Gopal Thinakaran - US grants

DESCRIPTION (From the Applicant's Abstract): Alzheimer's disease (AD), the most common type of progressive dementia in the elderly, is characterized by the deposition of beta-amyloid peptides (Abeta) in the brain parenchyma and cerebral vessels. A subset of AD, classified as familial early-onset AD (FAD), is inherited as an autosomal dominant disorder. Mutations in genes encoding polytopic membrane proteins, termed presenilin 1 (PS1) and presenilin 2 (PS2), account for the majority of early-onset cases of AD. Presenilins (PS) play an important role in the generation of Abeta peptides. Abeta production is abrogated in PS1-deficient (PS-1-) cells. Moreover, FAD-linked mutant PS1 increases the production of highly fibrillogenic Abeta42 peptides. The precise role of PS1 in Abeta production, and the molecular mechanisms by which FAD-linked PS1 mutations lead to elevations in Abeta42 production have not been defined. Understanding these issues is of central importance to AD research. It is our view that molecular and structural domain analysis of PS1 will provide information critical for a clear understanding of how genetic mutations in PS1 might influence the normal function(s) of PS1, and confer pathogenic properties to mutant PS1 polypeptides. At present, very little is known regarding the molecular and structural domains of PS1. To address this issue, we will generate a series of PS1 polypeptides harboring experimental deletions and assess the influence on: PS1 endoproteolysis, "gamma-secretase" processing of amyloid precursor protein, the intramembranous cleavage of Notch1, as well as evaluate the potential of the deletion polypeptides harboring FAD-linked missense mutations to elevate the levels of Abeta42. It is known that PS1 expression is tightly regulated at the post-translational level by complex formation with other proteins; however the mechanism(s) responsible for this regulation have not been defined. To gain insights regarding the regulation of PS1 protein accumulation, we will perform a functional screen based on a novel retroviral expression cloning strategy to identify proteins that participate in regulating PS1 accumulation. Because little or no Abeta is produced in the absence of PS1, identity(ies) of proteins that regulate PS levels is critical for the design of rational therapeutic strategies aimed at reducing Abeta burden. Finally, we have outlined transgenic strategies to examine the in vivo role of the hydrophilic domain of PS1, which is the domain least conserved between PS1 and PS homologues. Recent studies have predicted important function(s) for this domain based on phosphorylation, caspase cleavage, and protein interactions. Our efforts will focus on the role played by PS1 hydrophilic loop domain during mammalian embryonic development, and in the process of amyloid production/deposition in the brains of transgenic mice.

[unreadable] DESCRIPTION (provided by applicant): Cerebral deposition of beta-amyloid peptides (AB) is one of the pathological hallmarks of Alzheimer's disease. AB is generated by sequential proteolysis of amyloid precursor protein (APP) by BACE1 and g-secretase. g-secretase is a multimeric protein complex made of four main subunits, namely PS1 (or PS2), nicastrin, PEN2 and APH-1, and a few regulatory subunits including CD147 and p23 (also referred to asTMP21). PS1 is thought to function as the catalytic subunit of g-secretase complex. Post-translational maturation and stability of the four core subunits of g-secretase are mutually regulated, and each of them is indispensable for functional enzyme activity. On the other hand, diminution of CD147 or p23 expression increases AB production, suggesting that these proteins negatively regulate g-secretase processing of APP. The specific mechanisms by which p23 modulates g-secretase cleavage of APP remain undetermined. Studies outlined in this proposal address the function of p23 in trafficking and g-secretase processing of APP in cultured cells and in mouse brains Specifically, we propose to elucidate the functional interaction between p23 and g-secretase subunits so that we can better understand the mechanisms by which p23 negatively regulates AB production. We will define the structural domains essential for p23's influence on APP trafficking and AB production, and investigate p23 modulation of AB deposition using p23 transgenic mice and p23 conditional knockout mice. Finally, we will investigate the details on p23's role in secretory and endocytic trafficking of APP and examine the relationship between Golgi morphology and AB production. Our investigation uses a combination of biochemical, molecular and cell biology techniques to accomplish the following specific aims. Aim 1: To study the functional interaction between p23 and g-secretase. Aim 2: To investigate p23 regulation of AB production and deposition in mouse brain. Aim 3: To determine the mechanisms linking p23 function with APP trafficking and g-secretase processing. Our studies address issues that are central to molecular Alzheimer's disease pathogenesis. We seek to investigate a novel aspect of g-secretase modulation by p23 that impacts on AB production. Our studies have the potential to uncover significant insights on p23 regulation of APP trafficking and AB production, and may lead to the development of p23-based novel therapeutic strategies aimed reducing AB burden by selective inactivation of g-secretase function in APP processing. