Gwenn A. Garden, MD/PhD - US grants

DESCRIPTION (provided by applicant): One of the more devastating effects of HIV infection is the onset of neurologic dysfunction. A portion of patients with HIV will develop a syndrome of cognitive and motor decline known as HIV associated dementia (HAD). No specific agents for the prevention of treatment of HAD have been identified, perhaps because the pathophysiology of HAD is not completely understood. HIV infected immune cells invade the central nervous system (CNS) early during infection and establish a cascade of events leading to eventual neuronal dysfunction and degeneration. HIV infected monocytes and microglia in the brain release viral proteins, chemokines and cytokines that promote activation of uninfected microglia and astrocytes. The inflammatory response elicited in neuroglia leads to altered metabolism of excitatory amino acids (EAA's) and nitric oxide (NO). The cerebrospinal fluid of HAD patients contains elevated concentrations of EAA's and cytokines known to promote neuronal injury and apoptosis. However, the extent of neuronal apoptosis in HAD patients is not well correlated with cognitive dysfunction. Alternatively, loss of synaptic and dendritic markers is closely associated with behavioral declines. Caspase enzymes, a family of proteases involved in apoptosis, are activated in neurons of HAD patients and in animal models of HAD. Caspase enzyme inhibition also prevents dendrite degeneration in a HAD mouse model. These findings suggest that molecular mediators of apoptosis participate in generating the neuronal dysfunction observed in HAD prior to induction to irreversible apoptosis. Experiments proposed here will examine how the pro-apoptotic transcription factor p53 promotes neuronal injury or dysfunction in tissue culture and in vivo models of HAD. The cellular localization of p53 will be analyzed in HAD cases and several HAD models to determine which cell types respond to HIV with p53 activation. The effect of CNS HIV infection or the HIV coat protein gp120 on initiation of p53-mediated transcription will be assessed. The absence of p53 protects neurons from gp120-induced apoptosis, thus the mechanism of this neuroptrotection will be further defined. Neurotoxic factors released from activated neuroglia that requires p53 for the induction of neuronal injury will be identified. How p53 may promote neurodegeneration through the modulation of caspase enzyme activity will also be studied.

neoplasm /cancer; university

neoplasm /cancer; university

neoplasm /cancer; university

neoplasm /cancer; university

growth /development; neoplasm /cancer; university

[unreadable] DESCRIPTION (provided by applicant): This proposal requests funds to replace a standard confocal microscope that supports the research of investigators affiliated with two multi-disciplinary research centers at the University of Washington. The Center for Human Development and Disabilities and the Virginia Merrill Bloedel Hearing Research Center have jointly managed a microscopic imaging core facility since 1995. This facility has been well supported since its inception by NIH P30 grants, individual investigator grants, and institutional funds. The Bio-Rad MRC-1024UV confocal microscope, purchased in 1994, has been a workhorse for this large group of productive investigators. Despite its having out- of-date technology, this instrument has supported more than 25 research publications in the past 5 years. Unfortunately, this group of investigators can no longer depend on this early generation confocal system to reliably facilitate completion of funded research aims. Service and software upgrades are no longer possible, and down-time has led to a significant usage backlog and subsequent overuse of alternative equipment available to some investigators. The major and minor users on this application represent 13 departments in both clinical and basic sciences. The scientific questions they pursue range from identifying molecular determinants of early embryonic development to characterizing disease-modifying genes involved in infection, hearing loss, and neurological disorders. Several investigators in this group are conducting translational research projects in brain tumors, central nervous system injury, and neurodegeneration. The major scientific impetus behind this application is the need for reliable access to increased confocal microscopy time. However, research performed by this group will also be greatly enhanced by access to modern confocal technologies, including but not limited to, spectral unmixing, user- defined region of interest scanning, 360-degree scanning rotation, improved resolution in both time and space, and dramatic improvements in the efficiency of image acquisition. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Spinocerebellar Ataxia Type 7 (SCA7) is a dominantly inherited form of olivopontocerebellar atrophy caused by a CAG repeat expansion within the coding region of the gene for the ataxin-7 protein. Despite identification of the precise genetic abnormality associated with this disease, the mechanism by which the polyglutamine expansion in ataxin-7 causes selective neurotoxicity is not known. In a transgenic mouse model of SCA7 (polyglutamine expanded ataxin-7 expressed under the control of the prion protein promoter), degeneration of the cerebellar Purkinje cells (PC's) occurs in a non-cell autonomous fashion. Ultra-structural analysis of this SCA7 model revealed a pattern of PC degeneration observed when PC injury is caused by excitotoxic insult. The ultra-structural evaluation of SCA7 mice also revealed evidence for pathology in several cerebellar elements including loss of climbing fiber input and dramatic changes in the morphology of Bergmann glia. Additionally, ataxic SCA7 mice demonstrate loss of trophic factors normally supplied by climbing fibers and Bergmann glia. Taken together, these observations support the hypothesis that PC degeneration in SCA7 is secondary to an altered extra-cellular environment. In this proposal we plan to address the question of how mutant ataxin-7 alters the PC environment resulting in PC degeneration. First, we will perform comprehensive behavioral, histological and ultra structural analysis of the natural history of PC degeneration in SCA7 knock-in mice and mice expressing the mutant protein in specific cellular populations of the cerebellum using a conditional inactivation strategy. Second, two specific aspects of the extracellular environment will be examined: Neurotrophic factors and Neurotransmitter homeostasis. The specific alterations in the cerebellar environment that are associated with the onset of the disease phenotype in transgenic and knock-in mouse models of SCA7 will be further characterized. Additionally, experiments attempting to normalize the cerebellar environment in SCA7 mice will be initiated in order to determine if restoring neurotrophic support or preventing excitotoxicty will subsequently delay or prevent the disease phenotype. PUBLIC HEALTH RELEVANCE: The overriding objective of this proposal is to further define the pathogenesis of spinocerebellar ataxia type 7 (SCA7), an inherited neurodegenerative disease. The long-term aim is to identify potential targets for therapeutic intervention in this devastating neurodegenerative disease.

