0001-6064-3524", "clpid": "Krajmalnik-Brown-Rosa" }, { "family_name": "Wittung-Stafshede", "given_name": "Pernilla", "orcid": "0000-0003-1058-1964", "clpid": "Wittung-Stafshede-Pernilla" }, { "family_name": "Knight", "given_name": "Rob", "orcid": "0000-0002-0975-9019", "clpid": "Knight-Rob" } ], "abstract": "The intestinal microbiota influence neurodevelopment, modulate behavior, and contribute to neurological disorders. However, a functional link between gut bacteria and neurodegenerative diseases remains unexplored. Synucleinopathies are characterized by aggregation of the protein \u03b1-synuclein (\u03b1Syn), often resulting in motor dysfunction as exemplified by Parkinson's disease (PD). Using mice that overexpress \u03b1Syn, we report herein that gut microbiota are required for motor deficits, microglia activation, and \u03b1Syn pathology. Antibiotic treatment ameliorates, while microbial re-colonization promotes, pathophysiology in adult animals, suggesting that postnatal signaling between the gut and the brain modulates disease. Indeed, oral administration of specific microbial metabolites to germ-free mice promotes neuroinflammation and motor symptoms. Remarkably, colonization of \u03b1Syn-overexpressing mice with microbiota from PD-affected patients enhances physical impairments compared to microbiota transplants from healthy human donors. These findings reveal that gut bacteria regulate movement disorders in mice and suggest that alterations in the human microbiome represent a risk factor for PD.", "doi": "10.1016/j.cell.2016.11.018", "pmcid": "PMC5718049", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "2016-12-01", "series_number": "6", "volume": "167", "issue": "6", "pages": "1469-1480" }, { "id": "authors:p1jf2-4kh88", "collection": "authors", "collection_id": "p1jf2-4kh88", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20161128-093036959", "type": "article", "title": "Global Collaboration, Learning from Other Fields", "author": [ { "family_name": "Fairhall", "given_name": "Adrienne", "clpid": "Fairhall-A" }, { "family_name": "Svoboda", "given_name": "Karel", "clpid": "Svoboda-K" }, { "family_name": "Nobre", "given_name": "Anna C.", "clpid": "Nobre-A-C" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Nusser", "given_name": "Zoltan", "clpid": "Nusser-Z" }, { "family_name": "Ghosh", "given_name": "Anirvan", "clpid": "Ghosh-A" }, { "family_name": "Tsien", "given_name": "Richard", "clpid": "Tsien-Richard" } ], "abstract": "Neuroscience research is becoming increasingly more collaborative and interdisciplinary with partnerships between industry and academia and insights from fields beyond neuroscience. In the age of institutional initiatives and multi-investigator collaborations, scientists from around the world shared their perspectives on the effectiveness of large-scale collaborations versus single-lab, hypothesis-driven science.", "doi": "10.1016/j.neuron.2016.10.040", "issn": "0896-6273", "publisher": "Elsevier", "publication": "Neuron", "publication_date": "2016-11-02", "series_number": "3", "volume": "92", "issue": "3", "pages": "561-563" }, { "id": "authors:agxyk-a4992", "collection": "authors", "collection_id": "agxyk-a4992", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160920-092113773", "type": "article", "title": "Regional glutamine deficiency in tumours promotes dedifferentiation through inhibition of histone demethylation", "author": [ { "family_name": "Pan", "given_name": "Min", "clpid": "Pan-Min" }, { "family_name": "Reid", "given_name": "Michael A.", "clpid": "Reid-Michael-A" }, { "family_name": "Lowman", "given_name": "Xazmin H.", "clpid": "Lowman-Xazmin-H" }, { "family_name": "Kulkarni", "given_name": "Rajan P.", "clpid": "Kulkarni-Rajan-P" }, { "family_name": "Tran", "given_name": "Thai Q.", "clpid": "Tran-Thai-Q" }, { "family_name": "Liu", "given_name": "Xiaojing", "clpid": "Liu-Xiaojing" }, { "family_name": "Yang", "given_name": "Ying", "clpid": "Yang-Ying" }, { "family_name": "Hernandez-Davies", "given_name": "Jenny E.", "clpid": "Hernandez-Davies-Jenny-E" }, { "family_name": "Rosales", "given_name": "Kimberly K.", "clpid": "Rosales-Kimberly-K" }, { "family_name": "Li", "given_name": "Haiqing", "clpid": "Li-Haiqing" }, { "family_name": "Hugo", "given_name": "Willy", "clpid": "Hugo-Willy" }, { "family_name": "Song", "given_name": "Chunying", "clpid": "Song-Chunying" }, { "family_name": "Xu", "given_name": "Xiangdong", "clpid": "Xu-Xiangdong" }, { "family_name": "Schones", "given_name": "Dustin E.", "clpid": "Schones-Dustin-E" }, { "family_name": "Ann", "given_name": "David K.", "clpid": "Ann-David-K" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Lo", "given_name": "Roger S.", "clpid": "Lo-Roger-S" }, { "family_name": "Locasale", "given_name": "Jason W.", "orcid": "0000-0002-7766-3502", "clpid": "Locasale-Jason-W" }, { "family_name": "Kong", "given_name": "Mei", "clpid": "Kong-Mei" } ], "abstract": "Poorly organized tumour vasculature often results in areas of limited nutrient supply and hypoxia. Despite our understanding of solid tumour responses to hypoxia, how nutrient deprivation regionally affects tumour growth and therapeutic response is poorly understood. Here, we show that the core region of solid tumours displayed glutamine deficiency compared with other amino acids. Low glutamine in tumour core regions led to dramatic histone hypermethylation due to decreased \u03b1-ketoglutarate levels, a key cofactor for the Jumonji-domain-containing histone demethylases. Using patient-derived ^(V600E)BRAF melanoma cells, we found that low-glutamine-induced histone hypermethylation resulted in cancer cell dedifferentiation and resistance to BRAF inhibitor treatment, which was largely mediated by methylation on H3K27, as knockdown of the H3K27-specific demethylase KDM6B and the methyltransferase EZH2 respectively reproduced and attenuated the low-glutamine effects in vitro and in vivo. Thus, intratumoral regional variation in the nutritional microenvironment contributes to tumour heterogeneity and therapeutic response.", "doi": "10.1038/ncb3410", "pmcid": "PMC5536113", "issn": "1465-7392", "publisher": "Nature Publishing Group", "publication": "Nature Cell Biology", "publication_date": "2016-10", "series_number": "10", "volume": "18", "issue": "10", "pages": "1090-1101" }, { "id": "authors:e2n6h-2v807", "collection": "authors", "collection_id": "e2n6h-2v807", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160927-144217261", "type": "article", "title": "Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling", "author": [ { "family_name": "DePas", "given_name": "William H.", "clpid": "DePas-W-H" }, { "family_name": "Starwalt-Lee", "given_name": "Ruth", "clpid": "Starwalt-Lee-R" }, { "family_name": "Van Sambeek", "given_name": "Lindsey", "orcid": "0000-0002-7206-7410", "clpid": "Van-Sambeek-L" }, { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-S" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "abstract": "Physiological resistance to antibiotics confounds the treatment of many chronic bacterial infections, motivating researchers to identify novel therapeutic approaches. To do this effectively, an understanding of how microbes survive in vivo is needed. Though much can be inferred from bulk approaches to characterizing complex environments, essential information can be lost if spatial organization is not preserved. Here, we introduce a tissue-clearing technique, termed MiPACT, designed to retain and visualize bacteria with associated proteins and nucleic acids in situ on various spatial scales. By coupling MiPACT with hybridization chain reaction (HCR) to detect rRNA in sputum samples from cystic fibrosis (CF) patients, we demonstrate its ability to survey thousands of bacteria (or bacterial aggregates) over millimeter scales and quantify aggregation of individual species in polymicrobial communities. By analyzing aggregation patterns of four prominent CF pathogens, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus sp., and Achromobacter xylosoxidans, we demonstrate a spectrum of aggregation states: from mostly single cells (A. xylosoxidans), to medium-sized clusters (S. aureus), to a mixture of single cells and large aggregates (P. aeruginosa and Streptococcus sp.). Furthermore, MiPACT-HCR revealed an intimate interaction between Streptococcus sp. and specific host cells. Lastly, by comparing standard rRNA fluorescence in situ hybridization signals to those from HCR, we found that different populations of S. aureus and