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is the major cause of dementia in the elderly, afflicting more than 50% of the population over 80 years of age; presently 5.1 million Americans suffer from this devastating disorder. AD patients as well as aged individuals accumulate beta-amyloid peptides as deposits in brain, called senile plaques. Using cultured cells, transgenic mice, and conditional knockout mice as experimental models we investigate the function a protein called p23 in regulating beta-amyloid production and deposition. Our studies will be critical to develop novel rational AD therapeutics aimed at reducing beta-amyloid burden in the brain. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): One of the pathological hallmarks of Alzheimer's disease is the cerebral deposition of beta-amyloid peptides (Abeta), generated by sequential proteolysis of amyloid precursor protein (APP) by BACE1 and gamma-secretase. There has been considerable epidemiological interest in the relationship between cholesterol and susceptibility to Alzheimer's disease. Evidence from a variety of in vitro and in vivo studies indicates that specialized detergent-insoluble membrane (DIM) microdomains, termed lipid rafts, which are rich in cholesterol and sphingolipids, might be a critical link between cellular cholesterol levels and amyloidogenic processing of APP. Indeed, BACE1 is modified by S-palmitoylation and targeted to lipid rafts. Recently, we reported that each of the gamma-secretase subunits and APP C-terminal fragments (CTF) are enriched in DIM isolated from brain and cultured cells. The mechanisms that regulate recruitment of gamma-secretase and APP into raft microdomains remains to be determined. Moreover, the significance of amyloidogneic processing in raft vs. non-raft environment has not been investigated. Finally, it remains unclear whether familial Alzheimer's disease-linked mutations in APP, PS1, and PS2 modulate Abeta42 production by affecting their localization and processing of APP in lipid rafts. Studies outlined in this proposal address the functional implication of co-residence of BACE1, y-secretase, and APP CTF in specialized cholesterol-rich membrane microdomains. Specifically, we propose to investigate the mechanisms regulating the recruitment of gamma-secretase, APP, and APP CTFs to lipid rafts, and elucidate the functional significance of rafts in amyloidogenic processing of APP. Recently, we identified post-translational S-palmitoylation at a single Cysteine residue in nicastrin and at three Cysteine residues in APH-1. It is known that palmitoylated proteins are preferentially targeted to lipid rafts. Here, we outline our strategy to investigate functional role of intrinsic raft-targeting signals in subunits of the gamma-secretase, and explore gamma-secretase function in raft and non-raft environment. We also propose to identify structural and sequence determinants that target APP and APP CTFs to rafts, and characterize how raft lipid components modulate residence and trafficking of gamma-secretase and APP, and influence amyloidogenic processing. The following are the Specific Aims of this investigation. Aim 1: To study the mechanisms underlying association and function of gamma-secretase in lipid rafts. Aim 2: To investigate the mechanisms underlying the association of APP with lipid raft microdomains. Aim 3: To investigate lipid raft regulation of PS1 and APP trafficking, and amyloidogenic processing. Our studies will uncover significant insights on gamma-secretase biology and amyloidogenic processing of APP that will be invaluable for the development of novel therapeutic strategies aimed at selective inactivation of gamma-secretase function in APP processing, in an attempt to reduce Abeta burden. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Disturbance of Ca2+ homeostasis is a major cause of neuronal injury in transient ischemia and several neurodegenerative disorders such as Alzheimer's disease (AD), polyglutamine diseases, and Parkinson's disease. The endoplasmic reticulum (ER) is a major organelle for Ca2+ storage. The ER regulates multiple cellular functions through Ca2+ signaling, and provides a specialized environment for post-translational folding and maturation of transmembrane and luminal proteins. Loss of Ca2+ from intracellular stores causes elevation of cytosolic Ca2+, which can lead to neuronal vulnerability resulting from activation of intrinsic cell death pathways. In addition, since protein chaperones use Ca2+ as a cofactor, release of ER Ca2+ also causes cell injury through an accumulation of misfolded proteins. Physiological, pathological and experimental conditions that perturb ER function cause accumulation of misfolded proteins within the ER, and as a result of the ensuing ER stress, activate compensatory signaling pathways collectively known as the unfolded protein response (UPR). In this proposal we outline our strategy to develop a mouse model for neuroprotection against Ca2+ deregulation, based on overexpression of stanniocalcin 2 (STC2). We recently identified STC2 as a gene induced by the mammalian UPR, hypoxia, and cerebral ischemia. We further demonstrated that in cultured cells expression of STC2 is essential and sufficient to offer cytoprotection against cell death induced by disruption of ER Ca2+ homeostasis. STC2 is a secreted glycoprotein hormone highly conserved in fish and mammals. We postulate that STC2 carries out a distinct function in mammals as a critical survival component of the UPR, defending cells from injury caused by disruption of Ca2+ homeostasis under pathological conditions. The following are the specific aims of this proposal: Aim 1. To generate transgenic mice with spatial/temporal control of STC2 expression. Aim 2. To characterize the neuroprotective function of STC2. Our investigation will develop a valuable resource for the scientific community - transgenic mice with spatial and temporal control of STC2 expression, and perform in vitro and in vivo feasibility studies that will provide the proof of principle to demonstrate neuroprotective properties of STC2 against cell death initiated by deregulation of Ca2+ homeostasis. Outcome of our investigation will be critical for the future development of STC2 based therapeutic strategy to prevent neuronal death. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Microscopes have improved over the years to permit ever greater sensitivity and precision, yet the focal spot diffraction limit of optical lenses has ot been broken until very recently. Stimulated Emission Depletion (STED) technology is a physical structuring of the illumination focus to produce effective illumination points 3-4 times smaller than the diffraction limit. Just as confocal microscopy provides 2-3 times the resolution of standard microscopy, STED superresolution outperforms confocal microscopy by at least this margin and closer approaches the resolution of the electron microscope. The Integrated Light Microscopy Facility (ILMF) at The University of Chicago serves more than 160 NIH-funded researchers in the Biological Sciences Division, as well as interdisciplinary researchers from the Physical Sciences Division and neighboring Institutions. During the past decade the ILMF has witnessed an explosive growth in the use of confocal microscopy. Particularly popular is the most advanced instrument in the ILMF: a Leica SP5 multiphoton confocal microscope, which is typically booked to capacity and must be reserved far in advance. The Leica SP5 II STED-CW microscope requested in this proposal is an enhanced version of the SP5 that includes greater sensitivity, and, most significantly, provides real-time superresolution capability. The instrument includes the (newer) continuous-wave depletion laser scheme that is better suited for live-cell probes. The system includes the latest technology, a hybrid detector that offers APD-like sensitivity with lower noise and wider dynamic range. The researchers at the University of Chicago require the resolution enhancement as well as the increased capacity, and have undertaken a lease to obtain these tools while replacing a failing, older SP2 confocal microscope in a Core serving 162 PIs and over 750 users, an action that demonstrates strong Institutional Commitment for this proposal. The seamlessly integrated STED capability is a major technical advance for researchers at The University of Chicago, and is a novel regional resource also available to researchers from other Institutions. PUBLIC HEALTH RELEVANCE: This request is to buyout an interim lease that the University of Chicago has undertaken to provide continued basic and newly-advanced high-end research microscopy, replacing a failing older system. The requested superresolution microscope has undergone extensive in-house testing, is housed in an established Core Facility, and provides novel capabilities that will significantly enhance the research programs of a large NIHfunded user community.