DESCRIPTION (provided by applicant): Inflammation in the nervous system occurs during injury, stroke and several neurodegenerative diseases. Some degenerative diseases of the central nervous system (CNS) such as multiple sclerosis occur primarily due to abnormal inflammation. How inflammation impacts neurodegeneration is not completely understood but likely involves important roles for resident inflammatory cells known as microglia. Microglia exposed to infectious agents, injured CNS tissue, or abnormal protein accumulations (such as the beta-amyloid protein that accumulates in Alzheimer's disease), release inflammatory mediators that are often toxic to CNS cells and may produce a vicious cycle of chronic inflammation. Long term exposure to an inflammatory state can lead to the accumulation of reactive oxygen species (ROS) which may damage a variety of cellular components including DNA. This can lead to activation of cellular systems evolved to induce a cell suicide program called apoptosis. During experiments using an in vitro model of inflammation induced neuronal injury we observed that an important regulator of apoptosis, p53 is required in both neurons and microglia for inflammatory neurotoxicity. To determine how p53 is involved in the microglia response to a pro-inflammatory stimulus we examined global gene expression profiles from cultured microglia obtained from wild type and p53 deficient mice. The difference in gene expression between these two genotypes of microglia in conjunction with additional data on cytokine release from p53 deficient microglia led to the hypothesis that p53 participates in determining the activation phenotype in microglia cells in response to inflammatory signals. Microglia are cells in the macrophage linage, and several types of macrophage activation have been characterized. Two types of macrophage activation include "classical" tissue destructive activation and "alternative" activation in the down regulatory phase of the inflammatory response when macrophages participate in tissue repair. In this we will address the question of whether p53 acts during myeloid differentiation to influence to potential for polarized microglia activation or concurrently with the microglia response to a specific activation signal. We will also examine how p53 may interact with c-Maf, a transcription factor repressed by p53 in microglia, which has a previously described role in determining macrophage activation patterns. Finally, we will determine if p53 transits to microglia mitochondria in response to inflammatory stimuli, thereby impacting mitochondrial integrity and ROS generation. PUBLIC HEALTH RELEVANCE: Inflammation is a key pathological component of many common disease of the nervous system including Alzheimer's disease, Parkinson's disease and Stroke. Microglia are inflammatory cells that reside in the nervous system and we do not currently have a complete understanding of how these cells respond to injury or disease. The proposed experiments will enhance our understanding of how p53, a key regulator of cell division and survival may also be a fundamental determinate of how microglia respond to stimuli that initiate or perpetuate inflammation.

During the past grant cycle, the Neuroscience Core consisted of two components: Brain Imaging and Cellular Morphology. These components were related by their support of Research Affiliates studying the neurobiology of neurodevelopmental and neurodegenerative disorders. However, as new technologies and investigators were assimilated into both components, it became clear that these two components had divergently expanded to a point that justified splitting into two separate Cores. The now separate Cellular Morphology Core has been growing extensively during the past grant cycle, acquiring new equipment, incorporafing new technologies, and assisting a large number of new Research Affiliates. Advances in the molecular genetics of human disease in combination with burgeoning new technologies in mammalian genetic engineering have resulted in a large number of novel cell, tissue, and animal models of human disease. This has lead to increased demand for the availability of research methodologies needed to carefully characterize these new models and provide quantifiable readouts of phenotypes that can be employed to study the efficacy of therapeufic interventions in pre-clinical trials.