A. xylosoxidans grow slowly overall yet exhibit growth rate heterogeneity over hundreds of microns. These results demonstrate the utility of MiPACT-HCR to directly capture the spatial organization and metabolic activity of bacteria in complex systems, such as human sputum.", "doi": "10.1128/mBio.00796-16", "pmcid": "PMC5040109", "issn": "2150-7511", "publisher": "American Society for Microbiology", "publication": "mBio", "publication_date": "2016-09-27", "series_number": "5", "volume": "7", "issue": "5", "pages": "e00796-16" }, { "id": "authors:npxan-57878", "collection": "authors", "collection_id": "npxan-57878", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160628-085406782", "type": "article", "title": "Single-molecule RNA detection at depth via hybridization chain reaction and tissue hydrogel embedding and clearing", "author": [ { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-Eric" }, { "family_name": "Schwarzkopf", "given_name": "Maayan", "orcid": "0000-0001-8128-1059", "clpid": "Schwarzkopf-Maayan" }, { "family_name": "He", "given_name": "Ting-Fang", "clpid": "He-Ting-Fang" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-Alon" }, { "family_name": "Sohn", "given_name": "Chang Ho", "clpid": "Sohn-Chang-Ho" }, { "family_name": "Lignell", "given_name": "Antti", "orcid": "0000-0001-7664-5583", "clpid": "Lignell-Antti" }, { "family_name": "Choi", "given_name": "Harry M. T.", "orcid": "0000-0002-1530-0773", "clpid": "Choi-Harry-M-T" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas, from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA, imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (PAssive CLARITY Technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving \u223c20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.", "doi": "10.1242/dev.138560", "pmcid": "PMC5004914", "issn": "0950-1991", "publisher": "Company of Biologists", "publication": "Development", "publication_date": "2016-08-01", "series_number": "15", "volume": "143", "issue": "15", "pages": "2862-2867" }, { "id": "authors:wcraa-mcj63", "collection": "authors", "collection_id": "wcraa-mcj63", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160712-101339180", "type": "article", "title": "Extracting structural and functional features of widely distributed biological circuits with single cell resolution via tissue clearing and delivery vectors", "author": [ { "family_name": "Treweek", "given_name": "Jennifer Brooke", "orcid": "0000-0002-5601-9646", "clpid": "Treweek-Jennifer-B" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "The scientific community has learned a great deal from imaging small and naturally transparent organisms such as nematodes and zebrafish. The consequences of genetic mutations on their organ development and survival can be visualized easily and with high-throughput at the organism-wide scale. In contrast, three-dimensional information is less accessible in mammalian subjects because the heterogeneity of light-scattering tissue elements renders their organs opaque. Likewise, genetically labeling desired circuits across mammalian bodies is prohibitively slow and costly via the transgenic route. Emerging breakthroughs in viral vector engineering, genome editing tools, and tissue clearing can render larger opaque organisms genetically tractable and transparent for whole-organ cell phenotyping, tract tracing and imaging at depth.", "doi": "10.1016/j.copbio.2016.03.012", "pmcid": "PMC4975678", "issn": "0958-1669", "publisher": "Elsevier", "publication": "Current Opinion in Biotechnology", "publication_date": "2016-08", "volume": "40", "pages": "193-207" }, { "id": "authors:qk83w-f4y03", "collection": "authors", "collection_id": "qk83w-f4y03", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160527-092723542", "type": "article", "title": "Using Cre-Dependent In Vivo Selection to Identify AAV Variants That Enable Efficient and Widespread Gene Transfer to the Adult Central Nervous System", "author": [ { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Pravdo", "given_name": "Piers L.", "clpid": "Pravdo-P-L" }, { "family_name": "Simpson", "given_name": "Bryan P.", "clpid": "Simpson-B-P" }, { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-S" }, { "family_name": "Luo", "given_name": "Yicheng", "orcid": "0000-0003-3704-2389", "clpid": "Luo-Yicheng" }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Banerjee", "given_name": "Abhik", "clpid": "Banerjee-A" }, { "family_name": "Wu", "given_name": "Wei-Li", "orcid": "0000-0003-2610-1881", "clpid": "Wu-Wei-Li" }, { "family_name": "Yang", "given_name": "Bin", "clpid": "Yang-Bin" }, { "family_name": "Huber", "given_name": "Nina", "clpid": "Huber-Nina" }, { "family_name": "Pasca", "given_name": "Sergiu P.", "clpid": "Pasca-S-P" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Recombinant adeno-associated viruses (rAAVs) are commonly\nused vehicles for in vivo gene transfer. However, the tropism\nrepertoire of naturally occurring AAVs is limited, prompting the\ndevelopment of novel AAV capsids with more desirable transduction\ncharacteristics. We have developed a capsid selection method, called\nCre-recombination-based AAV targeted evolution (CREATE), that\nenables the identification of AAV capsids that more efficiently\ntransduce defined cell populations in vivo (Deverman et al. in press,\nNature Biotechnology). We generated AAV capsid libraries and\nused CREATE to identify variants that cross the blood brain barrier\nand efficiently and widely transduce astrocytes in the mouse central\nnervous system (CNS) after intravenous injection. One variant, AAVPHP.\nB, transfers genes throughout the adult CNS with an efficiency\nthat is 40- to 92-fold greater (depending on the CNS region) than that\nof the current standard, AAV9. It transduces the majority of astrocytes\nand neurons across multiple CNS regions, and in vitro, it transduces\nhuman neurons and astrocytes more efficiently than does AAV9. We\nare now evolving AAV-PHP.B for even greater transduction of specific\nCNS cell types as a means to both develop more effective vectors\nand to gain insight into the mechanism of enhanced transduction.\nOur identification of AAV-PHP.B and several other enhanced vectors\nafter only two rounds of selection establishes CREATE as a powerful\nmethod to customize AAV vectors for biomedical applications.", "doi": "10.1016/S1525-0016(16)33061-1", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2016-05", "series_number": "S1", "volume": "24", "issue": "S1", "pages": "S99" }, { "id": "authors:px9eg-37y87", "collection": "authors", "collection_id": "px9eg-37y87", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160420-090626768", "type": "article", "title": "Cholinergic Mesopontine Signals Govern Locomotion and Reward through Dissociable Midbrain Pathways", "author": [ { "family_name": "Xiao", "given_name": "Cheng", "orcid": "0000-0001-9649-7450", "clpid": "Xiao-Cheng" }, { "family_name": "Cho", "given_name": "Jounhong Ryan", "orcid": "0000-0001-9542-716X", "clpid": "Cho-Jounhong-Ryan" }, { "family_name": "Zhou", "given_name": "Chunyi", "clpid": "Zhou-Chunyi" }, { "family_name": "Treweek", "given_name": "Jennifer B.", "orcid": "0000-0002-5601-9646", "clpid": "Treweek-Jennifer-B" }, { "family_name": "Chan", "given_name": "Ken", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "McKinney", "given_name": "Sheri L.", "clpid": "McKinney-Sheri-L" }, { "family_name": "Yang", "given_name": "Bin", "clpid": "Yang-Bin" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "The mesopontine tegmentum, including the pedunculopontine and laterodorsal tegmental nuclei (PPN and LDT), provides major cholinergic inputs to midbrain and regulates locomotion and reward. To delineate the underlying projection-specific circuit mechanisms, we employed optogenetics to control mesopontine cholinergic neurons at somata and at divergent projections within distinct midbrain areas. Bidirectional manipulation of PPN cholinergic cell bodies exerted opposing effects on locomotor behavior and reinforcement learning. These motor and reward effects were separable via limiting photostimulation to PPN cholinergic terminals in the ventral substantia nigra pars compacta (vSNc) or to the ventral tegmental area (VTA), respectively. LDT cholinergic neurons also form connections with vSNc and VTA neurons; however, although photo-excitation of LDT cholinergic terminals in the VTA caused positive reinforcement, LDT-to-vSNc modulation did not alter locomotion or reward. Therefore, the