Alison M. Goate, PhD - US grants

I have recently joined the faculty of the Psychiatry department at Washington University and am in the process of establishing my own independent research group working on the molecular genetics of Alzheimer's disease (AD). During the last six years I have successfully used a genetic approach to identify mutations at codon 717 of the amyloid precursor protein gene in some cases of familial Alzheimer's disease (FAD). In the intermediate term, my goal is to continue to use a this approach to unravel the causes of FAD and other familial dementias which show Mendelian transmission. In the longer term I plan to use a similar approach to study other psychiatric disorders which exhibit familial clustering but do not show Mendelian inheritance, such as alcoholism and schizophrenia. Receiving a career development award will make a substantial difference to me as I am expected to raise 95% of my salary from grant support. This award would give me a stable source of support which would allow me devote my full attention to the research rather then spending a substantial amount of time writing grants to support my salary and will also permit me the discretion to elect to assume only those teaching and/or administrative duties I wish. I have a tenure track associate professor position jointly held between the Departments of Psychiatry and Genetics. Within the Psychiatry department there are a substantial number of faculty who share my interest in the genetics of psychiatric disorders, whilst in other departments within the medical school there are many people who share my interest in molecular genetics and in transgenic approaches to the study of inherited diseases and gene expression and regulation. Finally, there are many faculty throughout the medical school who are part of the Alzheimer's Disease Research Center and who contribute to a co-ordinate multidisciplinary approach to understanding the pathogenesis of Alzheimer's. My principal research interest is understanding the pathogenesis of AD. We are testing the hypothesis that mutations in the amyloid precursor protein (APP) gene can cause AD. To test this hypothesis we are introducing the APP717 mutations into mice via homologus recombination in embryonic stem cells to determine whether they are sufficient to cause disease. In addition we are collaborating with Dr. Frank Ashall who is using cell lines derived from patients with the APP717 mutations to determine the biochemical effect to these mutations upon the regulation of APP expression and proteolytic processing. Secondly, we are using positional cloning to identify the gene on chromosome 14 which predisposes to another form of early onset FAD. Thirdly, we are doing an association study with samples from control and AD individuals to determine whether alleles of the association study with samples from control and AD individuals to determine whether alleles of the APP gene may predispose to AD in the elderly. It is hoped that these studies will help to determine whether premature b-amyloid deposition is central to the pathogenesis of all cases of AD or only those which are caused by mutations within the APP gene.

This is a resubmission of a proposal to develop a small animal model of Alzheimer's disease (AD) using gene targeting in embryonic stem (ES) cells to introduce mutations to the mouse amyloid precursor protein (APP) gene. Despite much research the only animal model currently available is the aging non-human primate. The lack of a small animal model has slowed not only the progress in our understanding of the molecular pathogenesis of AD but also the development of rational therapeutic strategies for the treatment of the disease. The identification of several mutations within the APP gene in a number of families with an autosomal dominant form of AD has opened up the possibility of creating a genetic model of AD in mice. Three of these mutations are caused substitutions in the same codon, APP717. The primary aim of this research project is to test the hypothesis that these mutations are sufficient to cause AD. To test this hypothesis we plan to use gene targeting via homologous recombination in ES cells to introduce a point mutation into codon 717 of the mouse APP gene. Introduction of these ES cells into mouse blastocysts and the subsequent breeding of chimeras derived will produce animals heterozygous for the condon 717 mutation. It is these animals which will provide the test of the hypothesis that the codon 717 mutation cause AD. If indeed these mutations are sufficient to cause disease these animals will develop a spontaneous neurodegenerative disorder. Unlike aging humans, aging mice do not develop beta-amyloid plaques. Several hypotheses have been proposed which address this issue. These are: that mice do not live long enough to develop senile plaques; that APP is processed differently in mouse brain or that there are sequence differences between mouse and human beta-amyloid which confer different physical properties upon the two proteins making mouse beta-amyloid less prone to fibril formation. To control for this last possibility, we have made constructs in which three point mutations have been introduced into exon 16 in addition to the exon 17 mutation at codon 717. The result of these three point mutations is that the beta-amyloid sequence derived from these constructs is identical to the human beta-amyloid sequence. To minimize the effects of the first possibility we will engineer a construct containing both the Swedish and a 717 mutation, this should accelerate b-amyloid deposition. If any of the constructs produce AD neuropathology, these animals will be bred to establish a colony which will be used for studies to understand the pathogenic mechanisms of disease, to characterize the phenotype and ultimately to test therapeutic strategies.

DESCRIPTION: (From Abstract) Molecular genetic studies of familial Alzheimer's disease (FAD) have led to the identification of three genes which, when mutated can cause Alzheimer's disease (AD); beta-amyloid precursor protein (APP) and two related genes presenilin 1 (PS1) and presenilin 2 (PS2). The normal function of each of these molecules remains unknown, however, FAD mutations in all three genes result in an increase in levels of Ab42, an amyloidogenic fragment derived from APP by proteolytic processing. The dominant inheritance of the FAD phenotype suggests that these mutations either cause a gain of function or a loss of function via a dominant negative mechanism. PS1 knock-out mice are not viable and show multiple developmental abnormalities. Neurons derived from PS1 knock-out mice show a decrease in Ab levels suggesting that PS1 is required for normal APP processing and that the FAD mutations are not loss of function alleles. Genetic experiments have demonstrated that two presenilin homologs exist in C. elegans: sel-12 and hop-1, and that these molecules undergo similar endoproteolytic processing to their mammalian counterparts. Mutations that reduce sel-12 activity or sel-12 and hop-1 activity cause phenotypes that are consistent with a reduction in signaling through the lin-12 and glp-12 (Notch homologs) pathways. The phenotype of constitutively active lin-12 mutants lacking the extracellular and transmembrane domains are not modified by reduction of sel-12 activity suggesting that the sel-12 affects Notch signaling upstream of Notch activation. PS are membrane proteins with multiple membrane spanning domains that are primarily located within the endoplasmic reticulum. To determine the normal function of PS1 we will examine Notch maturation and processing in the presence and absence of functional PS1. We will extend our observations to the effect of PS function on APP maturation and processing and finally we will examine the effect of FAD mutations on the normal function of PS1 with respect to both Notch and APP. Specifically, we hypothesize that loss of PS function leads to reduced Notch signaling and reduced Ab because PS is required for normal Notch and APP maturation within the secretory pathway. We also hypothesize that FAD-causing mutations modify the normal activity of PS1 and that this change will have no effect on Notch signaling but will subtly alter APP processing. Through these studies we hope to determine the normal function of PS1 and to gain a better understanding of the role of PS1 in AD pathogenesis. We also anticipate that these studies will increase our understanding of the Notch signaling, a process that is important not only for many developmental decisions but for cancer as well.