? DESCRIPTION (provided by applicant): BACE1 is a type I transmembrane protein that initiates Alzheimer's disease Aß production by cleavage of amyloid precursor protein (APP). Accumulating evidence demonstrates that synaptic activity dynamically regulates Aß release production near synapses. The molecular and cellular mechanisms underlying activity- dependent increase of Aß production remain largely unknown. In addition to APP, BACE1 cleaves a number of synaptic transmembrane substrates. Recently, we discovered unidirectional dendritic retrograde transport of internalized BACE1 in hippocampal neurons and found evidence that BACE1 undergoes long-range transport from somatodendritic compartment to axon, in a process termed transcytosis. Very few studies have been published on the regulation of neuronal BACE1 trafficking, and also on protein transcytosis in neurons. BACE1 undergoes post-translational S-palmitoylation, phosphorylation, and ubiquitylation. These modifications on synapse-associated proteins occur in response to synaptic activity, which, in turn, regulate dynamic protein trafficking. We hypothesize that synaptic activity modulates dynamic trafficking at the synapse and transcytosis of internalized BACE1, thus providing a crucial mechanism by which synaptic activity could promote amyloidogenic processing of APP at or near synaptic sites. The Specific Aims of this proposal are: Aim 1) To test the hypothesis that synaptic activity regulates BACE1 trafficking and transcytosis. We will use advanced live-cell imaging and FRAP methods to characterize how synaptic activity affects BACE1 localization in dendritic spines and presynaptic terminals and the local dynamics of BACE1 internalization and recycling at the synapse. In parallel, we will assay transcytosis of internalized BACE1 using microfluidic culture system. Aim 2) To test the hypothesis that synaptic activity dynamically modulates BACE1 post-translational modifications. We will investigate how synaptic activity modulates dynamic modifications within the cytosolic tail of endogenous BACE1. We will confirm and extend the findings by performing trafficking studies (as in Aim 1) in neurons expressing BACE1 bearing mutations within the sites of S-palmitoylation, phosphorylation, and ubiquitylation. Our proposal is timely, unique, and highly innovative because we were the first to describe BACE1 transcytosis in recycling endosomes. We now propose to extend our findings to study how synaptic activity modulates this highly unusual mode of protein trafficking. Our goal to investigate synaptic regulation of BACE1 is also highly significant because endogenous BACE1 and transgene expressed BACE1-YFP predominantly localize to presynaptic terminals in vivo, and BACE1 accumulates in dystrophic presynaptic terminals near senile plaques in the brains of individuals with AD. Thus, investigating how synaptic activity is coupled to BACE1 trafficking and axonal targeting is highly relevant to mechanisms underlying Alzheimer's disease pathogenesis as well as for BACE1 processing of its multiple neuronal substrates under physiological and pathological conditions.