selective targeting of projection-specific mesopontine cholinergic pathways may offer increased benefit in treating movement and addiction disorders.", "doi": "10.1016/j.neuron.2016.03.028", "pmcid": "PMC4840478", "issn": "0896-6273", "publisher": "Cell Press", "publication": "Neuron", "publication_date": "2016-04-20", "series_number": "2", "volume": "90", "issue": "2", "pages": "333-347" }, { "id": "authors:ezhsc-jnr62", "collection": "authors", "collection_id": "ezhsc-jnr62", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160606-133730058", "type": "article", "title": "Tools for Anatomical and Functional Analysis of Widely Distributed Brain Networks", "author": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Our research interests focus on developing tools and methods for neuroscience (optogenetic actuators and sensors; tissue clearing and imaging) as well as on investigating the mechanisms underlying deep brain stimulation (DBS) and on the long-term effects of DBS on neuronal health, function, and ultimately behavior. Methods such as Optogenetics (for precise function control) and CLARITY (for intact circuit mapping) enable scientists to understand nervous system circuit anatomy, function, and dysfunction at a depth previously impossible, vastly expanding knowledge of the nervous system and associated psychiatric, neurological, and peripheral organs disorders. CLARITY renders tissue transparent for visualization and identification of cellular components and their molecular identity without slicing therefore improving the likelihood to detect and accurately map sparse populations or projections. This method complements Optogenetics in that it can reveal circuit-wide effects of optogenetic manipulations and also aid in mapping novel circuits that need tuning in disease. The mastery, improvement, and implementation of both these methods require a large-scale cross-disciplinary effort. To this end, our projects range from optimizing CLARITY for non-brain tissue, to developing actuators and sensors of electrical activity via protein engineering and mining of natural sources, to studying the impact of neuromodulation of selected pathways in the central and peripheral nervous system on behavior.", "doi": "10.1016/j.bpj.2015.11.1965", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2016-02-16", "series_number": "3", "volume": "110", "issue": "3", "pages": "364A-365A" }, { "id": "authors:j19ev-9mb91", "collection": "authors", "collection_id": "j19ev-9mb91", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160603-153016574", "type": "article", "title": "Rhodopsin Engineering through Structure-Guided Recombination", "author": [ { "family_name": "Rice", "given_name": "Austin J.", "clpid": "Rice-A-J" }, { "family_name": "Bedbrook", "given_name": "Claire N.", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-C-N" }, { "family_name": "Yang", "given_name": "Kevin K.", "orcid": "0000-0001-9045-6826", "clpid": "Yang-Kevin-K" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Arnold", "given_name": "Frances H.", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "abstract": "Membrane proteins assume key roles in environmental sensing, metabolite uptake, and product efflux. Given the vital connections they facilitate between a cell and its environment, there is great potential to engineer these proteins to modulate cellular activity and productivity. For this project, we focus our efforts on rhodopsins, light-sensing transporters and channels that have applications in optical biosensors, neuronal monitoring and control, and bioenergy. Using structure-guided recombination we have built a library of chimeric rhodopsins. We used a novel assay for membrane protein localization to pre-screen a fraction of the library. A diverse selection of well-expressing variants has been further characterized via electrophysiology. Through machine learning we can associate rhodopsin expression, localization, and functional properties to blocks of structure and make predictions on the properties of untested chimeras.", "doi": "10.1016/j.bpj.2015.11.947", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2016-02-16", "series_number": "3", "volume": "110", "issue": "3", "pages": "170A" }, { "id": "authors:sgthf-gne05", "collection": "authors", "collection_id": "sgthf-gne05", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160201-131537838", "type": "article", "title": "Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain", "author": [ { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-Benjamin-E" }, { "family_name": "Pravdo", "given_name": "Piers L.", "clpid": "Pravdo-Piers-L" }, { "family_name": "Simpson", "given_name": "Bryan P.", "clpid": "Simpson-Bryan-P" }, { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-Sripriya" }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Banerjee", "given_name": "Abhik", "orcid": "0000-0001-9127-5461", "clpid": "Banerjee-Abhik-K" }, { "family_name": "Wu", "given_name": "Wei-Li", "orcid": "0000-0003-2610-1881", "clpid": "Wu-Wei-Li" }, { "family_name": "Yang", "given_name": "Bin", "clpid": "Yang-Bin" }, { "family_name": "Huber", "given_name": "Nina", "clpid": "Huber-Nina" }, { "family_name": "Pasca", "given_name": "Sergiu P.", "orcid": "0000-0002-3216-3248", "clpid": "Pa\u0219ca-Sergiu-P" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination\u2013based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9, and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.", "doi": "10.1038/nbt.3440", "pmcid": "PMC5088052", "issn": "1087-0156", "publisher": "Nature Publishing Group", "publication": "Nature Biotechnology", "publication_date": "2016-02", "series_number": "2", "volume": "34", "issue": "2", "pages": "204-209" }, { "id": "authors:vj1b1-mdj56", "collection": "authors", "collection_id": "vj1b1-mdj56", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160202-092152118", "type": "article", "title": "Fluorescent boost for voltage sensors", "author": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-N-C" } ], "abstract": "The development of a voltage sensor in which a microbial rhodopsin protein is fused with a fluorescent protein enables the neuronal activity of single cells in live animals to be measured with unprecedented speed and accuracy.", "doi": "10.1038/529469a", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2016-01-28", "series_number": "7587", "volume": "529", "issue": "7587", "pages": "469-470" }, { "id": "authors:yx9xd-rk111", "collection": "authors", "collection_id": "yx9xd-rk111", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160108-103050704", "type": "article", "title": "Genomic Reconstruction of an Uncultured Hydrothermal Vent Gammaproteobacterial Methanotroph (Family Methylothermaceae) Indicates Multiple Adaptations to Oxygen Limitation", "author": [ { "family_name": "Skennerton", "given_name": "Connor T.", "orcid": "0000-0003-1320-4873", "clpid": "Skennerton-C-T" }, { "family_name": "Ward", "given_name": "Lewis M.", "orcid": "0000-0002-9290-2567", "clpid": "Ward-L-M" }, { "family_name": "Michel", "given_name": "Alice", "orcid": "0000-0002-0273-4097", "clpid": "Michel-Alice-J" }, { "family_name": "Metcalfe", "given_name": "Kyle", "orcid": "0000-0002-2963-765X", "clpid": "Metcalfe-K-S" }, { "family_name": "Valiente", "given_name": "Chanel", "clpid": "Valiente-C" }, { "family_name": "Mullin", "given_name": "Sean", "orcid": "0000-0002-6225-3279", "clpid": "Mullin-S-W" }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "abstract": "Hydrothermal vents are an important contributor to marine biogeochemistry, producing large volumes of reduced fluids, gasses, and metals and housing unique, productive microbial and animal communities fueled by chemosynthesis. Methane is a common constituent of hydrothermal vent fluid and is frequently consumed at vent sites by methanotrophic bacteria that serve to control escape of this greenhouse gas into the atmosphere. Despite their ecological and geochemical importance, little is known about the ecophysiology of uncultured hydrothermal vent-associated methanotrophic bacteria. Using metagenomic binning techniques, we recovered and analyzed a near-complete genome from a novel gammaproteobacterial methanotroph (B42) associated with a white smoker chimney in the Southern Lau basin. B42 was the dominant methanotroph in the community, at \u223c80x coverage, with only four others detected in the metagenome, all on low coverage contigs (7x\u201312x). Phylogenetic placement of B42 showed