Studying the genetics of Alzheimer's Disease (AD) has added significantly to our understanding of the disease. During the last six years it has been established that familial early onset Alzheimer's disease (FAD) is a genetically heterogenous disease that can be caused by mutations in at least three different genes: the beta-amyloid protein precursor (APP) gene on chromosome 21, the presenilin 1 (PS-1) gene on chromosome 14 and the presenilin 2 (PS-2) gene on chromosome 1 (1-3). Since other families exist that do not carry mutations within any of these genes it is very likely that there are other as yet unidentified FAD genes. In vitro experiments suggest that mutations in each of the known genes cause AD through changes in APP processing that lead to elevated levels of total Abeta or specifically increase Abeta42 (4). This provides strong support for the "Amyloid Hypothesis" of AD pathogenesis. The study of the genetics of late onset AD has also led to the identification of the first genetic risk factor for AD. The epsilon 4 allele of the apolipoprotein E (ApoE) gene has been shown to increase risk for AD in every population studied although the magnitude of the increase in risk varies between populations. Although it is still uncertain how the ApoE4 allele increases risk for AD there is some evidence to support the hypothesis that ApoE4 modifies amyloid deposition by an unknown mechanism. However, since there are many individuals with AD who have no ApoE4 alleles there must be other risk factors for late onset AD. We hypothesize that at least some of these risk factors are genetic and that they may also modify amyloid deposition. The aim of this proposal is to use a genetic linkage strategy to identify new genetic risk factors for late onset AD. As our test sample we will use three hundred caucasian sib pairs with an age of onset of AD over the age of sixty five years to perform a 20cM genome-wide screen using microsatellite markers. Genomic regions showing evidence of linkage will be followed up with flanking markers in the same sample and in a second sample of equivalent size also selected on the basis of racial origin and age of onset. It is anticipated that by restricting the racial origin and age of onset of our initial samples we will reduce the likely genetic heterogeneity and increase our chances of detecting a second risk factor. Candidate genes in genomic regions that continue to show evidence of linkage will then be followed up using a case control association approach in caucasians and in other ethnic groups. Finally, we will complete a 20cM genome screen in the replication sample increasing the effective sample size to 600 sib pairs, enabling us to look for genes of smaller effect size.

disease /disorder onset; genetic mapping; Alzheimer's disease; presenilin; family genetics; nucleic acid sequence; autosomal dominant trait; linkage mapping; clinical research; human subject;

Alzheimer's disease (AD) is the most common neurodegenerative disease in the U.S., affecting over 4 million people over the age of 65 years. Current medications treat the symptoms but not the underlying causes of disease. There is therefore an urgent need to understand the underlying pathogenic mechanisms of disease to enable rational drug design. During the last twenty years genetic studies of familial early onset AD have demonstrated that mutations in three genes cause AD via a common biochemical pathway involving Ass metabolism. Studies of late onset AD (LOAD) have implicated genotype at the apolipoprotein E (APOE) locus as a major risk factor that also acts via an Ass dependent mechanism. However, only 50% of LOAD cases carry a risk allele at the APOE locus. The goal of this study is to combine quantitative trait locus (QTL) studies of biochemical measures in cerebrospinal fluid and case control data to identify and validate novel genetic risk factors for LOAD. In the current proposal we will use publicly available existing genome-wide associate study (GWAS) data in case control datasets and newly acquired GWAS data (through the AD Genetics Consortium (ADGC)) in samples with biomarker measurements to identify SNPs/genes that influence risk for LOAD via an Ass dependent mechanism. GWAS data in the Washington University/University of Washington (WU/UW) biomarker datasets will be generated during the next twelve months as part of the first phase of genotyping by the ADGC. GWAS data for the AD Neuroimaging Initiative (ADNI) series and from several case control datasets are already in hand (approx. 3000 cases and 3000 controls). In this study we propose three specific aims focused on the analysis of this existing data. First we will test in the WU/UW CSF series for genetic factors on chromosome 10 and elsewhere in the genome that influence CSF Ass levels. Second, we will try to replicate these findings in an independent series with CSF biomarker measurements, collected by the ADNI consortium. We will then compare our findings in the CSF series with the results of a combined GWAS dataset in AD cases and controls. This will allow us to identify the putative AD genes that influence risk by an Ass dependent mechanism, facilitating follow-up mechanistic studies to confirm the functional effects of these genes on Ass metabolism and AD risk. The use of case-control and endophenotype measures in CSF provides a powerful and novel approach to the genetics of LOAD.

[unreadable] DESCRIPTION (provided by applicant): During the last 10 yrs it has been established that familial early onset AD is a genetically heterogeneous disease that can be caused by mutations in at least three different genes: Beta-amyloid protein precursor (APP)and presenilins 1 and 2 (1-3). Most mutations in these genes cause AD through changes in APP processing that elevate levels of total Beta-amyloid (A-Beta) or specifically increase A-Beta42 (4), providing strong support for the "Amyloid Hypothesis" of AD pathogenesis. Study of the genetics of late onset AD (LOAD) has also led to the identification of the epsilon 4 allele of the apolipoprotein E (APOE) gene as a risk factor. It has been shown to increase risk for AD in every population studied although the magnitude of the increase in risk varies between populations (6). Recent studies in animal models of A-Beta deposition have demonstrated that APOE4 also increases risk for AD through an A-Beta-related mechanism (7). To identify additional genetic risk factors for LOAD we performed a genome screen in 450 affected sibling pairs. The strongest evidence for linkage was obtained on chromosome 10 with a peak multipoint Iod score of 3.9. Younkin and colleagues have also provided evidence for a quantitative trait locus (QTL) that influences plasma A-Beta levels in the same region of chromosome 10 suggesting that the chromosome 10 AD locus may also influence AD risk via an A-Beta-dependent mechanism. Nine other chromosomal locations gave an MLS >1, including chromosome 12 where we and others have previously reported evidence of linkage. Although the linkage to chromosome 12 is modest, sib pair analyses with covariates increased the Iod score from 1.35 to 4.51 in APOE4 negative sib pairs. Furthermore a genome screen on the same dataset using age of onset as a quantitative trait also provided good evidence for a QTL influencing age of onset in the same region of chromosome 12. In the current application we propose to use linkage and linkage disequilibrium analyses to identify the disease alleles on chromosomes 10 and 12. We will use both case-control and family-based association methods to analyze individual single nucleotide polymorphisms (SNPs) and SNP haplotypes in genes under the linkage peak. SNPs will be identified from the public databases and by sequencing the genes in AD cases. Putative risk alleles/haplotypes will be tested for a functional effect on A-Beta levels in an in vitro model. Risk alleles will be characterized in several populations to determine their importance. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): There is growing interest in the potential contribution of cholesterol to the pathogenesis of Alzheimer's disease (AD). Treatment with cholesterol-lowering statins appears to lower the risk of developing AD. Furthermore, Abeta production has been directly associated with cholesterol-rich domains (lipid rafts) and cholesterol levels have been shown to modulate APR processing and Abeta generation. The link between cholesterol and Abeta has recently been revealed in Niemann Pick Type C (NPC) diseases as well as AD. Deficiency in NPC1 protein causes intracellular accumulation of unesterified cholesterol in late endosomal/lysosomal compartments that is accompanied by a significant increase in Abeta production and a shift in presenilin 1 (PS1) localization to early/late endosomes. Contradictory results were reported on whether changes in cholesterol content may directly affect gamma-secretase cleavage of APP. The goal of this project is to elucidate the mechanisms by which cholesterol affects APP metabolism. We hypothesize that cholesterol modulates APP processing in a similar manner in wild-type and NPC1 knock-out cells. We also hypothesize that cholesterol levels regulate intracellular trafficking of APP, BACE1 and PS1, three key players in the pathogenesis of Alzheimer's disease. We speculate that cholesterol-dependent association with lipid rafts of the three proteins regulates their endocytosis and thus generation of Abeta. In addition, we hypothesize that levels of cholesterol and/or apolipoprotein E (apoE) modulate APP processing directly regulating gamma-secretase activity. To analyze the parallels between cholesterol and APP processing in wild-type and NPC1-/- cells we will test whether an increase or a decrease in cholesterol levels modulates APP metabolism in a similar manner. Comparing cellular localization and association with lipid rafts of APP, BACE1 and PS1 in wt and in NPC1 Knock out cells under sterol-starved and sterol-fed conditions we will elucidate whether the cholesterol-effect on aberrant APP processing is mediated via specific subcellular or lipid raft compartment(s). To test whether gamma-secretase activity per se can be regulated by cholesterol and/or apoE levels we will perform in vitro gamma-assay. If gamma-secretase activity is regulated by cholesterol levels and/or apoE we will test whether this feature is specific for gamma-cleavage of APP or is common among other gamma-secretase substrates (e.g. Notch 1). This research will be done primarily in Croatia at the Rudjer Boskovic Institute in collaboration with Dr. Silva Hecimovic as an extension of NIH grant #R01AG016208. Through these studies we will elucidate the molecular mechanisms of cholesterol action in Alzheimer's disease. Finding the molecular link(s) between cholesterol and AD is important both for treating Alzheimer's disease as well as for understanding normal APP function.

