2018 — 2021 |
Ideker, Trey (co-PI) [⬀] Kampmann, Martin Krogan, Nevan J [⬀] Willsey, Arthur Jeremy (co-PI) [⬀] |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Psychiatric Cell Map Initiative: Connecting Genomics, Subcellular Networks, and Higher Order Phenotypes @ University of California, San Francisco
SUMMARY The global burden of mental illness, including autism spectrum disorders (ASD), intellectual disability, epilepsy, Tourette disorder, schizophrenia and bipolar disorder, is enormous, whether measured in health care expenditures, lost productivity, or personal suffering. Unfortunately, there is a striking lack of insight into the underlying molecular biology of these syndromes. However, recent advances in gene discovery are setting the stage for a transformation in the understanding of these psychiatric disorders. Understanding pathobiology and developing novel treatments is becoming increasingly dependent on knowledge of biological networks of multiple types, including physical interactions among proteins and syntheticlethal and epistatic interactions among genes. Here we seek support for a new effort, the Psychiatric Cell Map Initiative (PCMI, www.pcmi.ucsf.edu), aimed at comprehensively understanding these complex interactions in psychiatric disorders and how they differ between diseased and healthy states. While we will focus on ASD in this proposal, this work will establish a paradigm to investigate other psychiatric disorders in future work. The PCMI is a multicampus initiative of the University of California, involving UC San Francisco, UC San Diego and UC Berkeley, which leverages genomics, proteomics, highthroughput sequencing, advanced network mapping, computational analysis, and research platforms developed by multiple PCMI investigators over the past decade. Thus primed, these platforms will be tuned to efficiently generate, assemble, and analyze molecular networks linked to ASD, in relevant cell types, with a view towards pathway and networkbased personalized therapy. Specifically, over the next five years the PCMI will seek to catalyze major phase transitions in ASD research and therapy by (1) Comprehensively mapping the networks of physical interactions among proteins linked to ASD, revealing the protein complexes and higherorder molecular units underlying ASD in multiple cell types of the human brain? (2) Mapping the parallel networks of syntheticlethal and epistatic interactions among ASD genes using CRISPRbased approaches? (3) Establishing the robust computational methodology, enduser software, and databases for assembly and use of ASD cell network maps in both basic and clinical modalities? (4) Translating molecular insights into an understanding of higher order phenotypes? (5) Building a critical mass of leading investigators focused on psychiatric disorders worldwide to expand PCMI into a global coordinated partnership? and (6) Training the current and nextgeneration of scientists in Network Biology and its applications to research focused on psychiatric disorders.
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1 |
2019 — 2020 |
Kampmann, Martin |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Core D: Crispri/a Core @ Weill Medical Coll of Cornell Univ
PROJECT SUMMARY The CRISPRi/a core will support research of Projects 1, 2, and 3 by enabling knockdown and overexpression of endogenous genes in human iPSC-derived neurons. The CRISPRi/a technology, which we co- developed, enables highly specific, inducible and reversible control of gene expression in mammalian cells. We use a catalytically inactive version of the bacterial Cas9 protein (dCas9) to recruit transcriptional repressors (for CRISPRi) or transcriptional activators (for CRISPRa) to endogenous genes, as directed by single guide RNAs (sgRNAs). We have established the use of this technology in two modes: to investigate the function of individual genes of interest (reverse genetics), and to conduct genome-wide screens to uncover genes relevant for a biological process of interest (forward genetics). We will support the research of Projects 1, 2, and 3 by enabling CRISPRi/a-based forward and reverse genetics in human iPSC-derived neurons. First, we will generate and validate stable CRISPRi and CRISPRa cell lines from the isogenic human iPSCs expressing wild-type tau or V337M tau that are used by Projects 1, 2, and 3. Then, we will generate and validate sgRNAs targeting axon initial segment (AIS) proteins to enable the investigation of their effects on plasticity and excitability of V337M tau neurons (for Project 1), sgRNAs targeting key autophagy pathways to address the question if modulation of these pathways can restore neuronal excitability of V337M tau neurons (for Projects 1, 2, and 3), and sgRNAs targeting proteins selectively interacting with V337M tau that could underlie the abnormality in neuronal activity and autophagy pathways induced by pathogenic seeding (for Projects 1 and 3). We will also conduct two genome-wide CRISPRi screens. First, we will aim to identify cellular pathways controlling tau uptake, which will then be further characterized by Project 2. Second, we will aim to identify cellular pathways controlling templates tau aggregation. These forward genetics approaches will complement the hypothesis-driven reverse-genetics approaches and provide an unbiased survey of relevant cellular pathways. We will work with the Data core to integrate our datasets with those generated by the MS core, with the goal to identify convergent results from the proteomic and genetic approaches, and to work with the Human core to validate the relevance of our cell-based findings in human patients.