it is a member of the Methylothermaceae, a family currently represented by only one sequenced genome. Metabolic inferences based on the presence of known pathways in the genome showed that B42 possesses a branched respiratory chain with A- and B-family heme copper oxidases, cytochrome bd oxidase and a partial denitrification pathway. These genes could allow B42 to respire over a wide range of oxygen concentrations within the highly dynamic vent environment. Phylogenies of the denitrification genes revealed they are the result of separate horizontal gene transfer from other Proteobacteria and suggest that denitrification is a selective advantage in conditions where extremely low oxygen concentrations require all oxygen to be used for methane activation.", "doi": "10.3389/fmicb.2015.01425", "pmcid": "PMC4688376", "issn": "1664-302X", "publisher": "Frontiers Research Foundation", "publication": "Frontiers in Microbiology", "publication_date": "2015-12-23", "volume": "6", "pages": "Art. No. 1425" }, { "id": "authors:e4qmn-syf18", "collection": "authors", "collection_id": "e4qmn-syf18", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150914-123933415", "type": "article", "title": "Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping", "author": [ { "family_name": "Treweek", "given_name": "Jennifer B.", "clpid": "Treweek-J-B" }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-N-C" }, { "family_name": "Yang", "given_name": "Bin", "clpid": "Yang-Bin" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-A" }, { "family_name": "Lignell", "given_name": "Antti", "orcid": "0000-0001-7664-5583", "clpid": "Lignell-Antti" }, { "family_name": "Xiao", "given_name": "Cheng", "orcid": "0000-0001-9649-7450", "clpid": "Xiao-Cheng" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Ladinsky", "given_name": "Mark S.", "orcid": "0000-0002-1036-3513", "clpid": "Ladinsky-M-S" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Fowlkes", "given_name": "Charless C.", "orcid": "0000-0002-2990-1780", "clpid": "Fowlkes-C-C" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1\u20132 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.", "doi": "10.1038/nprot.2015.122", "pmcid": "PMC4917295", "issn": "1754-2189", "publisher": "Nature Publishing Group", "publication": "Nature Protocols", "publication_date": "2015-11", "series_number": "11", "volume": "10", "issue": "11", "pages": "1860-1896" }, { "id": "authors:746rc-19032", "collection": "authors", "collection_id": "746rc-19032", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150728-090618874", "type": "article", "title": "Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo", "author": [ { "family_name": "Bedbrook", "given_name": "Claire N.", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-Claire-N" }, { "family_name": "Kato", "given_name": "Mihoko", "orcid": "0000-0003-3827-8879", "clpid": "Kato-Mihoko" }, { "family_name": "Ravindra Kumar", "given_name": "Sripriya", "orcid": "0000-0001-6033-7631", "clpid": "Ravindra-Kumar-Sripriya" }, { "family_name": "Lakshmanan", "given_name": "Anupama", "orcid": "0000-0002-6702-837X", "clpid": "Lakshmanan-Anupama" }, { "family_name": "Nath", "given_name": "Ravi D.", "clpid": "Nath-Ravi-D" }, { "family_name": "Sun", "given_name": "Fei", "clpid": "Sun-Fei" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Arnold", "given_name": "Frances H.", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering.", "doi": "10.1016/j.chembiol.2015.06.020", "pmcid": "PMC4546540", "issn": "1074-5521", "publisher": "Cell Press", "publication": "Chemistry and Biology", "publication_date": "2015-08-20", "series_number": "8", "volume": "22", "issue": "8", "pages": "1108-1121" }, { "id": "authors:70kmm-8py90", "collection": "authors", "collection_id": "70kmm-8py90", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140730-113211664", "type": "article", "title": "Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons", "author": [ { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-N-C" }, { "family_name": "Bedbrook", "given_name": "Claire N.", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-C-N" }, { "family_name": "Chiu", "given_name": "Hui", "clpid": "Chiu-Hui" }, { "family_name": "Engqvist", "given_name": "Martin K. M.", "clpid": "Engqvist-M-K-M" }, { "family_name": "Xiao", "given_name": "Cheng", "orcid": "0000-0001-9649-7450", "clpid": "Xiao-Cheng" }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Arnold", "given_name": "Frances H.", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Probing the neural circuit dynamics underlying behaviour would benefit greatly from improved genetically encoded voltage indicators. The proton pump Archaerhodopsin-3 (Arch), an optogenetic tool commonly used for neuronal inhibition, has been shown to emit voltage-sensitive fluorescence. Here we report two Arch variants with enhanced radiance (Archers) that in response to 655\u2009nm light have 3\u20135 times increased fluorescence and 55\u201399 times reduced photocurrents compared with Arch WT. The most fluorescent variant, Archer1, has 25\u201340% fluorescence change in response to action potentials while using 9 times lower light intensity compared with other Arch-based voltage sensors. Archer1 is capable of wavelength-specific functionality as a voltage sensor under red light and as an inhibitory actuator under green light. As a proof-of-concept for the application of Arch-based sensors in vivo, we show fluorescence voltage sensing in behaving Caenorhabditis elegans. Archer1's characteristics contribute to the goal of all-optical detection and modulation of activity in neuronal networks in vivo.", "doi": "10.1038/ncomms5894", "pmcid": "PMC4166526", "issn": "2041-1723", "publisher": "Nature Publishing Group", "publication": "Nature Communications", "publication_date": "2014-09-15", "series_number": "9", "volume": "5", "issue": "9", "pages": "Art. No. 4894" }, { "id": "authors:7hzm0-wtx83", "collection": "authors", "collection_id": "7hzm0-wtx83", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140825-213535516", "type": "article", "title": "Directed evolution of a far-red fluorescent rhodopsin", "author": [ { "family_name": "McIsaac", "given_name": "R. Scott", "orcid": "0000-0002-5339-6032", "clpid": "McIsaac-R-Scott" }, { "family_name": "Engqvist", "given_name": "Martin K. M.", "clpid": "Engqvist-Martin-K-M" }, { "family_name": "Wannier", "given_name": "Timothy", "clpid": "Wannier-Timothy-M" }, { "family_name": "Rosenthal", "given_name": "Adam Z.", "orcid": "0000-0002-6936-3665", "clpid": "Rosenthal-Adam-Z" }, { "family_name": "Herwig", "given_name": "Lukas", "clpid": "Herwig-Lukas" }, { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-Nicholas-C" }, { "family_name": "Imasheva", "given_name": "Eleonora S.", "clpid": "Imasheva-Eleonora-S" }, { "family_name": "Lanyi", "given_name": "Janos K.", "clpid": "Lanyi-Janos-K" }, { "family_name": "Balashov", "given_name": "Sergei P.", "clpid": "Balashov-Sergei-P" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Arnold", "given_name": "Frances H.", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "abstract": "Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with live-cell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK_a of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at \u223c620 nm/730 nm).", "doi": "10.1073/pnas.1413987111", "pmcid": "PMC4246972", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2014-09-09", "series_number": "36", "volume": "111", "issue": "36", "pages": "13034-13039" }, { "id": "authors:k57mg-mbq97", "collection": "authors", "collection_id": "k57mg-mbq97", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140801-121634734", "type": "article", "title": "Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing", "author": [ { "family_name": "Yang", "given_name": "Bin", "clpid": "Yang-Bin" }, { "family_name": "Treweek", "given_name": "Jennifer B.", "clpid": "Treweek-J-B" }, { "family_name": "Kulkarni", "given_name": "Rajan P.", "clpid": "Kulkarni-R-P" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Chen", "given_name": "Chun-Kan", "clpid": "Chen-Chun-Kan" }, { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-E" }, { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.", "doi": "10.1016/j.cell.2014.07.017", "pmcid": "PMC4153367", "issn": "0092-8674", "publisher": "Elsevier", "publication": "Cell", "publication_date": "2014-08-14", "series_number": "4", "volume": "158", "issue": "4", "pages": "945-958" }, { "id": "authors:f6stc-5k993", "collection": "authors", "collection_id": "f6stc-5k993", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140714-121054059", "type": "article", "title": "Bioelectronic