During the last two decades many genes have been shown to cause autosomal dominant forms of early onset dementing illnesses. These rare disorders have provided enormous insight into the pathogenesis of more common variants of the same diseases. Several of the most promising new therapeutics are based on the Afi hypothesis, a hypothesis largely supported by the causative mechanisms of disease mutations in autosomal dominant families. As these putative therapeutics are tested in clinical trials there is a growing need to use the FAD kindreds both to understand the natural history of the earliest clinical and preclinical phases of the disease and to test the efficacy of the therapeutics in a setting, where if the Afi hypothesis is correct, they should have a dramatic effect on prognosis. The first step in this overarching goal is to bring together a network of centers to characterize a large series of FAD kindreds with known disease-causing mutations. The goal of the Genetics Core of the DIAN initiative is to provide genetic information and useful biological materials to the research community for the study of AD. We will collect blood samples for DMA extraction from all study participants (n=300). Since these individuals are part of FAD kindreds with known causative mutations we will screen each sample for the known mutation in that family and genotype all families for known disease modifying alleles such as APOE e4. Blood from each individual will be sent to NCRAD for transformation to develop lymphoblastoid cell lines that will provide the largest resource of cell lines from FAD kindreds with known disease-causing mutations anywhere in the world.

2-Pyrrolidinone, 1-methyl-5-(3-pyridinyl)-, (S)-; Active Follow-up; Affect; African American; Afro American; Afroamerican; Alleles; Allelomorphs; Alternate Splicing; Alternative Splicing; American Cancer Society; Assay; Bio-Informatics; Bioassay; Bioinformatics; Biologic Assays; Biological Assay; Black Populations; Black or African American; Blood; Blood Plasma; CYP2A6; CYP2A6 protein, human; Candidate Disease Gene; Candidate Gene; Cells; Cessation of life; Cholinergic Receptors; Cholinoceptive Sites; Cholinoceptors; Chromosome Mapping; Classification; Code; Coding System; Cotinine; Data; Data Set; Dataset; Death; Dependence; Dependence, Nicotine; Development; Enzymes; FAD-monooxygenase; Family; Funding; GWAS; Gene Expression; Gene Localization; Gene Mapping; Gene Mapping, Total Human and Non-Human; Genes; Genetic; Genetics, Gene Mapping; Genome; Genotype; Glucuronic Transferase; Glucuronosyltransferase; Glucuronyltransferase; Goals; Haplotypes; Individual; Intermediary Metabolism; Lead; Linkage Mapping; METBL; Maps; Measures; Metabolic Processes; Metabolism; N,N-dimethylaniline N-oxidase; Nicotine; Nicotine Dependence; Nicotinic Acetylcholine Receptors; Nicotinic Receptors; Oocytes; Ovocytes; Pb element; Phenotype; Plasma; Polymorphism, Single Base; Pyridine, 3-(1-methyl-2-pyrrolidinyl)-, (S)-; RNA Splicing; RNA Splicing, Alternative; Receptors, ACh; Receptors, Acetylcholine; Reticuloendothelial System, Blood; Reticuloendothelial System, Serum, Plasma; Risk; SNP; SNP Map; SNPs; Scotine; Series; Serum, Plasma; Single Nucleotide Polymorphism; Single Nucleotide Polymorphism Map; Smoke; Smoker; Smoking; Smoking Behavior; Splicing; Staging; Survey Instrument; Surveys; Systematics; Testing; Tobacco Consumption; Tobacco use; Twin Studies; UDP Glucuronosyltransferase; UDP Glucuronyl Transferase; UDPglucuronate beta-D-glucuronosyltransferase (acceptor-unspecific); Variant; Variation; black American; case control; cytochrome P-450 2A6; cytochrome P-450 CYP2A6 (human); dimethylaniline monooxygenase (N-oxide forming); disability; endophenotype; flavin-containing monooxygenase; follow-up; gene function; genetic mapping; genome wide association scan; genome wide association studies; genome wide association study; genome-wide scan; genomewide association scan; genomewide association studies; genomewide association study; genomewide scan; heavy metal Pb; heavy metal lead; in vivo; member; mutant; nicotine C-oxidase; nicotine addiction; novel; oxidation; protein function; receptor sensitivity; whole genome association studies; whole genome association study

DESCRIPTION (provided by applicant): This grant proposal is in response to RFA-OD-09-005, "Recovery Act limited Competition: Supporting New Faculty Recruitment to Enhance Research Resources through Biomedical Research Core Centers" and is entitled "Translational Neuroscientist and the Hope Center Program on Protein Folding and Neurodegeneration (HCPPFN)". The goal of this newly formed center is to develop better ways to diagnose and treat neurodegenerative diseases through an understanding of the underlying molecular mechanisms of disease. A common theme of these disorders is the central role of protein mis-folding and aggregation leading to neuronal cell death. To the scientific goals of the center we have identified 15 faculty within the institution who are currently using a variety of approaches to understanding the molecular basis of these disorders. Many of these individuals already collaborate on projects but through the center we are fostering an interdisciplinary approach and encourage collaboration between center laboratories. The goal of the current application is to recruit a junior faculty with expertise in the biochemical analysis of protein folding/aggregation into this interdisciplinary program, to enable this individual to develop an independent research program on the biochemistry of protein folding and aggregation in relation to neurodegenerative disease and to enhance the research of the center through new collaborative projects enabled by this recruitment. Six faculty from this center, including the new recruit, will be located in a new state-of-the-art research facility, the BJC Institute for Health, located in the center of the medical school campus. This facility is the centerpiece of the Biomed 21 initiative to promote interdisciplinary translational research. The HCPPFN will work with the Depts. of Biochemistry and Neurology to recruit and mentor a junior faculty with appropriate expertise and to encourage collaborative projects through regular "chalk talks" among center faculty and targeted pilot funding from both internal and external sources. It is anticipated that these pilot projects will develop into larger collaborative projects such new program projects.

