2000 — 2003 |
Huang, Yadong |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Apoe Expression Level Modulates Vldl Metabolism @ J. David Gladstone Institutes
Apolipoprotein (apo) E serves as a ligand to mediate the clearance of triglyceride-rich lipoproteins, such as very low density lipoproteins (VLDL) and their remnants, from circulation. Paradoxically, however, we have recently found that overexpression of apoE3 causes hypertriglyceridemia in transgenic mice or combined hyperlipidemia in transgenic rabbits by directly stimulating hepatic VLDL production and by impairing VLDL lipolysis. The goal of this proposal is to investigate the molecular mechanisms by which apoE expression levels determine VLDL metabolism. The first aim is to establish quantitatively the effects of increasing apoE expression levels on VLDL production, lipolysis, and clearance in mice. We will test the hypothesis that all three effects of apoE on VLDL metabolism are dependent on apoE expression levels. We will determine the range of apoE levels that support normal VLDL and remnant lipoprotein clearance and that inhibit lipolytic processing by increasing plasma apoE levels from extrahepatic sources. We will also determine the effect of increasing hepatic apoE synthesis on the induction of VLDL production. The second aim is to investigate the cellular and molecular mechanisms responsible for the stimulation of hepatic VLDL production by apoE. Hepatic VLDL production appears to involve two steps and different regulatory factors. We will determine the effects of apoE on each step and on several regulatory factors in transfected rat hepatoma (McA-RH7777) cells expressing different levels of human apoE. We will address the following three major questions: Does apoE stimulate triglyceride synthesis in the cells? Does apoE transport the newly synthesized triglycerides within the cells? Does apoE colocalize with apoB in the McA-RH7777 cells? Recently, three new polymorphisms (-491A/T, -427T/C, and -219T/G) have been found in the transcriptional regulatory region of the apoE gene. The third aim is to test the hypothesis that the apoE promoter polymorphisms may affect plasma levels of apoE and VLDL metabolism in humans. We will determine the allelic frequency of apoE promoter polymorphisms in normolipidemic humans by using restriction fragment length polymorphism analysis and the effect of apoE promoter polymorphisms on plasma levels of apoE and lipoproteins in normolipidemic and hyperlipidemic subjects.
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0.904 |
2004 |
Huang, Yadong |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Apolipoprotein Synthesis @ J. David Gladstone Institutes
apolipoproteins; blood lipoprotein biosynthesis; genetic regulatory element; nucleic acid sequence; microglia; astrocytes; liver; disease /disorder model; glia; transcription factor; macrophage; cholesterol; chromatin; gene expression; genetic transcription; RNase protection assay; tissue /cell culture; laboratory mouse; genetically modified animals; transfection; complementary DNA; in situ hybridization; immunocytochemistry; DNA footprinting; affinity chromatography;
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0.904 |
2007 |
Huang, Yadong |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Tissue Culture @ J. David Gladstone Institutes
The tissue culture core (Core B) will serve all projects in this program. Many studies proposed in these projects will require cultured neuronal cell lines or primary neurons. The personnel of Core B has considerable experience in growing and maintaining many different cell lines and strains. Our cell culture inventory consists of over 100 hybridoma cell lines that produce monoclonal antibodies to apolipoproteins, lipoprotein receptors, and other proteins, and about 50 cell lines that have been transfected with constructs that express different isoforms of apolipoprotein E, amyloid precursor protein, tau, or huntingtin or variants of each of these proteins. Core B will prepare the media and reagents used in culture studies, provide and maintain the equipment required for the preparation and growth of neuronal cultures, assist in transfection of different cell lines (e.g., Neuro-2a, B 103, C6, U87, HEK293T, and COS) with DNA constructs, perform the assay for quality control of recombinant apoE preparations, and assist in culturing primary neurons. The smooth operation of this core and the support it will provide for the proposed tissue culture studies is of fundamental importance to the successful outcome of this program project.