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0.93 |
2019 — 2021 |
Kampmann, Martin |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Systematic Elucidation of Endosomal Trafficking as a Therapeutic Opportunity in Ad Using Crispr-Based Functional Genomics @ University of California, San Francisco
PROJECT SUMMARY / ABSTRACT An effective, disease-modifying treatment for Alzheimer's Disease (AD) is an urgent, unmet need. For an AD treatment to be effective, it will likely have to target early events in AD pathogenesis. Two lines of evidence point to a central and early role for changes in endolysosomal trafficking in AD pathogenesis: First, several risk genes associated with late-onset AD (LOAD) function in endocytosis and endolysosomal trafficking. Intriguingly, a variant in the trafficking factor gene RAB10 recently co-discovered by the Karch lab, which lowers RAB10 expression, confers resilience to AD. Second, pathological changes in the endolysosomal system, such as enlarged early endosomes and upregulation of lysosomal enzymes, are some of the earliest pathological hallmarks of human AD brains. Therefore, our central hypothesis is that endolysosomal trafficking is a therapeutic target for early intervention in AD. The goal of the proposed research is to elucidate specific therapeutic targets to correct endolysosomal defects associated with LOAD risk genes in neurons, and to therapeutically recapitulate protection from LOAD conferred by variants of the RAB10. The Kampmann lab co- developed a genetic screening platform enabling inducible and reversible repression (CRISPRi) and activation (CRISPRa) of genes in human cells for genome-wide loss- and gain-of-function screens, and implemented it in human iPSC-derived neurons. The Karch lab has established a large collection of patient-derived fibroblasts and iPSCs, and generated CRISPR-corrected isogenic control lines that have enabled us to uncover phenotypes in iPSC-derived neurons linked to disease variants, including endolysosomal defects. We propose to combine our innovative approaches for two Specific Aims. The goal of Aim 1 is to identify therapeutic targets for AD that recapitulate the mechanism of protective RAB10 variants. We hypothesize that the protective variants in RAB10 counteract the endolysosomal defects associated with AD. We will test this hypothesis in iPSC-derived neurons and human brains. We found that protective variants in RAB10 reduce RAB10 expression, and conversely RAB10 expression is elevated in LOAD brains. We will conduct unbiased genome- wide CRISPRi/a screens in WT iPSC-derived neurons to identify genes that control RAB10 levels and may therefore be therapeutic targets. In parallel, we will conduct genome-wide screens to identify other therapeutic targets that phenocopy protective variants in RAB10. We will focus on hits that show epistasis with the RAB10 protective variant, which are most likely to phenocopy the effect of the protective RAB10 allele in human individuals at risk for AD. The goal of Aim 2 is to use our genetic interaction mapping approach to elucidate connections between LOAD risk genes, the endolysosomal pathway, and associated therapeutic targets. We will validate the potential of the identified therapeutic targets in a panel of AD patient-derived iPSC-derived neurons and isogenic controls for an extensive array of endolysosomal and APP processing phenotypes.