Alzheimer's disease (AD) is the most common neurodegenerative disease, afflicting over 4 million people over the age of 65 years, in the U.S. Current medications treat the symptoms but not the underlying causes of disease. There is therefore an urgent need to understand the pathogenic mechanisms of disease to enable rational drug design. During the last twenty years genetic studies of familial early onset AD have dramatically changed our understanding of the disease by demonstrating that mutations in three different genes cause disease via a common biochemical pathway involving B-amyloid (Ali) metabolism. Genetic epidemiology has demonstrated that late onset AD (LOAD) also has a strong genetic component. However, to date only the e4 allele of apolipoprotein E, present in only 50% of LOAD cases, has been convincingly demonstrated to influence risk for LOAD. There is therefore a clear need for new approaches to understanding the genetics of LOAD. We will use intermediate traits, or endophenotypes to identify novel genetic risk factors for LOAD. Endophenotypes may be continuous variables that are correlated with disease but measurable in many or all individuals, avoiding the heterogeneity associated with clinical diagnoses and allowing the use of quantitative statistical methods. Endophenotypes may also provide a biological model of disease and the possible effects of the associated genetic variation. Several promising endophenotypes are protein biomarkers found in cerebrospinal fluid (CSF) including amyloid-beta (A(3), tau, serpin peptidase inhibitor, clade C (antithrombin), member 1 (ATI11), serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (ACT), carnosine dipeptidase 1 (CNDP1) andA-2- glycoprotein 1, zinc (ZAG). These proteins are present in all individuals, show variability amongnon- demented individuals and change with disease. The goal of this study is to identify cis-acting genetic variation that is associated with CSF levels of these AD biomarkers, and to test in independent datasets whether this variation also influences age at onset of AD or risk for AD. Functional studies will be employed to determine the biological effects of the associated genetic variation. These data will inform our models of age at onset of AD, AD diagnosis (project 2) and our studies of the interaction between preclinical AD and post-stroke dementia (project 1). As a proof of principle regarding this approach we have already identified genetic variants in A/MPTthat show significant association with both CSF tau and ptau181 levels. Further study shows that this association is limited to individuals with evidence of A(3deposition. Genetic variation in this region also appears to be associated with expression levels of tau mRNA in individuals with amyloid deposition and age at onset of LOAD.

DESCRIPTION (provided by applicant): Several genome-wide association studies (GWAS) for late-onset Alzheimer's disease (LOAD) have now been published. While all of these studies detected the association of APOE 54 with risk for LOAD only the two largest studies, with over 10,000 cases and controls provided genome-wide significant evidence for any novel loci. To develop larger datasets we and others have formed large collaborative groups, such as the Alzheimer's Disease Genetics Consortium (ADGC). We have also developed an innovative program using cerebrospinal fluid biomarker levels as endophenotypes for our genetic studies of LOAD. Our preliminary data demonstrate the power and novelty of this approach in identifying genes that alter biomarker levels and modify LOAD risk, age at onset or rate of disease progression. This endophenotype approach also has the advantage of pin-pointing specific biological hypotheses regarding the effects of associated variants that can be tested using simple cell culture assays. The goal of this proposal is to combine and analyze existing LOAD GWAS data, then use a novel approach that incorporates quantitative intermediate traits, re-sequencing, bioinformatics, expression and functional studies to facilitate the identification and characterization of genetic variants that modulate risk for LOAD, age at onset or rate of disease progression. To accomplish this we will 1) combine and analyze LOAD GWAS data, 2) use a novel method, the Genomic Information Network, to systematically incorporate biological information to prioritize single nucleotide polymorphisms (SNPs) for follow-up, 3) examine top SNPs from the LOAD GWAS for association with cerebrospinal fluid amyloid-beta and tau levels to establish specific hypotheses of mechanism, 4) use novel genetic and bioinformatic methods to identify putative causal variants from the replicated SNPs, 5) use re-sequencing to identify novel variants in the regions surrounding replicated SNPs, 6) examine the top hits for effects on gene expression. Finally, we will use information from these efforts to test specific amyloid-beta or tau related hypotheses for replicated SNPs in cell culture models. This proposal combines the unique resources and skills of our research team with the vast wealth of publicly available resources into a novel approach to the identification and characterization of genetic risk factors for LOAD.

One hundred years ago dementing illnesses were classified based upon their clinical presentation and neuropathology. T he promise of the twenty first century is that w e w ill be able to classify these same diseases by t he genetic cause or genetic risk factors, a classification based upon etiology not symptomatology. During the last two decades mutations in many genes have been shown to cause Inherited forms of early onset dementing illnesses. These rare disorders have provided enormous insight into the pathogenesis of more common variants of the same diseases. A growing realization of the importance of genetic risk factors for common diseases has led the research community to assess the role of genetic as well as environmental risk factors in susceptibility for late onset Alzheimer's disease. In 1993, polymorphism in t he apolipoprotein E (APOE) gene w as shown to be the first identified risk factor for AD. A dose-dependent effect o f t he AP0E4 allele has been observed in al most every population studied. APOE genotype has now become an important variable in clinical and pathological studies of AD. Indeed, all clinical trials now evaluate putative AD drugs in AP0E4 positive and AP0E4 negative patient subgroups. The goal of the Genetics Core of the Washington University ADRC is to provide genetic information and useful materials on all ADRC participants. In the case of late onset AD we will obtain family history data, APOE genotypes and bank plasma and serum samples. This data will be stored in the master ADRC database and provided to investigators upon request and thus specifically will support Projects 1 (Perrin) and 3 (Bateman) of this competing renewal application. When new genetic risk factors are identified we will also provide this information where possible. In the case of multiplex kindreds we will collect family history data and screen individuals for mutations in the known dementia causing genes. Cell lines and brains from these individuals will be available for molecular studies on the effects of specific mutations.

No abstract provided

Core G: Genetics Project Summary/Abstract One hundred years ago, dementing illnesses were classified based upon their clinical presentation and neuropathology. The promise of the twenty-first century is that we will be able to classify these same diseases by the genetic cause or genetic risk factors, a classification based upon etiology not symptomatology. During the last two decades many genes have been shown to cause autosomal dominant forms of early onset dementing illnesses. These rare disorders have provided enormous insight into the pathogenesis of more common variants of the same diseases. In 1993, a polymorphism in the apolipoprotein E (APOE) gene was identified as the first genetic risk factor for AD. A dose-dependent effect of the APOE4 allele has now become an important variable in all studies of AD. During the last five years genome-wide association studies and next generation sequencing studies have begun to identify many novel risk factors for AD. The goal of the Genetics Core of the Knight ADRC is to provide genetic information and useful biological materials on all ADRC participants. We will obtain family history data, plasma, serum, DNA, RNA, and APOE genotypes on all ADRC participants. Blood samples on all participants will be sent to NCRAD. Many participants will also have GWAS, exome array and whole exome/genome sequence data through national and international initiatives and ADRC affiliated projects held by Drs. Goate and Cruchaga. These data will be stored in the master ADRC database and provided to investigators upon request. We will identify and assess novel multiplex kindreds. Families that meet criteria for other funded projects such as DIAN, NIA-LOAD and LEFFTDS will be invited to participate in these studies. The Core will support Projects 1, 2 and 3 of the Knight ADRC and will continue to support the Health Aging and Senile Dementia and Adult Children Study Program Project Grants (JC Morris, PI) during the next five years. New in this application we will collect skin biopsies from targeted individuals carrying specific genetic risk factors for AD and we will begin to collect blood annually for RNA expression and DNA methylation studies to enable novel biomarker programs in peripheral tissues.