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0.904 |
2008 — 2012 |
Huang, Yadong |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Tissue Culture Core @ J. David Gladstone Institutes
APOE [{C0003595}]; Amyloid A4 Protein Precursor; Amyloid Protein Precursor; Amyloid beta-Protein Precursor; Apo-E; ApoE; Apolipoprotein E; Apolipoproteins; California; Cell Line; Cell Lines, Strains; CellLine; Cells; Cultured Cells; Equipment; Equipment and supply inventories; Glia; Glial Cells; HD protein, human; Huntingtin; Huntingtin Protein; Huntingtin protein, human; Huntington disease protein, human; Huntington's disease gene product; Hybridomas; IT15 protein, human; Inventory; Investigators; Isoforms; Kolliker's reticulum; Lentivirinae; Lentivirus; Lipoprotein Receptor; Liquid substance; Moab, Clinical Treatment; Monoclonal Antibodies; N element; N2 element; Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; Nitrogen; Non-neuronal cell; Operation; Operative Procedures; Operative Surgical Procedures; Outcome; Postdoc; Postdoctoral Fellow; Production; Programs (PT); Programs [Publication Type]; Protein Isoforms; Proteins; Reagent; Research Associate; Research Personnel; Researchers; Subfamily lentivirinae; Surgical; Surgical Interventions; Surgical Procedure; Transfection; Universities; Variant; Variation; Virus-Lenti; Work; amyloid precursor protein; cell type; cultured cell line; experience; fluid; gene product; human Huntingtin protein; liquid; monoclonal antibody production; nerve cement; neuronal; post-doc; post-doctoral; programs; surgery; tissue culture
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0.904 |
2008 — 2012 |
Huang, Yadong |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Apolipoprotein E in Alzheimer's Disease: Cellular Mechanisms @ J. David Gladstone Institutes
Apolipoprotein (apo) E4 is a major risk factor or susceptibility gene for Alzheimer's disease (AD). Although the pathogenic mechanisms are unclear, our findings during the preceding funding period, and findings reported by others, suggest that apoE4[unreadable]with its multiple cellular origins and multiple structural and biophysical properties[unreadable]contributes to AD by interacting with different factors through various pathways, some of which are amyloid-(3 (A|3) dependent and others are not. Although the A|3-dependent roles of apoE4 in AD pathogenesis have been widely studied and much valuable information generated, the A|3-independent roles of apoE4[unreadable]the focus of the current proposal[unreadable]have drawn less attention and have been understudied. In the central nervous system (CNS), apoE is produced by several types of cells, including astrocytes, activated microglia, and injured neurons. Emerging evidence suggests that neuron-generated apoE and astroycte-generated apoE have distinct roles in physiological and pathophysiological pathways, including AD pathogenesis. Thus, understanding how apoE expression is regulated in CNS neurons should provide fundamental insights into the varied effects of apoE isoforms in neurobiology and neurodegeneration. This proposal builds on four findings during the preceding funding period. First, CNS neurons express apoE in response to injury. Second, neuronal expression of apoE after injury is regulated by an astrocytederived factor (or factors) that controls intron-3 retention/splicing of the apoE gene. Third, apoE4 is more susceptible than apoES to neuron-specific proteolysis, and the resulting fragments cause AD-like neurodegeneration and behavioral deficits in transgenic mice. Fourth, in transgenic mice, pan-neuronal expression of apoE4 or its fragment causes learning and memory deficits and early neuronal deficits in the entorhinal cortex, subiculum, and hippocampus, suggesting selective vulnerability of specific CNS neurons. The goal of this project is to study the regulation of apoE expression in CNS neurons and to explore Apindependent, isoform-specific roles of apoE in the pathogenesis of AD. Specifically, we will explore the regulation of apoE expression in CNS neurons (Aim 1);determine if inhibition of proteolysis reduces or abolishes apoE4-related detrimental effects in transgenic mice (Aim 2);and explore the mechanisms underlying the selective vulnerability of different types of CNS neurons to apoE4 and its fragments (Aim 3). These studies, involving both in vitro and in vivo approaches, will provide insights into the regulation and role of apoE4 in both health and disease and may identify new therapeutic targets for apoE4-associated neurodegenerative disorders, particularly AD.
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0.904 |
2013 — 2016 |
Gao, Fen-Biao (co-PI) [⬀] Huang, Yadong Miller, Bruce L (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Frontotemporal Dementia Induced Pluripotent Stem Cell Consortium @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): We propose to establish a comprehensive, validated repository of adult human dermal fibroblasts and human induced pluripotent stem cell (hiPSC) lines from frontotemporal dementia (FTD) patients with genetically defined mutations and familial, non-mutation carrying controls. hiPSCs hold tremendous promise for the development of in vitro FTD models for studying disease pathogenesis in relevant human cell types that would otherwise be impossible to obtain, such as human neurons. Using an established, collaborative, multi- institutional approach, we will bank adult human dermal fibroblasts from FTD patients carrying common mutations in the genes currently known to cause FTD: tau (MAPT), C9ORF72, and progranulin (GRN). In Aim 1, we will recruit both FTD patients with defined genetic mutations and control subjects. Comprehensive and longitudinal clinical evaluations will be linked to each cell line, allowing us to correlate disease characteristics with molecular phenotypes. In Aim 2, we will reprogram fibroblasts into hiPSCs by non-DNA-integrating technologies with which we have had recent success. In addition, we will further create EGFP reporter lines for monitoring and standardizing differentiation protocols in FTD-relevant cell types such as forebrain neurons. We will also correct selective mutations to create isogenic control lines so that we can precisely differentiate mutation-specific phenotypes from the noise of inter-individual variability. In Aim 3, we will derive and validate human neurons to model and study FTD pathogenesis in culture and to deliver hiPSC lines with robust phenotypes for FTD research and drug development. Based on our previous research experience in RNA and Tau biology and pathophysiology, we will focus on human neurons with GGGGCC repeat expansions in C9ORF72 and MAPT mutations. All cell lines will be banked at the Coriell Institute and will be accessible to the worldwide FTD research and drug development community. These resources should significantly alter the FTD research landscape by accelerating discovery.