PROJECT SUMMARY This proposal focuses on BIN1, one of the recently identified common risk genes within the major susceptibility loci for late-onset Alzheimer's disease (LOAD) (second only to APOE). BIN1 (Bridging INtegrator-1) is a member of a family of adaptor proteins that regulate membrane dynamics in the context of endocytosis and membrane remodeling. Alternate splicing of the 20 exons in BIN1 gene generates ubiquitous and tissue- specific isoforms, which differ in their tissue distribution, subcellular localization, and function. These functions include cell cycle progression, apoptosis, cytoskeletal organization, and DNA repair. An increase of BIN1 expression and alternate splicing of BIN1 have been reported in the brains of individuals with LOAD but how these observations translate to increased risk for AD is entirely not clear. In preliminary studies, we have characterized prominent BIN1 expression in mature oligodendrocytes in the gray and white matter in rodent and the human brain. By generating oligodendrocyte-specific Bin1 conditional knock out (cKO) mice, we confirmed that BIN1 is mainly expressed in mature oligodendrocytes. Interestingly, we observe aberrant BIN1 expression near human senile plaques and BIN1 accrual in amyloid deposits of AD transgenic mouse models. Based on these novel findings, we hypothesize that BIN1 functions in mature oligodendrocytes, and that alteration in BIN1 expression and/or function in oligodendrocytes plays a role in AD-related pathogenic processes and neurodegeneration in a non-cell autonomous manner. We propose the following specific aims to test novel hypothesis related to the role of BIN1 in AD. The Specific Aims of this proposal are: Aim 1: To test the hypothesis that BIN1 cellular expression and localization are altered in AD brain. We will investigate the BIN1 expression and alternate splicing in normal and diseased human brain. We will determine the subcellular localization of BIN1 by fractionation and confocal microscopy, and clarify the ultrastructural BIN1 localization using immunoelectron microscopy. Aim 2: To test the hypothesis that downregulation of BIN1 expression will attenuate amyloid and tau pathology in transgenic mice. We will ascertain whether cell-type specific loss of BIN1 expression in neurons or mature oligodendrocytes will attenuate AD-related neuropathology and behavior deficits in transgenic AD mouse models. Aim 3: To test the hypothesis that BIN1 plays a role in oligodendrocyte differentiation, survival, and maturation, as well as myelination in vivo. We will utilize Bin1 cKO mouse models and cultured oligodendrocytes to test this hypothesis. This proposal is timely, unique, and highly innovative. We believe that our investigation will uncover significant insights on BIN1's function in the brain, characterize novel Bin1 cKO mouse models of interest to the AD field, establish whether attenuation of BIN1 expression provides a novel strategy for disease intervention, and lay the foundation for characterization of BIN1 functional variants linked to AD, and guide future functional characterization of biological pathways and pathogenic mechanisms regulated by this major LOAD risk gene.

PROJECT SUMMARY Proteostasis dysfunction of pancreatic ?-cells results in accumulation of oligomerized amylin within pancreatic islets, which is a hallmark of type-2 diabetes. We recently showed that oligomerized amylin also incorporates into the brain blood vessel walls, forms neuritic deposits and co-localizes with Alzheimer's disease (AD) ?- amyloid peptides (A?) as mixed A?-amylin plaques in brains of individuals with late-onset AD as well as familial early-onset AD. It is unknown whether cerebral amylin deposits are a consequence of AD or type-2 diabetes, or a ?hidden? trigger of AD. Moreover, we found that overexpressing human amylin within pancreatic islets in rats induces systemic amylin dyshomeostasis, brain amylin accumulation, microglia activation and behavior changes. Furthermore, elevated human amylin in the periphery greatly accelerates behavior changes in a rat model of AD pathogenesis. These findings suggest the hypothesis that amylin dyshomeostasis in pancreatic islets and subsequent secretion of oligomerized amylin in the blood can affect the progression of AD by compromising the ability of microglia to efficiently clear A? and by inducing mixed A?-amylin pathology. Thus, ameliorating amylin dyshomeostasis in the periphery can limit the progression of AD. The overarching goal of this proposal is to test our hypothesis and investigate molecular mechanisms underlying the interaction of amylin with A? pathology. We will accomplish this goal by characterizing novel transgenic mice with inducible and reversible expression of human amylin in the pancreas (HuAmy line). We will carefully dissect proteostasis dysfunction of pancreatic ?-cells in the periphery and the consequent liability it imposes on the central nervous system. We will also cross HuAmy line and 85Dbo line to generate APP/PS1 transgenic mice with regulated expression of human amylin in the periphery. We predict that turning off the human amylin transgene expression at early time points during aging mitigates the disease progression in HuAmy:APP/PS1 mice by limiting the blood-brain barrier injury and rescuing the ability of microglia to clear A?. This investigation using novel regulated amylin expression represents the most direct in vivo approach to rigorously test the involvement of peripheral amylin in the biological pathways of AD pathogenesis. The completion of these aims will elucidate the interplay of amylin dyshomeostasis with the progression of A? pathology and whether ameliorating amylin dyshomeostasis in the periphery can reduce or reverse AD in a preclinical model. We believe that the proposed investigation fills significant gaps in our understanding of the molecular link between type-2 diabetes and AD.