? DESCRIPTION (provided by applicant): Genome-wide association, whole genome/exome sequencing and gene network studies have already enabled researchers to identify twenty loci influencing Alzheimer's disease (AD) risk and another half dozen genes carrying specific rare variants that influence disease risk. With the new whole-genome sequence (WGS) and whole-exome sequence (WES) data from 10,000+ AD cases and controls from the ADSP, combined with mRNA expression data from 3,500+ individuals from AMP, it is now possible to develop a more comprehensive picture of the genetic architecture of AD and associated risk. Beyond refining AD genetic architecture, our goal is to identify and validate therapeutic targets for AD b identifying genes that functionally drive or protect from AD and interrogating their respective gene networks for therapeutic targets. We will do this using the largest, most comprehensive data set, to date. Genetic and pathway-based analyses have strongly implicated a small number of networks including immune response, phagocytosis, lipid metabolism and endocytosis. We will integrate data from genetic studies and gene expression/regulation studies to identify risk and resilience genes to pinpoint key networks that functionally drive AD development and progression. We will take two complementary approaches to identify risk and resilience AD genes: (1) we will use a family-based approach to identify both risk and protective alleles using publicly available data and our own WGS/WES data from both NIALOAD and Utah families; and (2) we will use publicly available high-dimensional molecular data from AD cases and controls to construct global interaction and causal networks. We will then focus our analysis of ADSP case control sequence data on the most compelling networks, thereby reducing our search space and increasing power. To identify therapeutic targets, we will use network analysis to test known drugs that target networks identified in our sequence analysis of both family-based and case control data. We will then validate our findings by performing in vitro experiments based our in silico observations and determine the functional consequences of risk/resilience alleles identified from the AD sequence data. Together, the findings from this study will pinpoint key networks that functionally drive AD and will provide critical insight into therapeutic intervention

Alzheimer's disease (AD) is the only disease among the top ten killers in the U.S. without a disease modifying therapy. As a result it is also the only one that is increasing in prevalence. Human genetic studies provide a powerful means to identify genes and pathways that are causally linked to the etiology of disease, and to generate new therapeutic hypotheses for drug discovery. Technological advances in the last few years have enabled large-scale genome-wide association studies (GWAS) to identify common variants that modulate AD risk, and whole genome/whole exome sequencing to identify rare mutations associated with AD. This work has led to the discovery of more than 20 loci (in addition to APOE) that are causally linked to AD. Our systems- level analysis of genetic variants associated with AD in GWAS and sequencing studies implicate defective phagocytic clearance of cellular debris by myeloid cells (efferocytosis), as an important component of the etiology of AD as have analyses of gene regulatory networks in healthy and AD human brains by other investigators. Fine mapping of one of these GWAS loci led us to identify a common variant (rs1057233) in SPI1, which reduces SPI1 expression and risk for AD. Like ABCA7 and TREM2, two other established AD risk factors that play key roles in efferocytosis, SPI1 is expressed in immune cells of the myeloid lineage (e.g., monocytes, macrophages and microglia). SPI1 is a transcription factor (PU.1) that is critical for microglial development and regulates expression of many of the AD-associated genes implicated in efferocytosis. We hypothesize that modulation of SPI1 expression influences AD risk through global changes in gene expression within microglia that lead to altered efferocytosis. We will integrate computational and experimental approaches to define the mechanism(s) by which functional variation in SPI1 reduces SPI1 expression and risk for AD. To investigate this protective effect, we will first seek to replicate it in vitro using microglial cells derived from isogenic human iPSC cell lines with different rs1057233 genotypes (Aim 1). We will also genetically decrease/increase SPI1 expression in microglial cells of the mouse brain and measure the effect of these interventions on molecular, cellular and AD-related phenotypes in vivo (Aim 2 and 3 respectively). To enable these studies, we have recently developed a novel mouse model that can be used to profile the ribosome-bound transcriptome of microglial cells in the brain while also conditionally and specifically down- or up-regulating the expression of a gene of interest like SPI1 in microglia. Using the same model crossed with an AD mouse model, we will investigate AD-related outcomes like micro-gliosis and ß-amyloid deposition in the context of reduced or increased SPI1 expression in microglia.

Core F: Abstract One hundred years ago dementing illnesses were classified based upon their clinical presentation and neuropathology. The promise of the twenty first century is that we will be able to classify these same diseases by the genetic cause or genetic risk factors, a classification based upon etiology not symptomatology. During the last two decades many genes have been shown to cause autosomal dominant forms of early onset dementing illnesses. These rare disorders have provided enormous insight into the pathogenesis of more common variants of the same diseases. In 1993, a polymorphism in the apolipoprotein E (APOE) gene was identified as the first genetic risk factor for AD. A dose-dependent effect of the APOE4 allele has now become an important variable in all studies of AD. During the last five years genome-wide association studies and next generation sequencing studies have begun to identify many novel risk factors for AD. The goal of the Genetics and Genomics Core of the ISMMS ADRC is to provide genetic data and biospecimens on all ADRC participants. We will obtain longitudinal blood samples on ADRC participants. One blood sample will be sent to NCRAD, where it will be available to the entire community and will be included in national AD Genetics initiatives such as ongoing genome-wide association studies (GWAS) and whole genome/exome sequencing projects. The second tube will be retained locally and will be flow-sorted to generate specific blood cell populations. DNA and RNA will be generated from these specific cell types. Plasma and APOE genotype will also be available on all ADRC participants. Many participants will also have GWAS, exome array and/or whole exome/genome sequence data through national and international initiatives and ADRC affiliated projects. This data will be stored in the master ISMMS ADRC database and provided to investigators upon request. The Core will support projects and other cores as well as ADRC affiliated ageing and dementia projects. New in this application, we will begin to collect blood annually and isolate monocytes for future RNAseq/ DNA methylation studies to enable novel biomarker programs in peripheral tissues.