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0.904 |
2014 |
Huang, Yadong Kreitzer, Anatol |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Optogenetic Study of Apoe4-Related Alzheimers Disease @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): The complexity and multifactorial nature of Alzheimer's disease (AD) pose unique challenges for the development of effective therapies. Efforts to target specific AD-related pathways have shown promise in animal studies, only to fail during human trials. There is a pressing need to identify novel therapeutic targets for AD. Apolipoprotein (apo) E4 increases the risk and lowers the age of onset for developing AD in a gene dose- dependent manner. In most clinical studies, apoE4 carriers account for 65-80% of all AD cases, highlighting the importance of apoE4 in AD pathogenesis. Emerging evidence from animal and clinical studies suggest that targeting some of the apoE4's detrimental effects for therapeutic intervention could be useful for treating AD. However, for many of these targets, a systemic validation is needed before further development. Mounting evidence from our lab suggests that hippocampal GABAergic neurotransmission is one such promising target for intervention in the setting of apoE4-positive AD. First, we found that expression of apoE4 in knock-in (KI) mice causes age-dependent impairment of GABAergic interneurons in the hilus of the hippocampus, which correlates with the extent of learning and memory deficits. Second, optogenetic inhibition of hilar GABAergic interneuron activity impairs spatial learning and memory in mice, indicating that hilar GABAergic interneuron impairment can directly cause cognitive deficits. Third, the GABAA receptor potentiator pentobarbital rescues the learning and memory deficits in apoE4-KI mice. Fourth, apoE4 impairs GABAergic interneurons in human iPSC-derived neuronal cultures and in the hippocampal hilus in AD patients. Fifth, hilar transplantation of mouse medial ganglionic eminence (MGE)-derived GABAergic interneuron progenitors restores normal learning and memory in apoE4-KI mice without or with A¿ accumulation. These results strongly implicate apoE4 in hilar GABAergic interneuron impairment, leading to learning and memory deficits, which could be a therapeutic target for AD. There are different subtypes of GABAergic interneurons in the hippocampal hilus, including those positive for somatostatin, parvalbumin, or neuropeptide Y. Using transgenic Cre driver lines, we will selectively express light-sensitive optogenetic proteins, including channelrhodopsin-2, halorhodopsin, and ArchaerhodopsinT, in these cells to control their activity and thus determine their contributions to normal learning and memory and to apoE4-induced learning and memory deficits in mice. Specifically, we propose to validate the application of optogenetic tools for the manipulation of different hilar GABAergic interneuron subtypes ex vivo and in vivo (Aim 1); to determine the contribution of different subtypes of hilar GABAergic interneurons to apoE4-induced learning and memory deficits using optogenetic tools (Aim 2); and to determine the underlying mechanisms by which hilar transplantation of mouse MGE-derived GABAergic interneuron progenitors restores normal learning and memory in aged apoE4-KI mice using optogenetic tools (Aim 3).
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0.904 |
2014 |
Huang, Yadong |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Studying Alzheimer's Disease Using Human Ips Cells @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease (AD). Remarkably, the lifetime risk estimate of developing AD for individuals with two copies of the apoE4 allele is ~70% by the age of 85. By comparison, the lifetime AD risk estimate for individuals with two copies of the apoE3 allele is ~10% by the age of 85. Although many apoE4 homozygotes do go on to develop early-onset AD (EOAD, <65 years) or late-onset AD (LOAD, >65 years), some of them (~20%) stay asymptomatic over age 85. Understanding the susceptibility of the former apoE4 group and the resistance of the latter apoE4 group to AD might allow for the development of strategies to prevent or delay the disease in people at risk for AD. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles (tauopathy). A major difficulty of studying AD-related tauopathy is that overexpression of wild-type (WT) tau in mice or cultured cells does not lead to tauopathy. Thus, almost all published studies used mice or cells overexpressing tau with different mutations to model tauopathy in AD. However, all those tau mutations cause frontotemporal dementia (FTD) but not AD, raising concerns on their mechanistic accuracy. Until recently, tau-A152T polymorphism was identified, which is associated with increased risk for both FTD and AD. Thus, studying tau-A152T-related pathophysiology should shed light on the pathogenesis of tauopathy in both AD and FTD. While animal models and post-mortem tissues have provided key insights into the pathogenesis of AD, they also pose strategic limitations. Induced pluripotent stem cells (iPSCs) derived from human somatic cells with AD-linked mutations or polymorphisms, together with genome-editing techniques, hold great promise as in vitro models for studying disease pathogenesis in relevant cell types, including human neurons. This proposal aims to capitalize on this promise by building on our preliminary studies showing that: (1) AD patient iPSC- derived neurons with an apoE4/4 genotype developed AD-related phenotypes in culture, including increased A¿ levels and elevated tau phosphorylation, compared to apoE3/3-iPSC-derived neurons; and (2) iPSC- derived neurons with tau-A152T developed tauopathy due to increased tau fragmentation. Specifically, we will determine the phenotypic differences among human neurons derived from apoE4/4- iPSC lines from EOAD patients, LOAD patients, and asymptomatic controls <65 and >85 years of age (Aim 1); explore the transcriptomic and proteomic differences among human neurons derived from the isogenic iPSC lines with an apoE3/3 or apoE4/4 genotype (Aim 2); and determine the contribution of tau fragmentation to the pathogenesis of tauopathy in AD versus FTD (Aim 3). These studies, using human neurons and astrocytes derived from isogenic iPSC lines, will likely identify genetic factors capable of modifying apoE4's detrimental effects in AD pathogenesis and may discover new therapeutic targets for apoE4-associated AD. These studies should also provide insight into the pathogenic differences of tauopathies in AD versus FTD.