This proposal focuses on BIN1, the most significant late-onset Alzheimer's disease (LOAD) susceptibility locus identified via genome-wide association studies. AD-associated BIN1 single-nucleotide polymorphisms increase BIN1 expression. Alternative splicing, as well as an increase in BIN1 expression, have also been reported in LOAD. BIN1 is an adaptor protein that regulates membrane dynamics in the context of endocytosis and membrane remodeling. Its function in the brain is not known, and there is much to learn about the relationship between BIN1 and the elevated risk for LOAD. BIN1 can directly bind to Tau, leading to the suggestion that BIN1 might influence tangle pathology in AD. We recently reported predominant BIN1 expression in mature oligodendrocytes in the human and rodent brain. Interestingly, unlike in non-AD brain, neurons in AD brain begin to express BIN1. In preliminary studies, we identified a selective increase in the levels of BIN1 isoform 9 (BIN1iso9) in LOAD. How these observations translate to increased risk for LOAD is not clear. Transgenic mouse models have been invaluable in investigating the disease mechanisms. The overarching goal of this proposal is to test a gain-of-function hypothesis using BIN1 transgenic mice and resolve whether the LOAD- associated changes in BIN1 per se are causal to the disease process or rather they are reactive to the disease. To accomplish this goal, we have generated a transgenic line and used Cre drivers to activate human BIN1iso9 expression in a cell-type specific manner. The proposed investigation will test our central hypothesis that an increase in BIN1iso9 expression will worsen AD-associated pathology and behavior abnormalities. The goal of Aim 1 is to perform targeted proteomics using select reaction monitoring to perform a comprehensive characterization of the BIN1 isoform diversity in LOAD. We will also use immunogold electron microscopy to localize BIN1 with ultrastructural precision in the human brain and AD mouse models. Aim 2 studies will test the hypothesis that transgenic expression of BIN1iso9, which recapitulates BIN1 upregulation in AD, will compromise neuronal endocytosis and synaptic transmission, as well as interfere with myelin protein trafficking in oligodendrocytes. Aim 3 studies will test the hypothesis that BIN1iso9 expression will exacerbate neuropathology and behavioral deficits in mouse models of amyloid and tau pathogenesis. This timely and unique proposal is highly innovative. This investigation using novel BIN1 transgenic mice represents the most direct in vivo approach to rigorously test BIN1's involvement in the biological pathways of AD pathogenesis. In addition, this investigation will use proteomics approaches to clarify altered BIN1 isoform expression in LOAD, precisely define BIN1 localization in the brain, and characterize Bin1 transgenic mice as a resource to the AD field. We believe that successful completion of the proposed investigation will fill significant gaps in our understanding of BIN1 as a risk factor for LOAD, and guide future functional characterization of biological pathways and pathogenic mechanisms regulated by this major LOAD risk gene.