medicines: a research roadmap", "author": [ { "family_name": "Birmingham", "given_name": "Karen", "clpid": "Birmingham-K" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Anikeeva", "given_name": "Polina", "clpid": "Anikeeva-P" }, { "family_name": "Grill", "given_name": "Warren M.", "clpid": "Grill-W-M" }, { "family_name": "Pikov", "given_name": "Victor", "clpid": "Pikov-V" }, { "family_name": "McLaughlin", "given_name": "Bryan", "clpid": "McLaughlin-B" }, { "family_name": "Pasricha", "given_name": "Pankaj", "clpid": "Pasricha-P" }, { "family_name": "Weber", "given_name": "Douglas", "clpid": "Weber-D" }, { "family_name": "Ludwig", "given_name": "Kip", "clpid": "Ludwig-K" }, { "family_name": "Famm", "given_name": "Kristoffer", "clpid": "Famm-K" } ], "abstract": "Realizing the vision of a new class of medicines based on modulating the electrical signalling patterns of the peripheral nervous system needs a firm research foundation. Here, an interdisciplinary community puts forward a research roadmap for the next 5 years.", "doi": "10.1038/nrd4351", "issn": "1474-1776", "publisher": "Nature Publishing Group", "publication": "Nature Reviews Drug Discovery", "publication_date": "2014-06", "series_number": "6", "volume": "13", "issue": "6", "pages": "399-400" }, { "id": "authors:mqyw4-6hn71", "collection": "authors", "collection_id": "mqyw4-6hn71", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130513-151857819", "type": "article", "title": "Structural and molecular interrogation of intact biological systems", "author": [ { "family_name": "Chung", "given_name": "Kwanghun", "orcid": "0000-0002-8167-3340", "clpid": "Chung-Kwanghun" }, { "family_name": "Wallace", "given_name": "Jenelle", "clpid": "Wallace-J" }, { "family_name": "Kim", "given_name": "Sung-Yon", "clpid": "Kim-Sung-Yon" }, { "family_name": "Kalyanasundaram", "given_name": "Sandhiya", "clpid": "Kalyanasundaram-S" }, { "family_name": "Andalman", "given_name": "Aaron S.", "clpid": "Andalman-A-S" }, { "family_name": "Davidson", "given_name": "Thomas J.", "clpid": "Davidson-T-J" }, { "family_name": "Mirzabekov", "given_name": "Julie J.", "clpid": "Mirzabekov-J-J" }, { "family_name": "Zalocusky", "given_name": "Kelly A.", "clpid": "Zalocusky-K-A" }, { "family_name": "Mattis", "given_name": "Joanna", "clpid": "Mattis-J" }, { "family_name": "Denisin", "given_name": "Aleksandra K.", "clpid": "Denisin-A-K" }, { "family_name": "Pak", "given_name": "Sally", "clpid": "Pak-Sally" }, { "family_name": "Bernstein", "given_name": "Hannah", "clpid": "Bernstein-H" }, { "family_name": "Ramakrishnan", "given_name": "Charu", "clpid": "Ramakrishnan-C" }, { "family_name": "Grosenick", "given_name": "Logan", "clpid": "Grosenick-L" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.", "doi": "10.1038/nature12107", "pmcid": "PMC4092167", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2013-05-16", "series_number": "7449", "volume": "497", "issue": "7449", "pages": "332-337" }, { "id": "authors:a043p-7sh38", "collection": "authors", "collection_id": "a043p-7sh38", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130513-151856843", "type": "article", "title": "Optogenetic Delay of Status Epilepticus Onset in an In Vivo Rodent Epilepsy Model", "author": [ { "family_name": "Sukhotinsky", "given_name": "Inna", "clpid": "Sukhotinsky-I" }, { "family_name": "Chan", "given_name": "Alexander M.", "clpid": "Chan-Alexander-M" }, { "family_name": "Ahmed", "given_name": "Omar J.", "clpid": "Ahmed-O-J" }, { "family_name": "Rao", "given_name": "Vikram R.", "clpid": "Rao-Vikram-R" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Ramakrishnan", "given_name": "Charu", "clpid": "Ramakrishnan-C" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" }, { "family_name": "Majewska", "given_name": "Ania K.", "clpid": "Majewska-A-K" }, { "family_name": "Cash", "given_name": "Sydney S.", "clpid": "Cash-S-S" } ], "abstract": "Epilepsy is a devastating disease, currently treated with medications, surgery or electrical stimulation. None of these approaches is totally effective and our ability to control seizures remains limited and complicated by frequent side effects. The emerging revolutionary technique of optogenetics enables manipulation of the activity of specific neuronal populations in vivo with exquisite spatiotemporal resolution using light. We used optogenetic approaches to test the role of hippocampal excitatory neurons in the lithium-pilocarpine model of acute elicited seizures in awake behaving rats. Hippocampal pyramidal neurons were transduced in vivo with a virus carrying an enhanced halorhodopsin (eNpHR), a yellow light activated chloride pump, and acute seizure progression was then monitored behaviorally and electrophysiologically in the presence and absence of illumination delivered via an optical fiber. Inhibition of those neurons with illumination prior to seizure onset significantly delayed electrographic and behavioral initiation of status epilepticus, and altered the dynamics of ictal activity development. These results reveal an essential role of hippocampal excitatory neurons in this model of ictogenesis and illustrate the power of optogenetic approaches for elucidation of seizure mechanisms. This early success in controlling seizures also suggests future therapeutic avenues.", "doi": "10.1371/journal.pone.0062013", "pmcid": "PMC3634849", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLoS ONE", "publication_date": "2013", "series_number": "4", "volume": "8", "issue": "4", "pages": "e62013" }, { "id": "authors:8hdv1-v7263", "collection": "authors", "collection_id": "8hdv1-v7263", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121206-140040487", "type": "article", "title": "Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins", "author": [ { "family_name": "Mattis", "given_name": "Joanna", "clpid": "Mattis-Joanna" }, { "family_name": "Tye", "given_name": "Kay M.", "orcid": "0000-0002-2435-0182", "clpid": "Tye-Kay-M" }, { "family_name": "Ferenczi", "given_name": "Emily A.", "clpid": "Ferenczi-Emily-A" }, { "family_name": "Ramakrishnan", "given_name": "Charu", "clpid": "Ramakrishnan-Charu" }, { "family_name": "O'Shea", "given_name": "Daniel J.", "clpid": "O'Shea-Daniel-J" }, { "family_name": "Prakash", "given_name": "Rohit", "clpid": "Prakash-Rohit" }, { "family_name": "Gunaydin", "given_name": "Lisa A.", "clpid": "Gunaydin-Lisa-A" }, { "family_name": "Hyun", "given_name": "Minsuk", "clpid": "Hyun-Minsuk" }, { "family_name": "Fenno", "given_name": "Lief E.", "clpid": "Fenno-Lief-E" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Yizhar", "given_name": "Ofer", "orcid": "0000-0003-4228-1448", "clpid": "Yizhar-Ofer" }, { "family_name": "Deisseroth", "given_name": "Karl", "orcid": "0000-0001-9440-3967", "clpid": "Deisseroth-Karl" } ], "abstract": "Diverse optogenetic tools have allowed versatile control over neural activity. Many depolarizing and hyperpolarizing tools have now been developed in multiple laboratories and tested across different preparations, presenting opportunities but also making it difficult to draw direct comparisons. This challenge has been compounded by the dependence of performance on parameters such as vector, promoter, expression time, illumination, cell type and many other variables. As a result, it has become increasingly complicated for end users to select the optimal reagents for their experimental needs. For a rapidly growing field, critical figures of merit should be formalized both to establish a framework for further development and so that end users can readily understand how these standardized parameters translate into performance. Here we systematically compared microbial opsins under matched experimental conditions to extract essential principles and identify key parameters for the conduct, design and interpretation of experiments involving optogenetic techniques.", "doi": "10.1038/NMETH.1808", "pmcid": "PMC4165888", "issn": "1548-7091", "publisher": "Nature Publishing Group", "publication": "Nature Methods", "publication_date": "2012-02", "series_number": "2", "volume": "9", "issue": "2", "pages": "159-172" }, { "id": "authors:rw2es-12329", "collection": "authors", "collection_id": "rw2es-12329", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121206-152726999", "type": "article", "title": "Leptin regulates the