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0.904 |
2014 — 2018 |
Huang, Yadong |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Project 3: Using Human Ips Cells to Study the Pathogenesis of Ad and Ftd @ University of California, San Francisco
PROJECT 3 - SUMMARY/ABSTRACT Alzheimer's disease (AD) is a complex neurodegenerative disorder caused by interactions among multiple genetic and environmental factors. Apolipoprotein (apo) E4 increases the risk and lowers the age of onset for AD in a gene dose-dependent manner. Remarkably, the lifetime risk estimate of developing AD for individuals with 2 copies of the apoE4 allele (~2% of the population) is ~70% by the age of 85. By comparison, the lifetime AD risk estimate for individuals with two copies of the apoE3 allele is ~10% by the age of 85. Although many of the apoE4 homozygotes develop early-onset AD (EOAD) or late-onset AD (LOAD), some of them (~20%) stay asymptomatic over age 85. Understanding the susceptibility of the former group and the resistance of the latter group to AD might allow for the development of strategies to prevent or delay AD in people at risk. Like AD, frontotemporal dementia (FTD) is also a multifactorial and heterogeneous neurodegenerative disorder. About 20-50% of FTD cases are inherited, and heterozygous mutations in the progranulin (PGRN) gene are one of the most common causes of the inherited forms of FTD. However, homozygous PGRN mutations unexpectedly cause neuronal ceroid lipofuscinosis (NCL) rather than FTD. Understanding how the same mutation within a single PGRN gene causes different phenotypes depending on gene dose is crucial for unraveling the pathogenesis of both FTD and NCL and for their therapeutic developments. Because neurons cannot be obtained directly from patients, induced pluripotent stem cells (iPSCs) derived from AD or FTD patients hold great promise as in vitro models for studying disease pathogenesis in human neurons. This proposal aims to capitalize on this promise by building on our previous efforts to generate a well- characterized repository of human iPSC (hiPSC) lines from AD and FTD patients and, by using them, to study disease phenotypes, reveal novel mechanisms, and identify new therapeutic targets. The goals of this project are to address the following two questions: (1) why are many apoE4 homozygotes vulnerable to AD pathogenesis at young ages when others (~20%) can stay asymptomatic by age 85 and over? (2) How do heterozygous and homozygous mutations within a single PGRN gene cause different clinical phenotypes, i.e., FTD and NCL, respectively? To answer these questions, we will compare the phenotypic differences (e.g., A? production/secretion and tau phosphorylation/fragmentation) among neurons derived from apoE4/4-hiPSC lines from EOAD patients, LOAD patients, and asymptomatic controls at different ages (Aim 1); determine the transcriptomic and proteomic differences among neurons derived from apoE4/4-hiPSC lines from EOAD patients, LOAD patients, and asymptomatic controls at different ages (Aim 2); and explore the underlying mechanisms by which heterozygous PGRN mutations cause FTD but homozygous PGRN mutations cause NCL (Aim 3). These studies should significantly accelerate AD and FTD research and related therapeutic development by enhancing our understanding of disease pathogenesis at the molecular and cellular levels.
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1 |
2015 — 2019 |
Huang, Yadong |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Apolipoprotein E in Alzheimer's Disease: Molecular Mechanism @ J. David Gladstone Institutes
? DESCRIPTION (provided by applicant): Apolipoprotein (apo) E4 increases the risk and lowers the age of onset for Alzheimer's disease (AD) in a gene dose-dependent manner. In most clinical studies, apoE4 carriers account for 60-75% of all AD cases, highlighting the importance of apoE4 in AD pathogenesis. Remarkably, the lifetime risk (LTR) estimate of developing AD by the age of 85 is ~60-70% for people with two copies of the apoE4 allele (~2% of the population) and ~30% for those with one copy of the apoE4 allele (~25% of the population), but is only ~10% in those with two copies of the apoE3 allele. Thus, apoE4 is considered a major gene, with semi-dominant inheritance, for AD. Although many hypotheses have been advanced, the exact mechanisms underlying the pathogenic actions of apoE4 remain unclear. This proposal builds on four novel findings from our studies of mouse models or human induced pluripotent stem cell (hiPSC)-derived neurons expressing different apoE isoforms. First, in apoE4-KI mice and mice expressing a neurotoxic apoE4 fragment, apoE4 and its fragment cause age- and tau-dependent impairment of GABAergic interneurons in the hilus of the hippocampus, and the extent of the impairment correlates with the extent of learning and memory deficits. Second, this apoE4-induced interneuron impairment also impairs adult hippocampal neurogenesis in mice. Third, the GABAA receptor potentiator pentobarbital rescues both the impaired neurogenesis and the deficits in learning and memory in apoE4-KI mice and apoE4 fragment transgenic mice. Fourth, apoE4 displays increased proteolysis and causes GABAergic interneuron death in hiPSC-derived neuronal cultures. These findings strongly suggest that apoE4 and its neurotoxic fragments cause hilar GABAergic interneuron impairment, contributing to learning and memory deficits and AD pathogenesis. The goal of this project is to determine how apoE4 impairs hilar GABAergic interneurons and how this process leads to spatial learning and memory deficits. Specifically, we will determine whether and how apoE4-induced impairments of GABAergic interneurons in the hilus of the dentate gyrus contribute to learning and memory deficits (Aim 1); explore the mechanisms by which apoE4 impairs hilar GABAergic interneurons (Aim 2); and determine whether and how apoE4 impairs human GABAergic interneurons derived from isogenic hiPSC lines with different apoE genotypes (Aim 3). These studies, involving in vitro, in vivo, and hiPSC approaches, will provide insights into the roles of apoE4 in both health and disease and may identify new therapeutic targets for apoE4-associated neurodegenerative disorders, particularly AD.