The National Institute of Aging Late Onset Alzheimer?s Disease Family Based Study (NIA-LOAD FBS) began in 2003, starting a trend of greater cooperation and sharing of clinical and biological resources among researchers. To date, a total of 1,454 multiplex late onset AD (LOAD) families have been recruited with 8,543 family members clinically assessed and DNA sampled. We have also recruited 1,030 controls. Genome-wide SNP arrays have been generated on 5,428 individuals, exome chip genotyping on 1,278 individuals, whole exome sequencing in 1,484 and whole genome in 928 family members and controls. The conversion rate of to LOAD among unaffected relatives in the NIA-LOAD FBS is three-fold higher than would be expected among individuals of similar age (see #67 Bibliography). All of these data have been placed in the public domain in NIAGADS and dbGaP. The NIA-LOAD FBS is widely used in Alzheimer disease genetics with 79 high level publications to support this claim (Bibliography). The NIA-LOAD FBS provides an excellent opportunity to improve our understanding of the clinical and biological impact of genetic variation in the elderly. Phenotypic information is continually updated in these families by regular cognitive evaluations and autopsy at the time of death to confirm the diagnosis of LOAD. We have begun to recruit additional family members with a particular emphasis on the offspring generation. We have been able to bank brain tissue from family members creating one of the largest collections of brain tissues for familial LOAD. We will now expand biological sampling to include RNA and peripheral blood mononuclear cells in selected families. As additional genes and variants are identified, the members of the NIA LOAD Family Study will again play a central role as we explore: What is the impact of these risk and protective variants on disease risk? Are the genetic variants highly penetrant? What is the risk of developing LOAD in offspring? Can the presence of variants be used for stratification of patients into specific subtypes for clinical trials? Can the family data be used to identify novel biomarkers of disease risk, age at onset onset or progression? The NIA-LOAD FBS dataset is uniquely poised to address these clinical and biological questions because of its large size, rigorous ascertainment criteria, standardized clinical assessment and lack of restriction to specific mutations. Our efforts have made it easy and seamless for the genetic data to be shared, allowing even more researchers to obtain the data and samples collected as part of the NIA-LOAD FBS for research studies. This is by far the largest collection of LOAD families available in the world. Virtually every major genetic study of Alzheimer?s disease has included patients and controls from the NIA-LOAD FBS dataset. The availability of dense phenotypic and genetic data will also position the NIA-LOAD FBS in to determine the impact of variants identified in whole genome and whole exome sequencing projects currently underway.

Project Summary Alzheimer's disease (AD) is the only disease among the top ten killers in the U.S. without a disease modifying therapy. Genetic studies provide a powerful means to identify genes and pathways that are causally linked to disease etiology. Technological advances have substantially reduced the cost of genomic analyses enabling the generation of large publicly available datasets that can be integrated to perform multi-scale analyses. Hypotheses generated from these data can then be validated in cell and animal models. A major problem encountered by genome-wide studies is power, particularly when searching for rare variants. One approach to this problem is to perform gene-based or gene-set-based analyses. Over the last three years it has become apparent that AD risk loci (both common and rare variants) are enriched for myeloid cell expressed genes, including APOE, TREM2, CD33, SORL1, ABCA7. Microglia are the resident phagocytic cells of the brain and share a common embryonic lineage with peripheral myeloid cells. We propose to use genomic and functional approaches to test the hypothesis that microglial function is modulated by AD risk and protective alleles in genes that are enriched within specific functional networks. This proposal will use publicly available whole genome/exome sequence data generated by the Alzheimer's Disease Sequencing Project (ADSP) and genome-wide association study (GWAS) data from the International Genomics of Alzheimer's Project (IGAP) and others together with gene expression data from purified macrophages and monocytes to identify myeloid expressed genes that carry rare or common variants that influence risk for AD (Aim 1). By integrating this data into co-expression networks in monocytes and macrophages we will determine whether AD loci lie within one or more regulatory networks (Aim 1). To validate these networks and determine the functional consequences of risk/protective alleles we will perform global transcriptomics and ATACseq in parallel with functional assays in microglial cells derived from isogenic human iPSC cell lines and mouse BV2 microglial cells, in which candidate gene expression is knocked-down or mutations are knock-in (Aim 2). Finally, we will use in vivo knock-down of gene expression specifically in adult microglia to test the physiological consequences of disrupting an AD-linked functional network (Aim 3). To enable these studies, we have developed a novel mouse model that can be used to profile the ribosome-bound transcriptome of microglial cells in the brain while also conditionally and specifically down-regulating the expression of a gene of interest like MS4A6A in microglia. Using the same model crossed with an AD mouse model, we will investigate AD-related outcomes like micro-gliosis and ß-amyloid deposition in the context of reduced MS4A6A expression in microglia. Together these studies will not only further our understanding of the genetic architecture of AD but also provide key information regarding the molecular mechanisms, setting the stage for novel therapeutic development.

Abstract Family-based approaches led to the identification of disease-causing Alzheimer?s Disease (AD) variants in the genes encoding amyloid-beta precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2). Subsequently, the identification of these genes led to the A?-cascade hypothesis and recently to the development of drugs that target that pathway. In this proposal, we will identify rare risk and protective alleles. In a recent study, we identified a rare coding variant in TREM2 with large effect size for risk for AD, confirming that rare coding variants play a role in the etiology of AD. We will use sequence data from families densely affected by AD, because we hypothesize that these families are enriched for genetic risk factors. We already have access to sequence data from 695 families (2,462 individuals), that combined with the ADSP data will lead to a very large family-based dataset: more than 805 families and 4,512 participants. Our preliminary results support the flexibility of this approach and strongly suggest that protective and risk variants with large effect size will be found. The identification of those variants and genes will lead to a better understanding of the biology of the disease.

Project Summary Alzheimer's disease (AD) is the only disease among the top ten killers in the U.S. without a disease modifying therapy. Genetic studies provide a powerful means to identify genes and pathways that are causally linked to disease etiology. Technological advances have substantially reduced the cost of genomic analyses enabling the generation of large publicly available datasets that can be integrated to perform multi-scale analyses. Hypotheses generated from these data can then be validated in cell and animal models. A major problem encountered by genome-wide studies is power, particularly when searching for rare variants. One approach to this problem is to perform gene-based or gene-set-based analyses. Over the last three years it has become apparent that AD risk loci (both common and rare variants) are enriched for myeloid cell expressed genes, including APOE, TREM2, CD33, SORL1, ABCA7. Microglia are the resident phagocytic cells of the brain and share a common embryonic lineage with peripheral myeloid cells. We propose to use genomic and functional approaches to test the hypothesis that microglial function is modulated by AD risk and protective alleles in genes that are enriched within specific functional networks. This proposal will use publicly available whole genome/exome sequence data generated by the Alzheimer's Disease Sequencing Project (ADSP) and genome-wide association study (GWAS) data from the International Genomics of Alzheimer's Project (IGAP) and others together with gene expression data from purified macrophages and monocytes to identify myeloid expressed genes that carry rare or common variants that influence risk for AD (Aim 1). By integrating this data into co-expression networks in monocytes and macrophages we will determine whether AD loci lie within one or more regulatory networks (Aim 1). To validate these networks and determine the functional consequences of risk/protective alleles we will perform global transcriptomics and ATACseq in parallel with functional assays in microglial cells derived from isogenic human iPSC cell lines and mouse BV2 microglial cells, in which candidate gene expression is knocked-down or mutations are knock-in (Aim 2). Finally, we will use in vivo knock-down of gene expression specifically in adult microglia to test the physiological consequences of disrupting an AD-linked functional network (Aim 3). To enable these studies, we have developed a novel mouse model that can be used to profile the ribosome-bound transcriptome of microglial cells in the brain while also conditionally and specifically down-regulating the expression of a gene of interest like MS4A6A in microglia. Using the same model crossed with an AD mouse model, we will investigate AD-related outcomes like micro-gliosis and ß-amyloid deposition in the context of reduced MS4A6A expression in microglia. Together these studies will not only further our understanding of the genetic architecture of AD but also provide key information regarding the molecular mechanisms, setting the stage for novel therapeutic development.