reward value of nutrient", "author": [ { "family_name": "Domingos", "given_name": "Ana I.", "clpid": "Domingos-A-I" }, { "family_name": "Vaynshteyn", "given_name": "Jake", "clpid": "Vaynshteyn-J" }, { "family_name": "Voss", "given_name": "Henning U.", "clpid": "Voss-H-U" }, { "family_name": "Ren", "given_name": "Xueying", "clpid": "Ren-Xueying" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Zhang", "given_name": "Feng", "clpid": "Zhang-Feng" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" }, { "family_name": "de Araujo", "given_name": "Ivan E.", "clpid": "de-Araujo-I-E" }, { "family_name": "Friedman", "given_name": "Jeffrey", "clpid": "Friedman-J" } ], "abstract": "We developed an assay for quantifying the reward value of nutrient and used it to analyze the effects of metabolic state and leptin. In this assay, mice chose between two sippers, one of which dispensed water and was coupled to optogenetic activation of dopaminergic (DA) neurons and the other of which dispensed natural or artificial sweeteners. This assay measured the reward value of sweeteners relative to lick-induced optogenetic activation of DA neurons. Mice preferred optogenetic stimulation of DA neurons to sucralose, but not to sucrose. However, the mice preferred sucralose plus optogenetic stimulation versus sucrose. We found that food restriction increased the value of sucrose relative to sucralose plus optogenetic stimulation, and that leptin decreased it. Our data suggest that leptin suppresses the ability of sucrose to drive taste-independent DA neuronal activation and provide new insights into the mechanism of leptin's effects on food intake.", "doi": "10.1038/nn.2977", "pmcid": "PMC4238286", "issn": "1097-6256", "publisher": "Nature Publishing Group", "publication": "Nature Neuroscience", "publication_date": "2011-12", "series_number": "12", "volume": "14", "issue": "12", "pages": "1562-1568" }, { "id": "authors:vcbsm-yey23", "collection": "authors", "collection_id": "vcbsm-yey23", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121206-085452439", "type": "article", "title": "Dynamics of Retrieval Strategies for Remote Memories", "author": [ { "family_name": "Goshen", "given_name": "Inbal", "clpid": "Goshen-I" }, { "family_name": "Brodsky", "given_name": "Matthew", "clpid": "Brodsky-M" }, { "family_name": "Prakash", "given_name": "Rohit", "clpid": "Prakash-R" }, { "family_name": "Wallace", "given_name": "Jenelle", "clpid": "Wallace-J" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Ramakrishnan", "given_name": "Charu", "clpid": "Ramakrishnan-C" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "Prevailing theory suggests that long-term memories are encoded via a two-phase process requiring early involvement of the hippocampus followed by the neocortex. Contextual fear memories in rodents rely on the hippocampus immediately following training but are unaffected by hippocampal lesions or pharmacological inhibition weeks later. With fast optogenetic methods, we examine the real-time contribution of hippocampal CA1 excitatory neurons to remote memory and find that contextual fear memory recall, even weeks after training, can be reversibly abolished by temporally precise optogenetic inhibition of CA1. When this inhibition is extended to match the typical time course of pharmacological inhibition, remote hippocampus dependence converts to hippocampus independence, suggesting that long-term memory retrieval normally depends on the hippocampus but can adaptively shift to alternate structures. Further revealing the plasticity of mechanisms required for memory recall, we confirm the remote-timescale importance of the anterior cingulate cortex (ACC) and implicate CA1 in ACC recruitment for remote recall.", "doi": "10.1016/j.cell.2011.09.033", "issn": "0092-8674", "publisher": "Elsevier", "publication": "Cell", "publication_date": "2011-10-28", "series_number": "3", "volume": "147", "issue": "3", "pages": "678-689" }, { "id": "authors:hx8md-xvw19", "collection": "authors", "collection_id": "hx8md-xvw19", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121206-133910388", "type": "article", "title": "Amygdala circuitry mediating reversible and bidirectional control of anxiety", "author": [ { "family_name": "Tye", "given_name": "Kay M.", "orcid": "0000-0002-2435-0182", "clpid": "Tye-K-M" }, { "family_name": "Prakash", "given_name": "Rohit", "clpid": "Prakash-R" }, { "family_name": "Kim", "given_name": "Sung-Yon", "clpid": "Kim-Sung-Yon" }, { "family_name": "Fenno", "given_name": "Lief E.", "clpid": "Fenno-L-E" }, { "family_name": "Grosenick", "given_name": "Logan", "clpid": "Grosenick-L" }, { "family_name": "Zarabi", "given_name": "Hosniya", "clpid": "Zarabi-H" }, { "family_name": "Thompson", "given_name": "Kimberly R.", "clpid": "Thompson-K-R" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Ramakrishnan", "given_name": "Charu", "clpid": "Ramakrishnan-C" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "Anxiety\u2014a sustained state of heightened apprehension in the absence of immediate threat\u2014becomes severely debilitating in disease states. Anxiety disorders represent the most common of psychiatric diseases (28% lifetime prevalence) and contribute to the aetiology of major depression and substance abuse. Although it has been proposed that the amygdala, a brain region important for emotional processing, has a role in anxiety, the neural mechanisms that control anxiety remain unclear. Here we explore the neural circuits underlying anxiety-related behaviours by using optogenetics with two-photon microscopy, anxiety assays in freely moving mice, and electrophysiology. With the capability of optogenetics to control not only cell types but also specific connections between cells, we observed that temporally precise optogenetic stimulation of basolateral amygdala (BLA) terminals in the central nucleus of the amygdala (CeA)\u2014achieved by viral transduction of the BLA with a codon-optimized channelrhodopsin followed by restricted illumination in the downstream CeA\u2014exerted an acute, reversible anxiolytic effect. Conversely, selective optogenetic inhibition of the same projection with a third-generation halorhodopsin (eNpHR3.0) increased anxiety-related behaviours. Importantly, these effects were not observed with direct optogenetic control of BLA somata, possibly owing to recruitment of antagonistic downstream structures. Together, these results implicate specific BLA\u2013CeA projections as critical circuit elements for acute anxiety control in the mammalian brain, and demonstrate the importance of optogenetically targeting defined projections, beyond simply targeting cell types, in the study of circuit function relevant to neuropsychiatric disease.", "doi": "10.1038/nature09820", "pmcid": "PMC3154022", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2011-03-17", "series_number": "7338", "volume": "471", "issue": "7338", "pages": "358-362" }, { "id": "authors:s6dqe-vm954", "collection": "authors", "collection_id": "s6dqe-vm954", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121211-085000915", "type": "article", "title": "Cholinergic Interneurons Control Local Circuit Activity and Cocaine Conditioning", "author": [ { "family_name": "Witten", "given_name": "Ilana B.", "clpid": "Witten-I-B" }, { "family_name": "Lin", "given_name": "Shih-Chun", "clpid": "Lin-Shih-Chun" }, { "family_name": "Brodsky", "given_name": "Matthew", "clpid": "Brodsky-M" }, { "family_name": "Prakash", "given_name": "Rohit", "clpid": "Prakash-R" }, { "family_name": "Diester", "given_name": "Ilka", "clpid": "Diester-I" }, { "family_name": "Anikeeva", "given_name": "Polina", "clpid": "Anikeeva-P" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Ramakrishnan", "given_name": "Charu", "clpid": "Ramakrishnan-C" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "Cholinergic neurons are widespread, and pharmacological modulation of acetylcholine receptors affects numerous brain processes, but such modulation entails side effects due to limitations in specificity for receptor type and target cell. As a result, causal roles of cholinergic neurons in circuits have been unclear. We integrated optogenetics, freely moving mammalian behavior, in vivo electrophysiology, and slice physiology to probe the cholinergic interneurons of the nucleus accumbens by direct excitation or inhibition. Despite representing less than 1% of local neurons, these cholinergic cells have dominant control roles, exerting powerful modulation of circuit