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0.904 |
2017 — 2021 |
Huang, Yadong Shi, Yanhong [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Modeling Alzheimer?S Disease Using Directly Reprogrammed Isogenic Neural Cells @ Beckman Research Institute/City of Hope
Project Summary Alzheimer's disease (AD) is the most common neurodegenerative disorder and a leading cause of disability and death. However, the precise mechanisms underlying AD pathogenesis remains to be elucidated. Although many transgenic mouse models have been generated for AD research and these models are important for our understanding of the pathological basis of the disease, none has captured the entire spectrum of the disease pathology, including considerable neuronal loss. This is likely due to significant species differences between mouse and human neural cells. Therefore, there is an urgent need to establish human disease modeling platforms to complement studies in animal models for AD research and drug development. Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, human iPSCs (hiPSCs) have been widely used for disease modeling and drug discovery. However, given the relative immaturity of cells differentiated from hiPSC, it is challenging to use them to model late-onset diseases, for which cellular aging is important in disease pathology. Direct reprogramming is an alternative cellular reprogramming technology, which allows direct conversion of one type of somatic cells, such as fibroblasts, into another type of somatic cells, such as neurons. It has been shown that direct reprogramming enables generation of human neurons that possess key elements of cellular aging, because this reprogramming process does not go through the rejuvenating iPSC stage. The objective of this proposal is to develop aging-relevant cellular models of late-onset AD (LOAD), using direct reprogramming technology in combination with CRISPR/Cas9-mediated gene editing and 3D neural culture, in order to recapitulate the age-associated phenotypes and uncover novel pathological mechanisms of LOAD. We propose to establish cellular models of LOAD using both neurons and astrocytes directly reprogrammed from patient fibroblasts or differentiated from induced neural stem cells (iNSCs) obtained through direct reprogramming. While the strongest risk factor for AD is aging, the strongest genetic risk factor of AD is apolipoprotein (apo) E4. We hypothesize that cellular aging and apoE genotype interact with each other to initiate and/or modulate LOAD pathologies. Accordingly, we propose three complementary aims to test this hypothesis. Aim 1: To generate isogenic human fibroblast lines with different apoE genotypes as cell sources for direct reprogramming. Aim 2: To model LOAD using directly reprogrammed human neurons and astrocytes. Aim 3: To model LOAD using directly reprogrammed NSC-derived neurons and astrocytes. The outcomes of the proposed studies will likely help to further define the roles of apoE4 in the development of age-associated AD pathological features, to uncover novel mechanisms for age and apoE4-related AD pathogenesis, and to design novel therapeutic strategies for AD.
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0.913 |
2017 |
Huang, Yadong |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Roles of Apoe4 in Brain Aging and Alzheimer's Disease @ J. David Gladstone Institutes
PROJECT SUMMARY The complexity and multifactorial nature of Alzheimer's disease (AD) pose unique challenges for mechanistic studies and developing therapies. Age is the number one risk factor for AD, with imaging and biomarker data suggesting that the pathophysiological processes of AD begin more than a decade prior to the clinical diagnosis of dementia. Apolipoprotein (apo) E4 is the major genetic risk factor for AD, which also lowers the age of onset of AD in a gene dose-dependent manner. In most clinical studies, apoE4 carriers account for 60? 75% of all AD cases, highlighting the importance of apoE4 in AD pathogenesis. Longitudinal studies in humans demonstrate that apoE4's detrimental effect on cognition depends on age and occurs before typical signs of AD arise. A challenge in AD research is to fully understand how the multiple etiologies, including apoE4, and age-related prodromal processes interactively contribute to the pathophysiology of AD. This proposal builds on three novel findings from our recent studies of mouse models. First, expression of apoE4 in female knock-in (KI) mice causes age-dependent impairment of GABAergic interneurons in the hilus of the hippocampus, which correlates with the severity of learning and memory deficits. Second, in vivo local field potential (LFP) recordings throughout the hippocampal circuit shows that compared to aged female apoE3-KI mice, aged female apoE4-KI mice have fewer sharp wave ripple (SWR) events?hippocampal network events critical for memory replay and consolidation?and have significantly reduced slow gamma activity during SWRs (see Preliminary Studies), which coordinates SWRs. Third, although most female apoE4- KI mice develop learning and memory deficits by 20 months of age, some of them (~25%) remain cognitively normal, similar to the proportion of apoE4/4 carriers who do not develop AD by 85 years of age. Understanding the neuronal and network mechanisms that differentiate the susceptible (pathological aging) and resistant (normal aging) groups of apoE4-KI mice might allow for the development of strategies to prevent or delay the disease in people at risk for AD. This proposal aims (1) to determine the effects of aging on apoE4 disruption of hippocampal network activity underlying memory replay and its relationship with GABAergic interneuron loss and cognitive decline in mice, (2) to explore whether apoE4 disruption of hippocampal network activity underlying memory replay occurs early and predicts the conversion of normal aging to AD-related pathological aging late in life in mice, and (3) to determine whether, during aging, persistent normal hippocampal network activity underlying memory replay predicts tenaciously normal cognition (normal aging) in apoE4-KI mice. The outcomes of the proposed studies will shed light on the interactive roles of apoE4 and aging in AD pathogenesis and could identify functional biomarkers capable of predicting the conversion of normal aging to AD-related pathological aging.