Alzheimer?s disease (AD) is a devastating neurodegenerative disorder without an effective cure. Recent genome- wide association studies (GWAS) on AD have identified >20 reproducible risk loci, providing an opportunity for understanding novel aspects of AD biology and developing effective interventions. However, each GWAS locus typically spans several genes and many equally associated genome-wide significant index/proxy single- nucleotide polymorphisms (SNPs) that are in strong linkage disequilibrium; it thus remains challenging to identify which genes and risk SNPs are causally involved in AD pathogenesis. Most GWAS risk variants are noncoding and likely regulate gene expression. Because accessible or open chromatin overlaps with cis-regulatory sequences, we hypothesize that many causal AD risk variants modulate chromatin accessibility to transcription factors, thereby altering molecular and cellular phenotypes relevant to AD. We have recently shown that open chromatin profiles in neurons derived from human induced pluripotent stem cells (hiPSCs) can help prioritize regulatory GWAS risk variants of schizophrenia. By directly comparing the quantitative measurements of open chromatin between the two alleles of a heterozygous SNP within the same sample, i.e., allele-specific open chromatin (ASoC) assay, we further showed that schizophrenia GWAS risk variants frequently exhibit ASoC in hiPSC-neurons. More importantly, we found that the neuronal ASoC variants are highly enriched for AD GWAS risk variants and can readily inform putatively regulatory risk variants at three leading AD risk loci (BIN1, CD2AP and CLU). Here, we will extend the ASoC approach to other hiPSC-derived AD-relevant cell types and also harness our expertise in CRISPR genome/epigenome editing and AD cell biology to address three specific questions: (1) Which AD GWAS risk variants are functional? For this, we will map open chromatin by ATAC-seq in glutamatergic and GABAergic neurons, astrocytes and microglia, and search for regulatory AD-risk variants that present ASoC; (2) Which genes are regulated by the putatively functional AD GWAS variants and are thus likely to be causal? For this, we will cost-effectively assay the regulatory effects of all putative AD-risk variants in hiPSC-derived neurons, astrocytes and microglia by combining multiplexed CRISPR/cas9 epigenome editing with single-cell RNA-seq; (3) What are the cellular phenotypic changes caused by the putatively causal AD variants/genes? For this, we will study the cis-regulatory effects of regulatory variants in BIN1, CD2AP and CLU (and other prioritized genes) on AD-relevant biochemical and cellular phenotypes in CRISPR-engineered hiPSC models. This project would impact the field by moving beyond GWAS to decipher causal mechanisms and develop effective treatments.

1) Project Summary/Abstract The estimated 20,000 genes in the human genome are variably expressed in the body?s organs and tissue, each made of heterogeneous cell types characterized by shared and cell-type-specific gene expression profiles. The biological function of a gene frequently depends on the spatial context within a healthy organ and tissue. Many human disorders, including developmental diseases, cancer, and neurodegenerative diseases, result from the deregulation of the spatial organization of cells within a tissue as well as their impaired gene expression. Systematic annotation of gene and protein expression is a crucial step in understanding the biological complexity, elucidating cellular identity, deciphering disease mechanisms, etc. However, most routine transcriptomics and proteomics technologies overlook the relationship between the disease state and spatially delineated alteration in gene and protein expression that exists in many human diseases. Preserving the spatial context of gene and protein expression is essential to gain deeper insights into tissue biology and the manifestation of disease pathology. Digital spatial profiling based on in situ RNA imaging and in situ sequencing has emerged as promising tools that could allow an analysis of cellular transcriptomes within their spatial context in tissue sections. The GeoMx Digital Spatial Profiling platform, introduced in 2019, represents a recent advancement that provides morphological context to high-plex protein or gene expression profiling from just one tissue section on a slide. In this system, spatial profiling of RNA and protein is performed on the GeoMx DSP platform, which includes imaging and fluidic components to capture spatial context as micropipette aspirates into 96-well plates. The samples are read on the nCounter, which provides a multiplexed measurement of transcripts and protein with a high level of precision while retaining spatial resolution using a direct, digital counting technology. Remarkably, the implementation of the technology allows one to profile up to 96 protein targets, ~800 RNA targets, or even multiplexing RNA and protein quantification with spatial resolution on the same formalin-fixed, paraffin-embedded tissue section on a microscope slide. Even within a year of its introduction, the GeoMx platform has been successfully used in a range of biological investigations, elucidating the versatility of the system for biomedical discovery and translational research. Implementation of this technology in a shared microscopy core at the University of South Florida Morsani College of Medicine will significantly benefit a large number of NIH-funded investigators engaged in biomedical research in fields as broad as Alzheimer?s disease, aging, stress and trauma, neuroinflammation, addiction, oncogenesis, innate immunity, infectious diseases, parasite-host interaction, computational and integrative biology, and gene therapy. The seamlessly integrated digital spatial profiling technology is a major technical advance for researchers at The University of South Florida. It will also be a unique regional resource available to researchers from other Institutions.