Mount Sinai ADRC (Sano): Genetics and Genomics Core (Core F) ? Research Summary One hundred years ago dementing illnesses were classified based upon their clinical presentation and neuropathology. The promise of the twenty first century is that we will be able to classify these same diseases by the genetic cause or genetic risk factors, a classification based upon etiology not symptomatology. During the last three decades many genes have been shown to cause autosomal dominant forms of early onset dementing illnesses. These rare disorders have provided enormous insight into the pathogenesis of more common variants of the same diseases. In 1993, a polymorphism in the apolipoprotein E (APOE) gene was identified as the first genetic risk factor for AD. A dose-dependent effect of the APOE4 allele has now become an important variable in all studies of AD. During the last ten years genome-wide association studies and next generation sequencing studies have begun to identify many novel risk factors for AD. The goal of the Genetics and Genomics Core of the ISMMS ADRC is to provide genetic data and biospecimens on all ADRC participants. We will obtain blood samples on ADRC participants. One blood sample will be sent to NCRAD, where it will be available to the entire community and will be included in national AD Genetics initiatives such as ongoing genome-wide association studies (GWAS) and whole genome/exome sequencing projects. The second tube will be retained locally. For DNA. Plasma and APOE genotype will also be available on all ADRC participants. Many participants will also have GWAS, exome array and/or whole exome/genome sequence data through national and international initiatives and ADRC affiliated projects. This data will be stored in the master ISMMS ADRC database and provided to investigators upon request. The Core will support projects and other cores as well as ADRC affiliated ageing and dementia projects. New in this application, we will begin to bank PBMCs and pilot plasma biomarker collection to enable novel biomarker programs in peripheral tissues.

Project Summary/Abstract Alzheimer's disease (AD) is a fatal neurodegenerative disease with a global prevalence close to 50 million people, which is expected to double by 2040. Finding an effective treatment for AD has proven difficult, as evidenced by numerous high profile Phase 3 clinical trial failures, most of which directly target the reduction of ß-amyloid. Thus, it is becoming increasingly urgent to develop new pharmacological strategies to combat AD. Drugs are twice as likely to successfully negotiate the drug development pipeline and obtain FDA approval when their targets are supported from human genetic studies of disease. Human genetic studies have revealed a critical role for microglia involvement in Alzheimer?s disease progression, and it has recently been discovered that the transcription factor PU.1 is a driver of the pro-neurodegenerative phenotype adopted by microglia during aging and disease. This proposal therefore aims to develop novel, newly-discovered PU.1 Inhibitory Modulators (PIMs) for preclinical development, with the long term goal of clinically testing the hypothesis that reducing PU.1 activity in microglia will safely delay the age of AD onset (AAO) in at-risk populations. The studies in this proposal leverage the interdisciplinary structure of the Neurodegeneration Consortium, a unique collaboration between basic science researchers and industry drug development veterans operating under a collaborative agreement to push forward novel therapeutics aimed at treating Alzheimer?s diseaes and other neurodegenerative diseases. Under Specific Aim 1, the in vivo safety and efficacy of PIMs will be determined in mouse models of AD. Under Specific Aim 2, parallel target engagement studies will be performed to identify the target of PIMs, and the identified targets will be used to develop assays to determine the efficacy of PIMs in AD and in ex vivo models. Under Specific Aim 3, selected PIMs will be optimized using PK/PD and ADMET screening to develop lead tool compounds into candidate compounds suitable for future Phase I studies. The combined biology, chemistry, and pharmacology expertise in the Neurodegeneration Consortium, spanning The University of Texas MD Anderson Cancer Center, the Massachussets Institute of Technology, and the Mt. Sinai School of Medicine, make this group of researchers ideally suited to execute the proposed aims.

PROJECT SUMMARY (PROJECT 1) Genetic association between the MAPT H1 haplotype and increased risk for multiple Tauopathies, including Frontotemporal Dementia (FTD) and Progressive Supranuclear Palsy (PSP), is well-established and replicated. Despite this, very little is known regarding the mechanisms behind the differences in disease risk associated with the H1 and H2 haplotypes. To date, comparison of these haplotypes has been restricted to studies of MAPT splicing, as well as associations with clinical phenotypes. However, there has been no investigation of the downstream functional implications of each MAPT haplotype. Our goal is to comprehensively assess the chromatin structure, gene expression and functional consequences of structural differences between the H1 and H2 haplotypes in cells from individuals of European and African ancestry, in order to better understand the mechanisms underlying risk for Tauopathy. The major MAPT haplotypes encompass a 970Kb inversion within the 17q21.31 locus and include many genes in addition to MAPT, as well as extensive genetic and structural variation. We hypothesize that the gross structural differences between haplotypes confer short- and long-range changes in chromatin structure, leading to altered regulation of gene expression within and outside the inversion, and that these changes in neurons and/or glia contribute to risk/protection for tauopathies. We will use 2D and 3D induced pluripotent stem cell (iPSC) models to examine multi-OMIC differences between H1/H1 and H2/H2 and human brain tissue derived from H1/H1 and H2/H2 carriers to validate these changes, followed by CRISPR-based functional genomic screens to test the impact of candidate causal variants on gene expression and function. We propose four specific aims: 1) Use single nuc sequencing, ISOseq and proteomics to characterize chromatin structure, gene/protein expression and splicing in human brain tissue from H1/H1 and H2/H2 carriers, 2) Use RNAseq, HiC, ATACseq, ISOseq and proteomics to characterize cell-autonomous changes in chromatin structure, gene expression and splicing in 2D neuron, astrocyte and microglial cultures from H1/H1 and H2/H2 individuals; 3) Use single cell ATAC/RNAseq to examine effects on gene expression in 3D assembloids from H1/H1 and H2/H2 individuals; 4) Use CRISPRa/i-based assays to test the functional impact of altering haplotype- and cell-specific gene enhancer regions on global gene expression and Tau- isoform expression and splicing. Understanding the mechanisms underlying the protective effects of the H2 haplotype has the potential to uncover new therapeutic strategies for FTD and other neurodegenerative diseases.