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0.904 |
2017 — 2021 |
Huang, Yadong |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Study Apoe4's Effects On Hippocampal Network Activity in Alzheimer's Disease @ J. David Gladstone Institutes
PROJECT SUMMARY The complexity and multifactorial nature of Alzheimer's disease (AD) poses unique challenges for mechanistic studies and developing therapies. Efforts to target AD-related pathways have shown promise in animal studies, only to fail during human trials. Thus, there remains a pressing need to identify novel mechanisms and therapeutic targets for treating or preventing AD. One of the earliest sites of AD pathology is the hippocampus, a brain structure critical for the learning and memory processes that falter early in AD. Decades of research have yielded insight into the genetics and cellular pathologies of the disease, but it is unclear how these pathologies disrupt hippocampal memory processes. The main genetic risk factor for AD is apolipoprotein (apo) E4, which lowers the age of onset of AD in a gene dose?dependent manner. In most clinical studies, apoE4 carriers account for 60?75% of all AD cases, highlighting the importance of apoE4 in AD pathogenesis. Although many hypotheses have been proposed, the cellular and network mechanisms underlying the pathophysiological actions of apoE4 are still unclear. This proposal builds on novel findings from our recent studies of mouse models. First, expression of apoE4 in knock-in (KI) mice causes age-dependent impairment of GABAergic interneurons in the hilus of the hippocampus, which correlates with the severity of learning and memory deficits. Second, deleting the apoE4 gene specifically in GABAergic interneurons prevents hilar interneuron loss and learning and memory deficits in LoxP-floxed apoE4-KI (apoE4-fKI) mice. Third, in vivo local field potential (LFP) recordings throughout the hippocampal circuit shows that compared to aged apoE3-KI mice, aged apoE4-KI mice have fewer sharp wave ripple (SWR) events?hippocampal network events critical for memory replay and consolidation?and have significantly reduced slow gamma activity during SWRs, which coordinates SWRs. Fourth, elimination of apoE4 in GABAergic interneurons rescues SWR-associated slow gamma activity but not SWR abundance in apoE4-fKI mice, suggesting that the disruption of interneuron-enabled slow gamma activity during SWRs is a critical mechanism of apoE4-mediated learning and memory impairments. This proposal aims (1) to determine the relative contribution of inhibitory interneuron subtypes to apoE4 disruption of hippocampal network activity underlying memory replay and (2) to determine whether apoE4 disruption of hippocampal network activity underlying memory replay depends on A?, tau, or both. The outcomes of the proposed studies will shed light on the pathogenesis of late-onset AD and could provide new targets for developing drugs treating or preventing AD.
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0.904 |
2017 — 2021 |
Huang, Yadong Sirota, Marina |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Apoe Genotype-Directed Drug Repositioning and Combination Therapy For Alzheimer's Disease @ J. David Gladstone Institutes
PROJECT SUMMARY Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder caused by interactions among multiple genetic and environmental factors. Apolipoprotein (apo) E4 has been identified as the major genetic risk factor for AD. It increases the risk and lowers the age of onset of AD in a gene dose?dependent manner. The genetic complexity and multifactorial nature of AD pose unique challenges for developing effective therapies and suggest the need for a precision medicine approach that takes into account individual variability. For the past several decades, new drug development efforts to target specific AD-related pathways have shown promise in animal studies, only to fail during human trials. Since the process of developing new drugs for AD is complicated and takes a long time and the related costs are extremely high, there is a pressing need to consider unconventional drug development strategies, such as repositioning drugs currently used for other conditions. The approach of drug repositioning has a number of advantages over the development of new drugs and has been applied successfully to various disease conditions. However, attempts at drug repositioning for AD treatment usually target specific AD-related pathways or mechanisms and have been largely unsuccessful or still under development. As we learn about the complexity of AD genetics and pathogenesis and the associated co-morbid conditions, it is becoming clear that efficacious treatments of AD will likely need to target multiple aspects of the disease and be directed towards several pathogenic processes, and as a result will likely require precision medicine and combination therapy of the repurposed drugs. The recent convergence of two factors presents an unprecedented opportunity to advance rational drug repositioning and data-driven development of drug combinations. First is the availability of public datasets from large-scale genomic, transcriptomic, and other molecular profiling databases. Second is the development of computational approaches and the network concept of drug targets and the power of phenotypic screening, which allows us to investigate the ability of one or more therapeutic agents to perturb entire molecular networks away from disease states. This proposal aims to capitalize on this promise by accomplishing three aims: (1) to analyze publically available, large-scale transcriptomic datasets of AD patients and age-matched controls to identify apoE genotype-specific gene expression signatures of AD, (2) to pursue drug repositioning based on apoE genotype-specific gene expression signatures of AD and validate the top drug candidates in apoE genotype-specific mouse models of AD, and (3) to explore combination therapy using drug repositioning based on apoE genotype-specific signatures of AD and validate the predicted combination candidates in apoE genotype-specific mouse models of AD. The outcomes of the proposed studies will shed light on the pathogenesis of AD and potentially identify existing drugs for treating or preventing AD.