activity. Furthermore, these neurons could be activated by cocaine, and silencing this drug-induced activity during cocaine exposure (despite the fact that the manipulation of the cholinergic interneurons was not aversive by itself) blocked cocaine conditioning in freely moving mammals.", "doi": "10.1126/science.1193771", "pmcid": "PMC3142356", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2010-12-17", "series_number": "6011", "volume": "330", "issue": "6011", "pages": "1677-1681" }, { "id": "authors:35z98-04g17", "collection": "authors", "collection_id": "35z98-04g17", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121206-103459856", "type": "article", "title": "Global and local fMRI signals driven by neurons defined optogenetically by type and wiring", "author": [ { "family_name": "Lee", "given_name": "Jin Hyung", "clpid": "Lee-Jin Hyung" }, { "family_name": "Durand", "given_name": "Remy", "clpid": "Durand-R" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Zhang", "given_name": "Feng", "clpid": "Zhang-Feng" }, { "family_name": "Goshen", "given_name": "Inbal", "clpid": "Goshen-I" }, { "family_name": "Kim", "given_name": "Dae-Shik", "clpid": "Kim-Dae-Shik" }, { "family_name": "Fenno", "given_name": "Lief E.", "clpid": "Fenno-L-E" }, { "family_name": "Ramakrishnan", "given_name": "Charu", "clpid": "Ramakrishnan-C" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "Despite a rapidly-growing scientific and clinical brain imaging literature based on functional magnetic resonance imaging (fMRI) using blood oxygenation level-dependent (BOLD) signals, it remains controversial whether BOLD signals in a particular region can be caused by activation of local excitatory neurons. This difficult question is central to the interpretation and utility of BOLD, with major significance for fMRI studies in basic research and clinical applications. Using a novel integrated technology unifying optogenetic control of inputs with high-field fMRI signal readouts, we show here that specific stimulation of local CaMKII\u03b1-expressing excitatory neurons, either in the neocortex or thalamus, elicits positive BOLD signals at the stimulus location with classical kinetics. We also show that optogenetic fMRI (ofMRI) allows visualization of the causal effects of specific cell types defined not only by genetic identity and cell body location, but also by axonal projection target. Finally, we show that ofMRI within the living and intact mammalian brain reveals BOLD signals in downstream targets distant from the stimulus, indicating that this approach can be used to map the global effects of controlling a local cell population. In this respect, unlike both conventional fMRI studies based on correlations and fMRI with electrical stimulation that will also directly drive afferent and nearby axons, this ofMRI approach provides causal information about the global circuits recruited by defined local neuronal activity patterns. Together these findings provide an empirical foundation for the widely-used fMRI BOLD signal, and the features of ofMRI define a potent tool that may be suitable for functional circuit analysis as well as global phenotyping of dysfunctional circuitry.", "doi": "10.1038/nature09108", "pmcid": "PMC3177305", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2010-06-10", "series_number": "7299", "volume": "465", "issue": "7299", "pages": "788-792" }, { "id": "authors:gpmkp-bkh25", "collection": "authors", "collection_id": "gpmkp-bkh25", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121206-083746393", "type": "article", "title": "Molecular and Cellular Approaches for Diversifying and Extending Optogenetics", "author": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Zhang", "given_name": "Feng", "clpid": "Zhang-Feng" }, { "family_name": "Ramakrishnan", "given_name": "Charu", "clpid": "Ramakrishnan-C" }, { "family_name": "Mattis", "given_name": "Joanna", "clpid": "Mattis-J" }, { "family_name": "Prakash", "given_name": "Rohit", "clpid": "Prakash-R" }, { "family_name": "Diester", "given_name": "Ilka", "clpid": "Diester-I" }, { "family_name": "Goshen", "given_name": "Inbal", "clpid": "Goshen-I" }, { "family_name": "Thompson", "given_name": "Kimberly R.", "clpid": "Thompson-K-R" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "Optogenetic technologies employ light to control biological processes within targeted cells in vivo with high temporal precision. Here, we show that application of molecular trafficking principles can expand the optogenetic repertoire along several long-sought dimensions. Subcellular and transcellular trafficking strategies now permit (1) optical regulation at the far-red/infrared border and extension of optogenetic control across the entire visible spectrum, (2) increased potency of optical inhibition without increased light power requirement (nanoampere-scale chloride-mediated photocurrents that maintain the light sensitivity and reversible, step-like kinetic stability of earlier tools), and (3) generalizable strategies for targeting cells based not only on genetic identity, but also on morphology and tissue topology, to allow versatile targeting when promoters are not known or in genetically intractable organisms. Together, these results illustrate use of cell-biological principles to enable expansion of the versatile fast optogenetic technologies suitable for intact-systems biology and behavior.", "doi": "10.1016/j.cell.2010.02.037", "pmcid": "PMC4160532", "issn": "0092-8674", "publisher": "Elsevier", "publication": "Cell", "publication_date": "2010-04-01", "series_number": "1", "volume": "141", "issue": "1", "pages": "154-165" }, { "id": "authors:7gzhg-qvg10", "collection": "authors", "collection_id": "7gzhg-qvg10", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121206-154946294", "type": "article", "title": "Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures", "author": [ { "family_name": "Zhang", "given_name": "Feng", "clpid": "Zhang-Feng" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Adamantidis", "given_name": "Antoine R.", "clpid": "Adamantidis-A-R" }, { "family_name": "Durand", "given_name": "Remy", "clpid": "Durand-R" }, { "family_name": "Airan", "given_name": "Raag D.", "clpid": "Airan-R-D" }, { "family_name": "de Lecea", "given_name": "Luis", "clpid": "de-Lecea-L" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "Elucidation of the neural substrates underlying complex animal behaviors depends on precise activity control tools, as well as compatible readout methods. Recent developments in optogenetics have addressed this need, opening up new possibilities for systems neuroscience. Interrogation of even deep neural circuits can be conducted by directly probing the necessity and sufficiency of defined circuit elements with millisecond-scale, cell type-specific optical perturbations, coupled with suitable readouts such as electrophysiology, optical circuit dynamics measures and freely moving behavior in mammals. Here we collect in detail our strategies for delivering microbial opsin genes to deep mammalian brain structures in vivo, along with protocols for integrating the resulting optical control with compatible readouts (electrophysiological, optical and behavioral). The procedures described here, from initial virus preparation to systems-level functional readout, can be completed within 4\u20135 weeks. Together, these methods may help in providing circuit-level insight into the dynamics underlying complex mammalian behaviors in health and disease.", "doi": "10.1038/nprot.2009.226", "pmcid": "PMC4503465", "issn": "1754-2189", "publisher": "Nature Publishing Group", "publication": "Nature Protocols", "publication_date": "2010-02-18", "series_number": "3", "volume": "5", "issue": "3", "pages": "439-456" }, { "id": "authors:gf61b-b4497", "collection": "authors", "collection_id": "gf61b-b4497", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121211-083527990", "type": "article", "title": "Optical Deconstruction of Parkinsonian Neural Circuitry", "author": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Mogri", "given_name": "Murtaza", "clpid": "Mogri-M" }, { "family_name": "Thompson", "given_name": "Kimberly R.", "clpid": "Thompson-K-R" }, { "family_name": "Henderson", "given_name": "Jaimie M.", "clpid": "Henderson-J-M" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "Deep brain stimulation (DBS) is a therapeutic option for intractable neurological and psychiatric disorders, including Parkinson's disease and major