PROJECT SUMMARY (APOE U19 Project 5) The most important Alzheimer?s disease (AD) risk gene is apolipoprotein E (APOE). APOE4 is associated with a dose dependent increase in risk, while APOE2 is associated with a dose dependent decrease in risk and delayed age at onset (AAO), relative to APOE3/3. Population-based studies have demonstrated that APOE4/4 homozygotes have ~60% life-time risk for AD by age 85 yrs compared to ~10% in APOE3/3 homozygotes. Despite this strong effect on AD susceptibility, there is huge variation (several decades) in AAO even within a single APOE genotype. We hypothesize that in human populations there are both risk and protective alleles in genes that modify AAO within a single APOE genotype by acting within the same cascade of events that modify AD risk and AAO downstream of APOE (see Overall section for detailed description of the ApoE Cascade Hypothesis). In this project we will analyze publicly available data to identify genes that modify AD risk in APOE4 carriers and APOE3/3 homozygotes. Preliminary analyses have identified rare TREM2 variants as modifiers of AAO in APOE3/3 homozygotes and rare EIF2B3 variants as modifiers of AAO in APOE4 carriers. ApoE is a secreted apolipoprotein that is primarily expressed in astrocytes. However, APOE expression is highly upregulated in subpopulations of microglia and other macrophages, particularly in response to tissue damage and lipid overload in the aged or diseased brain, in a manner that is at least in part dependent on TREM2. This is of particular relevance to AD because genetic studies have demonstrated that common risk alleles are specifically enriched in myeloid/microglial enhancers, suggesting that the effects of APOE genotype and its genetic modifiers on AD risk and AAO may be mediated by their effects on microglial cell function. We hypothesize that APOE genotype and its genetic modifiers alter AD risk/AAO through modulation of microglial cell function, particularly in response to brain tissue damage in aging and disease. To address this hypothesis we will use isogenic hiPSC-derived microglia (iMGL) to assess the impact of APOE genotype and its genetic modifiers (TREM2 R47H & EIF2B3 S404A) on microglial cell function in vitro and transplantation of isogenic hiPSC-derived hematopoietic progenitor cells (iHPCs) into mice to assess the impact of APOE genotype and its genetic modifiers (TREM2 R47H & EIF2B3 S404A) on microglial cell function in AD mouse brains. This project will be performed in collaboration with Cores C, E and G and addresses aims 2, 4 and 6 of the overall U19 project. Project 5 will identify novel modifiers of AAO of AD in the context of different APOE risk backgrounds and determine the molecular consequences of these novel risk genes on microglial function in vitro and in vivo. In so doing, Project 5, together with Projects 2-4, will provide insight into ApoE function in microglia leading to the identification of novel therapeutic targets for AD and generation of unique resources/data to be shared with the scientific community through Core A.

PROJECT SUMMARY (CORE A: ADMINISTRATIVE CORE) The purpose of the Administrative Core (Core A) is to ensure execution of our mission to conduct innovative, interdisciplinary research to identify and validate the molecular mechanisms contributing to tauopathy risk/protection associated with the H1/H2 haplotypes of the 17q21.31 region. The Center involves 3 geographically distributed sites in New York (Icahn School of Medicine at Mount Sinai), Los Angeles (UCLA) and San Francisco (UCSF). Interactions will occur at two levels: scientific (exchange of information and data, sharing of resources and specialized personnel) and administrative (organization of meetings and interactions within and outside the CWOW). The primary goal of Core A is to connect these physically separate sites by serving as a hub to facilitate interaction and communication between Core and Project leaders, investigators, and external scientific communities, and ensure efficient governance and oversight of the Center. This Core will ensure optimal utilization of center resources through maximization of institutional strengths and national and global opportunities to broaden knowledge about the molecular mechanisms underlying risk/protection for sporadic and familial tauopathy associated with H1/H2 haplotypes. This core is responsible for articulating the research agenda and ensuring that it is effectively accomplished. Core A will accomplish this through a structure that includes an Executive Steering Committee (ESC) led by the Director (Dr. Alison Goate) and the two associate directors (Drs. Geschwind and Kampmann) as well as Core leaders. The ESC will insure that the CWOW, national and international FTD resources are leveraged to the advantage of the CWOW and those of the wider FTD community. An External Advisory Committee will provide guidance and review to the CWOW leadership and communicate with NINDS Program Staff. The leadership will assure that the Center is aware of national and international commitments as well as of opportunities to maximize our effectiveness. In this capacity specific responsibilities include financial, administrative and regulatory management. This Core will also oversee the growth of early stage investigators within the center. We propose two topically related Projects supported by three Research Cores that leverage cutting-edge proteomic and transcriptomic approaches in brain tissue and induced pluripotent stem cell derived neurons and glia to determine the mechanisms contributing to tauopathy risk/protection associated with the H1/H2 haplotypes of the 17q21.31 region. The Data Core (Core D) will serve as a hub for integrating all data produced by Projects and Cores to be shared within and outside the CWOW. Core A will organize regular meetings to ensure progress, open communication and resources are available to serve Project and Core goals, that Projects and Cores have maximal opportunity to interact, and that Projects and Cores progress according to timelines and meet benchmarks of accomplishment. Core A and the Data Core will work together to develop and maintain a Center website.

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.

PROJECT SUMMARY (APOE U19: OVERALL) The overarching goal of this U19 project is to comprehensively understand the biology and pathobiology of apolipoprotein E (apoE) in aging and Alzheimer?s disease (AD) to inform therapeutic strategies. The ?4 allele of the APOE gene (APOE4) is the strongest genetic risk factor for AD impacting 50-70% of all AD patients, while the ?2 allele is protective compared to the common ?3 allele. APOE4 is also a strong risk factor for age-related cognitive decline and vascular cognitive impairment. To integrate existing knowledge and address critical gaps, we propose a unified ApoE Cascade Hypothesis that the structural differences and related biochemical properties among the three apoE isoforms initiate their differential effects on a cascade of events at the cellular and systems levels ultimately impacting aging-related pathogenic conditions including AD. Towards this, we have assembled a multi-disciplinary team to synergize expertise and resources across multiple institutions. By integrating five interactive Projects and seven robust Cores, we will create a nexus for apoE-related aging research, sharing the knowledge, expertise and resources with the broader scientific community. Project 1 will work closely with Core B to address the structural and biochemical properties of the three apoE isoforms to generate insights for functional outcomes. Projects 2, 3 and 4 will interactively study how apoE isoforms expressed in astrocytes, microglia, or vascular mural cells impact lipid metabolism, glial and vascular functions, AD-related pathologies, and cellular and molecular pathways using conditional mouse models and systems- based approaches. These studies will generate cell type-specific apoE/lipoprotein particles that will be collected through in vivo microdialysis for structural and biochemical studies. Project 5 will carry out genomic and genetic analyses to identify modifiers of APOE-related age at onset of AD. Studies in Projects 2-5 will be interactively supplemented by neuropathological studies using postmortem brains from healthy aging studies or with AD pathologies (Core C), biomarker studies using both human and mouse biospecimens (Core D), and functional studies using human iPSC-derived cellular and organoid models (Core E). This U19 proposal is supported by a comprehensive Multi-Omics Core (Core F) for centralized proteomics, lipidomics, and metabolomics studies on various animal and iPSC models, as well as human postmortem brains and fluid biospecimens. The Bioinformatics, Biostatistics, and Data Management Core (Core G) will provide critical supports for analyzing large datasets including those from single-cell RNA-seq and biostatistics supports to ensure scientific rigor. Core G will also work closely with the Administrative Core (Core A) to maintain an ApoE Web Portal designated as EPAAD where knowledge, resources, and data will be shared with the scientific community. Core A will also organize annual ApoE Symposium to promote collaboration and engage the ApoE Community. As such, this U19 will drive a team-based effort to generate essential knowledge to guide disease- modifying therapies for AD and other aging-related conditions.