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0.904 |
2020 — 2021 |
Huang, Yadong |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Study the Protective Roles of Apoe2 in Alzheimer's Disease Using Reprogrammed Isogenic Cells @ J. David Gladstone Institutes
The complexity and multifactorial nature of Alzheimer?s disease (AD) pose unique challenges for mechanistic studies and developing therapies. Although many transgenic mouse models have been generated for AD research and these models are important for our understanding of the pathological basis of the disease, none of them has captured the entire spectrum of the disease pathologies. This is likely due to significant species differences between mouse and human neural cells. Therefore, there is an urgent need to establish human disease modeling platforms to complement studies in animal models for AD research. Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, human iPSCs (hiPSCs) have been widely used for disease modeling and drug discovery. However, given the relative immaturity of cells differentiated from hiPSCs, it is challenging to use them for modeling late-onset diseases, such as AD, for which cellular aging is important in disease pathologies. Direct reprogramming is an alternative cellular reprogramming technology, which allows direct conversion of one type of cells, such as fibroblasts, into another type of cells, such as neural stem cells (NSCs) or neurons. It has been shown that direct reprogramming enables generation of human neurons that possess key elements of cellular aging, because this reprogramming process does not go through the iPSC stage involving extensive epigenetic modifications. While the strongest risk factor for AD is aging, the strongest genetic risk factor of AD is apolipoprotein E4 (apoE4). Among the three isoforms of human apoE (apoE2, apoE3, and apoE4), apoE4 increases AD risk, apoE3 is neutral, and apoE2 is protective. Although the roles of apoE4 in AD pathogenesis have been extensively studied, the protective roles of apoE2 in AD have been surprisingly understudied. Clearly, better understanding of the molecular and cellular mechanisms underlying apoE2?s protective roles in AD will likely provide novel targets or signaling pathways for anti-AD drug development, especially for late-onset AD. The objectives of this proposal are to develop aging-relevant human neuron models of late-onset AD, using direct reprogramming technology in combination with CRISPR/Cas9-mediated gene editing, and to dissect the underlying mechanisms of apoE2?s protective roles in AD. We propose three complementary aims to accomplish the goals. Aim 1: To generate isogenic human fibroblast lines with an apoE2/2 or apoE3/3 genotype from the parental human fibroblast lines with an apoE4/4 genotype as cell sources for direct reprogramming. Aim 2: To dissect the protective roles of apoE2 and their underlying mechanisms using directly reprogrammed human neurons and astrocytes. Aim 3: To dissect the protective roles of apoE2 and their underlying mechanisms using directly reprogrammed NSC-derived neurons and astrocytes. The outcomes of the proposed studies will promote our understanding of the molecular and cellular mechanisms underlying apoE2?s protective roles in AD, and will likely identify novel targets for anti-AD drug development.
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0.904 |
2021 |
Huang, Yadong |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neuronal Apoe Drives Selective Neurodegeneration in Alzheimer's Disease @ J. David Gladstone Institutes
PROJECT SUMMARY Selective neurodegeneration is a critical causal factor in Alzheimer?s disease (AD); however, the mechanisms that lead some neurons to perish while others remain resilient are unknown. There is regional susceptibility to AD-related neurodegeneration in the hippocampus and entorhinal cortex. Even within vulnerable neuronal populations, however, some cells are lost early while others prove more resilient. With recent technical improvements in single-cell analysis, we are able for the first time to examine the variability that drives regional and cellular differences in susceptibility to neurodegeneration. The major genetic risk factor for Alzheimer?s disease is apolipoprotein E4 (apoE4), which increases disease risk and decreases age of onset in carriers. Within the central nervous system, apoE is produced primarily in astrocytes but also in neurons following stress, injury, and aging. Neuronal apoE4 expression diminishes synaptic plasticity, impairs synaptogenesis, and decreases synaptic density both in vitro and in vivo. This proposal is based on intriguing preliminary studies. (1) Single-nucleus RNA-sequencing data from our lab have revealed a link between neuronal apoE and neuronal expression of the major histocompatibility complex class I (MHC-I). Like apoE, MHC-I is expressed in neurons following stress, injury, and aging. Neuronal MHC-I is localized to post-synaptic densities, where they limit long-term potentiation, enhance long term depression, and mediate synaptic pruning during development and, potentially, in neurodegenerative diseases. Our discovery of neuronal apoE upregulation of MHC-I provides insight into the mechanism by which both proteins potentially work in concert to contribute to synapse loss and eventually to selective neurodegeneration. (2) In AD patients, neuronal apoE expression correlates with neuronal MHC-I expression, which in turn predicts severity of Tau pathologies. (3) In AD model mice or cultured primary neurons, neuron-specific apoE4 knock-out decreases neuronal MHC-I expression and rescues neuronal and synaptic loss, establishing a causal relationship between neuronal apoE, upregulation of MHC-I, and selective neurodegeneration in AD. To capitalize on these novel findings and recent technical improvements in single-cell analyses, this proposal aims to determine the apoE-expression-high and MHC-I-expression-high neuron populations and explore their relationships with selective neurodegeneration across AD-susceptible and AD-resistant brain regions of apoE- KI mice with different apoE genotypes at different ages (Aim 1). We will also determine how apoE is regulating neuronal expression of MHC-I and how this expression leads to Alzheimer?s disease-related pathologies (Aim 2). Finally, we propose to determine the extent to which this apoE and MHC-I-mediated neuronal loss is caused by signaling to microglia (Aim 3), which has been heavily implicated in Alzheimer?s disease pathogenesis. The outcome of the proposed studies should shed light on the mechanisms underlying regional, cell-type-specific, and within-cell-type selective vulnerability to Alzheimer?s disease.