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1 |
2020 |
Ideker, Trey (co-PI) [⬀] Kampmann, Martin Krogan, Nevan J [⬀] Willsey, Arthur Jeremy (co-PI) [⬀] |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Proteomics Integration and Expansion of Downstream Analysis Capabilities Into the Cred Portal @ University of California, San Francisco
SUMMARY The global burden of mental illness, including autism spectrum disorders (ASD), intellectual disability, epilepsy, Tourette disorder, schizophrenia and bipolar disorder, is enormous, whether measured in health care expenditures, lost productivity, or personal suffering. Unfortunately, there is a striking lack of insight into the underlying molecular biology of these syndromes. However, recent advances in gene discovery are setting the stage for a transformation in the understanding of these psychiatric disorders. Understanding pathobiology and developing novel treatments is becoming increasingly dependent on knowledge of biological networks of multiple types, including physical interactions among proteins and syntheticlethal and epistatic interactions among genes. Here we seek support for a new effort, the Psychiatric Cell Map Initiative (PCMI, www.pcmi.ucsf.edu), aimed at comprehensively understanding these complex interactions in psychiatric disorders and how they differ between diseased and healthy states. While we will focus on ASD in this proposal, this work will establish a paradigm to investigate other psychiatric disorders in future work. The PCMI is a multicampus initiative of the University of California, involving UC San Francisco, UC San Diego and UC Berkeley, which leverages genomics, proteomics, highthroughput sequencing, advanced network mapping, computational analysis, and research platforms developed by multiple PCMI investigators over the past decade. Thus primed, these platforms will be tuned to efficiently generate, assemble, and analyze molecular networks linked to ASD, in relevant cell types, with a view towards pathway and networkbased personalized therapy. Specifically, over the next five years the PCMI will seek to catalyze major phase transitions in ASD research and therapy by (1) Comprehensively mapping the networks of physical interactions among proteins linked to ASD, revealing the protein complexes and higherorder molecular units underlying ASD in multiple cell types of the human brain? (2) Mapping the parallel networks of syntheticlethal and epistatic interactions among ASD genes using CRISPRbased approaches? (3) Establishing the robust computational methodology, enduser software, and databases for assembly and use of ASD cell network maps in both basic and clinical modalities? (4) Translating molecular insights into an understanding of higher order phenotypes? (5) Building a critical mass of leading investigators focused on psychiatric disorders worldwide to expand PCMI into a global coordinated partnership? and (6) Training the current and nextgeneration of scientists in Network Biology and its applications to research focused on psychiatric disorders.
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1 |
2021 |
Harari, Oscar Kampmann, Martin Karch, Celeste Marie (co-PI) [⬀] Pumiglia, Kevin M Temple, Sally Zuloaga, Kristen Leanne (co-PI) [⬀] |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Investigating the Functional Impact of Ad Risk Genes On Neuro-Vascular Interactions @ Regenerative Research Foundation
PROJECT SUMMARY/ABSTRACT Cerebrovascular pathology is present throughout stages of Alzheimer?s Disease and is correlated with cognitive changes. There is strong evidence that vascular dysfunction is a significant driver of neuropathology. Our long- term objective is to understand the function of Alzheimer?s Disease-associated risk genes in vascular cells, their contribution to the development of cerebrovascular pathology and the opportunities to use this information in therapeutic development. There are over 27 Alzheimer?s Disease-associated risk (AD-risk) loci encompassing numerous genetic variants in non-coding and coding regions and hundreds of linked genes. Our overarching hypothesis is that a subset of AD-risk genes impairs vascular function, causing release of inflammatory factors, blood brain barrier (BBB) impairment, and reduced perfusion, thus contributing to neurodegeneration. To address this, we have assembled a multi-disciplinary team with a proven track record of collaboration, including with ADSP and ADGP members, who bring expertise in vascular pathology in dementia, endothelial cell (EC) signaling and EC functional testing, Alzheimer?s Disease genomics, single cell and nuclear transcriptomics, bioinformatics, CRISPR-based gene editing for large scale screening and AD mouse models for in-depth functional assessment in vivo. Notably, we will address differences in gene effects related to the important biological variables, sex and metabolic disease. Men and women differ in their genetic risk for Alzheimer?s Disease, with sex-specific polygenic risk scores providing better prediction of onset, progression, and pathology than pooled-sex scores. Over 