depression. Because of the heterogeneity of brain tissues where electrodes are placed, it has been challenging to elucidate the relevant target cell types or underlying mechanisms of DBS. We used optogenetics and solid-state optics to systematically drive or inhibit an array of distinct circuit elements in freely moving parkinsonian rodents and found that therapeutic effects within the subthalamic nucleus can be accounted for by direct selective stimulation of afferent axons projecting to this region. In addition to providing insight into DBS mechanisms, these results demonstrate an optical approach for dissection of disease circuitry and define the technological toolbox needed for systematic deconstruction of disease circuits by selectively controlling individual components.", "doi": "10.1126/science.1167093", "pmcid": "PMC6744370", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2009-04-17", "series_number": "5925", "volume": "324", "issue": "5925", "pages": "354-359" }, { "id": "authors:stt3w-w5670", "collection": "authors", "collection_id": "stt3w-w5670", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121206-102214115", "type": "article", "title": "eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications", "author": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Thompson", "given_name": "Kimberly R.", "clpid": "Thompson-K-R" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "Temporally precise inhibition of distinct cell types in the intact nervous system has been enabled by the microbial halorhodopsin NpHR, a fast light-activated electrogenic Cl^\u2212 pump. While neurons can be optically hyperpolarized and inhibited from firing action potentials at moderate NpHR expression levels, we have encountered challenges with pushing expression to extremely high levels, including apparent intracellular accumulations. We therefore sought to molecularly engineer NpHR to achieve strong expression without these cellular side effects. We found that high expression correlated with endoplasmic reticulum (ER) accumulation, and that under these conditions NpHR colocalized with ER proteins containing the KDEL ER retention sequence. We screened a number of different putative modulators of membrane trafficking and identified a combination of two motifs, an N-terminal signal peptide and a C-terminal ER export sequence, that markedly promoted membrane localization and ER export defined by confocal microscopy and whole-cell patch clamp. The modified NpHR displayed increased peak photocurrent in the absence of aggregations or toxicity, and potent optical inhibition was observed not only in vitro but also in vivo with thalamic single-unit recording. The new enhanced NpHR (eNpHR) allows safe, high-level expression in mammalian neurons, without toxicity and with augmented inhibitory function, in vitro and in vivo.", "doi": "10.1007/s11068-008-9027-6", "pmcid": "PMC2588488", "issn": "1559-7105", "publisher": "Springer Verlag", "publication": "Brain Cell Biology", "publication_date": "2008-08", "series_number": "1-4", "volume": "36", "issue": "1-4", "pages": "129-139" }, { "id": "authors:jqjvd-qyq18", "collection": "authors", "collection_id": "jqjvd-qyq18", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121206-101209730", "type": "article", "title": "Controlling Neuronal Activity", "author": [ { "family_name": "Schneider", "given_name": "M. Bret", "clpid": "Schneider-M-B" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Zhang", "given_name": "Feng", "clpid": "Zhang-Feng" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "With a new technology called optogenetics, it is possible to turn neuronal activity on and off in distinct neuronal populations, using cell-type specific, optically-sensitive, molecular, neuronal activity \"switches.\" These \"switches\" are microbial, light-sensitive ion conductance-regulating proteins, exemplified by channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR). They are individually introduced into neuronal populations in the brain and become part of the cellular machinery. Ion flux-regulating activity of these \"switches\" can be controlled externally with light pulses. ChR2 is a cation channel that allows sodium ions to pass into a neuron after it has been activated by approximately 470 nm blue light (thereby increasing activity of the neuron and increasing action potentials). NpHR is a chloride pump that transfers chloride anions into the neuron after it has been activated by approximately 580 nm yellow light (thereby increasing accumulation of negative charge inside the cell and suppressing activity of the neuron). For application of this technology, light of the proper wavelength is delivered to the brain region of interest using a fiberoptic-based system or a light-emitting diode (LED). ChR2 and NpHR can be controlled independently to either increase action-potential firing of specific target neurons or to suppress neural activity, respectively, in intact tissue. In animal experiments, the LED or fiberoptic can be tethered to an external power source with lightweight flexible connectors, allowing stimulation during normal, freely moving behavior. The genes encoding these proteins are introduced into the brain with viral vectors and are expressed in distinct populations of neurons in vivo using specific DNA promoters fused to the gene, thereby guiding expression only in the cell type of choice.", "doi": "10.1176/appi.ajp.2008.08030444", "issn": "0002-953X", "publisher": "American Psychiatric Publishing", "publication": "American Journal of Psychiatry", "publication_date": "2008-05", "series_number": "5", "volume": "165", "issue": "5", "pages": "562-562" }, { "id": "authors:nfzmn-j2t87", "collection": "authors", "collection_id": "nfzmn-j2t87", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121206-095456619", "type": "article", "title": "Targeting and Readout Strategies for Fast Optical Neural Control In Vitro and In Vivo", "author": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Thompson", "given_name": "Kimberly R.", "clpid": "Thompson-K-R" }, { "family_name": "Zhang", "given_name": "Feng", "clpid": "Zhang-Feng" }, { "family_name": "Mogri", "given_name": "Murtaza", "clpid": "Mogri-M" }, { "family_name": "Kay", "given_name": "Kenneth", "clpid": "Kay-K" }, { "family_name": "Schneider", "given_name": "M. Bret", "clpid": "Schneider-M-B" }, { "family_name": "Deisseroth", "given_name": "Karl", "clpid": "Deisseroth-K" } ], "abstract": "Major obstacles faced by neuroscientists in attempting to unravel the complexity of brain function include both the heterogeneity of brain tissue (with a multitude of cell types present in vivo) and the high speed of brain information processing (with behaviorally relevant millisecond-scale electrical activity patterns). To address different aspects of these technical constraints, genetically targetable neural modulation tools have been developed by a number of groups (Zemelman et al., 2002; Banghart et al., 2004; Karpova et al., 2005; Lima and Miesenbock, 2005; Thompson et al., 2005; Chambers et al., 2006; Tan et al., 2006; Gorostiza et al., 2007; Lerchner et al., 2007; Szobota et al., 2007). One approach recently brought to neurobiology, combining both high speed and genetic targeting, is based on a family of fast light-responsive microbial opsins including halorhodopsins (e.g., NpHR) and channelrhodopsins (e.g., ChR2) (for review, see Zhang et al., 2007b). These microbial opsins are single-component transmembrane conductance regulators encompassing light sensitivity and fast membrane potential control within a single open reading frame, which can be used to achieve fast bidirectional control of specific cell types even in freely moving animals (Adamantidis et al., 2007; Zhang et al., 2007a). Although the basic functioning of these tools has been reviewed previously (Zhang et al., 2007b), here we describe a collection of targeting and readout strategies designed for rapid and flexible application of the microbial opsin system, and provide pointers to the relevant literature. Combinations of these multiple levels of targeting and readout define an evolving toolbox that may open up new possibilities for basic neuroscience investigation.", "doi": "10.1523/JNEUROSCI.3578-07.2007", "pmcid": "PMC6673457", "issn": "0270-6474", "publisher": "Society for Neuroscience", "publication": "Journal of Neuroscience", "publication_date": "2007-12-26", "series_number": "52", "volume": "27", "issue": "52", "pages": "14231-14238" } ]