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0.904 |
2021 |
Huang, Yadong |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 1: Differential Roles of Apoe Isoforms in Neural Network Dysfunction of Alzheimer's Disease @ J. David Gladstone Institutes
PROJECT 1 ? SUMMARY The complexity and multifactorial nature of Alzheimer?s disease (AD) pose unique challenges for mechanistic studies and developing therapies. Although the three major pathogenic factors of AD?amyloid-beta peptides, tau, and apolipoprotein E4 (apoE4)?have been extensively studied as independent entities in AD pathogenesis, efforts to target individual AD-related pathways, such as Ab production or clearance, have largely failed in human trials. Emerging evidence strongly suggests that AD is a consequence of age-dependent neural network dysfunction in brain regions that mostly affect cognition, likely through the interactive effects of multiple pathogenic factors. Thus, there is a pressing need to identify novel mechanisms and therapeutic targets, at a neural network level and in the context of interactions between multiple pathogenic factors?the focus of this proposal. APOE4, which encodes one of the three major isoforms of apoE in humans, is the major genetic risk factor for AD and lowers the age of onset in a gene dose-dependent manner. In most clinical studies, APOE4 carriers account for 60?75% of AD cases, highlighting the importance of APOE4 in AD pathogenesis. On the other hand, many clinical and population studies show that APOE2, encoding the rarest apoE isoform, is a strongly protective genetic factor in AD. A pathological hallmark of AD is the formation of neurofibrillary tangles (NFTs) consisting of hyperphosphorylated, insoluble tau, containing both 3R and 4R tau isoforms. In cell cultures and mouse models, apoE isoforms have tau-dependent differential effects, and tau has apoE isoform-dependent effects, suggesting interactive roles in AD pathogenesis. However, almost all of these studies of apoE isoforms and tau drew conclusions based either on mouse tau, which is present almost exclusively as 4R tau, or on mutant human tau, which is associated with frontotemporal dementia but not AD. In Project 1, we propose to determine cell- type-specific, differential roles of apoE isoforms in age-dependent neural network dysfunction and behavioral deficits, in the context of wildtype (WT) human tau (both 3R and 4R tau isoforms), in novel mouse models of AD. In Aim 1, we will determine differential roles of apoE isoforms in neural network dysfunction and behavioral deficits, using apoE-isoform-floxed knock-in (APOEfE2/fE2 [fE2], APOEfE3/fE3 [fE3], and APOEfE4/fE4 [fE4]) mice expressing WT human tau (MAPT knock-in or TAUWT) at different ages. In Aim 2, we will determine cell-type- specific, differential roles of apoE isoforms in neural network dysfunction and behavioral deficits, using fE2/TAUWT, fE3/TAUWT, and fE4/TAUWT mice expressing cell-type-specific Cre recombinase (neuron-specific Cresyn1, astrocyte-specific CreGFAP, and microglia-specific CreCx3cr1) in order to delete the APOE allele in a cell- type-specific manner. The studies in this project, together with those of the three other projects of this program project grant, will yield new insights into the multifactorial pathogenesis of AD and may identify neural network- based targets for apoE isoform?dependent and cell type-specific therapies to treat or prevent AD.
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0.904 |
2021 |
Huang, Yadong Mucke, Lennart [⬀] |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Decoding the Multifactorial Etiology of Neural Network Dysfunction in Alzheimer's Disease @ J. David Gladstone Institutes
OVERALL ? SUMMARY Alzheimer?s disease (AD) is a major unresolved public health problem. Efforts to prevent or stall this disease have failed, in good part because of inadequate understanding of its complex pathogenesis. Mounting evidence suggest that neural network dysfunction may underlie or promote AD-related cognitive deficits and contribute to disease progression. Yet, the causes and consequences of this dysfunction and the therapeutic potential of counteracting it remain sorely understudied. Therefore, the overarching goal of this program project is to decode the multifactorial etiology of AD-related neural network dysfunction and to leverage the novel mechanistic insights we will gain toward the development of better therapeutic strategies. Through collaborative interactions among four projects and two cores, our program will use systems neuroscience (neurophysiology and behavior) in combination with systems biology (single-cell transcriptomics and epigenomics), as well as neuropathology and improved mouse models, to determine how copathogenic interactions among apolipoprotein (apo) E4, amyloid-b (Ab), and tau cause neural network dysfunctions and cognitive decline in AD. An Administrative Core will coordinate all activities. Projects 1?3 will use novel mouse models of sporadic and familial AD to study interactions of different apoE isoforms with wildtype (WT) human tau (Project 1) or APP/Ab (Project 2), or among apoE4, Ab, and tau that is WT or bears disease-associated amino acid substitutions (Project 3). Project 4 will carry out single-nucleus transcriptomic and epigenomic analyses on postmortem brain tissues from deeply phenotyped human AD cases to gain novel insights into the multifactorial etiology of the human condition, validate leads from mouse studies, and encourage backtranslation into the models. An Integrative Data-Science Core will help us integrate results from all projects through innovative statistical modeling. This approach will reveal which aspects of human AD are most faithfully reproduced in the mouse models and help establish the causal drivers of cell-specific alterations in the human tissues, increasing the mechanistic resolving power of the latter studies. Therapeutic interventions in mouse models will determine whether reducing apoE4 expression in specific cell types can block copathogenic effects of apoE4 and tau on brain functions (Project 1), modulating the activity of specific interneurons can counteract copathogenic effects of apoE4 and APP/Ab (Projects 2 and 4), and knocking down tau can prevent and reverse brain dysfunction in models expressing all three pathogenic factors (Project 3). Through these highly cohesive efforts, our program will dissect the multifactorial interactions among AD-related pathogenic factors, define their relative contributions to the complex pathogenesis of brain dysfunctions, and help distinguish among neuropathological alterations that cause, result from, or are coincidental to neural network dysfunctions and cognitive decline. Sharing the diverse data sets we will generate and disseminating the novel integrative approaches we plan to develop for their analysis could enhance the progress of many other groups working in AD research and drug development or biomedicine in general.
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0.904 |