80% of individuals with Alzheimer?s Disease have co-morbid metabolic disease, which exacerbates vascular pathology. We have identified the top 50 AD-risk SNPs and 600 AD-associated genes, and these will be targeted for induced pluripotent stem cell (iPSC)-derived endothelial cell (EC) screens by prime editing and CRISPR-based gene inhibition and activation approaches respectively. iPSC-based production of human ECs and mural cells in 2D and 3D models has been optimized and scaled to enable efficient functional testing of the impact of gene changes, including on neuro-vascular interactions in cerebral organoids. Discoveries made in these human cell systems will be validated by an in-depth investigation of gene expression changes in individual ECs and mural cells across a large collection of Alzheimer?s Disease brain samples using single nuclear sequencing. The EC translatome will also be obtained from mouse Alzheimer?s Disease models that incorporate sex and metabolic disease. These diverse datasets will be harmonized and integrated in order to map vascular phenotypes of AD-risk genes and identify critical molecular pathways that are targetable drivers of AD cerebrovascular pathology. These data will add to the breadth of knowledge being gathered by other groups to further elucidate underlying neuronal, glial, microglial, endothelial and mural cell-cell interactions that contribute in a substantial way to the complex architecture of Alzheimer Disease pathology. 1
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0.91 |
2021 |
Geschwind, Daniel H Goate, Alison M [⬀] Kampmann, Martin |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Uncovering the Genetic Mechanisms of the Chromosome 17q21.31 Tau Haplotype On Neurodegeneration Risk in Ftd and Psp @ Icahn School of Medicine At Mount Sinai
PROJECT SUMMARY (OVERALL) Understanding the pathophysiology of dementia is often confounded by the uncertain causal roles of observed pathological phenotypes, even when highly correlated with disease. Genetic findings overcome these limitations by providing a causal anchor from which to begin mechanistic studies. In this regard, the genetic association between chromosome 17q21.31 and increased risk for tauopathies, including Frontotemporal Dementia (FTD) and Progressive Supranuclear Palsy (PSP), is well-established and striking. Despite this well-replicated association, little is known regarding mechanisms driving the differences in risk between the two major haplotypes, H1 and H2. This is in large part because this complex locus encompasses a genomic inversion of 970 KB, leading to an approximately 1.5Mb region where strong LD has confounded the identification of causal variants and understanding of the gene regulatory mechanisms contributing to disease. Here, we capitalize on recent advances in genomics to comprehensively characterize the genetic mechanisms by which this region, and the multiple loci within it, impart disease risk, thus identifying new targets for future therapeutic development. Our central hypothesis is that haplotype and cell type specific differences in gene expression and regulation, resulting from the H1/H2 genomic inversion lead to differences in risk for sporadic Tauopathies and differences in the effects of MAPT mutations associated with inherited forms of FTD. To test this hypothesis, we propose a multi-site, interdisciplinary center composed of two highly synergistic projects (P1, P2) and 4 cores (Proteomics, Human Tissue Validation, Data, Admin) integrating a highly complementary group of investigators with a strong history of collaboration and data sharing to connect multiple levels of function: a) genotype to b) chromatin structure to c) RNA expression and d) splicing, to protein and e) cell biological consequences to elucidate disease mechanisms. P1 will apply cutting edge multi-OMICs approaches in human induced pluripotent stem cell (iPSC)-derived neural cells and human brain tissue to determine the molecular and cellular mechanisms associated with the H1 and H2 haplotypes in individuals of European and African descent. Predicted regulatory element variation between haplotypes will be validated using a pooled CRISPR screen in assembloids. P2 uses parallel approaches to dissect the genetic mechanisms, cell types and molecular pathways involved in dominant forms of FTD-tau, and their modulation by the H1 and H2 haplotypes. Project 2 will use similar approaches to test whether H1/H2-associated differences in gene expression and regulation modulate the impact of FTD-associated MAPT mutations on disease-associated phenotypes and validate the impact of key haplotype specific enhancer/repressor regions using pooled Crispr i/a screens. Data and results generated from these projects will be integrated with existing publicly available data and distributed broadly to the research community. Understanding the mechanisms that lead from abnormal gene expression and protein modification, to tau aggregation and neurodegeneration will enable us to identify novel targets for drug discovery.
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0.913 |