DESCRIPTION (provided by applicant): We discovered mutations in the Senataxin gene (SETX) as the molecular basis of a juvenile-onset, autosomal dominant (AD) form of familial amyotrophic lateral sclerosis (ALS). This form of ALS, known as ALS4, is an essentially pure motor systems disorder characterized by limb weakness, severe muscle wasting, pyramidal signs, and slow disease progression. Intriguingly, recessive mutations of SETX cause a second disease, a form of ataxia known as Ataxia-Oculomotor Apraxia type 2 (AOA2). In AOA2, morbidity is also high with loss of ambulation occurring ~15-20 years following onset which normally ranges from 10-22 years of age. Together these findings demonstrate genotype-phenotype correlation for SETX mutations and confirm senataxin as an important neuronal protein. Senataxin is a large protein at 2677 amino acids and its precise function is largely unknown. It contains a conserved DNA/RNA helicase domain towards the C-terminal suggesting a function in RNA processing. Such important RNA processing functions have been characterized for the yeast orthologue, Sen1p. There is also a potential protein-protein interaction domain in the extreme N-terminal which is supported by studies in yeast. Our studies will focus on mutant forms of Senataxin associated with ALS4 and AOA2 and address the following broad aims: (1) We will characterize murine gene targeted and transgenic (Tg) models we have produced for ALS4 and test if they develop neuropathies allowing further detailed study of neuronal degeneration pathways;(2) Using gene trap technology we will develop a murine knock-out model for AOA2;and (3) we have hypothesized that ALS4 mutant Senataxin (L389S) may lead to toxic gain-of-function such as aberrant protein-protein interaction. To test this we undertook yeast two-hybrid (Y2H) screens with wt and ALS4 mutant N-terminal senataxin of a human brain expression library. We identified a ALS4 specific interaction with the kinesin, KIF1B, that was validated with further Y2H re-testing and mammalian 2-H assays. We will further examine this interaction and its potential pathogenic effects in ALS4. PUBLIC HEALTH RELEVANCE: The potential knowledge gained from this research is an improved understanding of the causes of neuronal death in familial forms of ALS and Ataxia. Murine models being developed as part of the study could also prove highly useful and would eventually be shared with other interested investigators. Since there is currently no cure or preventive treatment for these conditions, any knowledge gained has the potential to provide future diagnostic and therapeutic interventions for these patients.

DESCRIPTION (provided by applicant): Neuronal injury and degeneration are accompanied by inflammatory responses from the innate immune system. Central nervous system tissues have highly specialized innate immune responses mediated primarily by microglia, the resident macrophage population. Microglia invade the CNS early in embryonic development and are a highly specialized cell of myeloid origin with unique features and functions. One of those unique aspects of microglia behavior is that are normally quiescent, expressing few markers associated with other specialized macrophage behaviors. Like their macrophage cousins however, microglia can respond to changes in the CNS environment by adopting a variety of behaviors. When microglia respond to changes in their environment, they can perform functions associated with "classical" macrophage activation that evolved as effective responses to bacterial and viral pathogens as well as those involved in quelling an inflammatory response or participating in the process of tissue repair. Currently, there is debate regarding which microglia functions dominate during acute or chronic injuries and no clear method of experimentally inducing microglia to adopt a specific set of behaviors. We have identified p53 as a transcriptional regulator that promotes behaviors associated with classical activation in microglia while p53 deficiency yields gene expression patterns associated with anti-inflammatory and tissue repair functions in microglia. One mechanism by which p53 influences microglia behavior was identified as negative regulation of a second transcription factor, c-Maf. The c-Maf transcription factor is a known regulator of both lymphocyte and myeloid cell differentiation, generally observed to promote the anti-inflammatory/tissue repair arm of both the innate and adaptive immune system. This proposal will further explore these discoveries by first demonstrating that the function of c-Maf in macrophages is recapitulated in microglia, second identifying the molecular mechanisms by which p53 influences c-Maf expression and third determining if p53 modulates microglia behaviors in vivo using the Cre/lox system to inactivate p53 in a time and cell type specific fashion. PUBLIC HEALTH RELEVANCE: A wide variety of nervous system disease and injury states are associated with a robust inflammatory response. Currently there is no clear understanding of the molecules that regulate whether the cells responsible for this inflammatory response will behave in a manner that helps protect nervous system tissue or exacerbates the injury process. This proposal aims to study a molecular pathway that regulates inflammatory behavior outside of the nervous system to determine if it could be harnessed as a method of maximizing recovery or preventing further degeneration in a variety of diseases including stroke, Alzheimer's disease and vascular dementia.

DESCRIPTION (provided by applicant): Recent progress in genetics has identified a new class of RNA molecules called microRNAs (miRNA) involved in gene regulation. miRNAs are single-stranded RNA molecules of ~22 nucleotides complementary to a site in the 3'untranslated region (UTR) of mRNAs. The annealing of the miRNA to the mRNA inhibits protein translation and sometimes facilitates cleavage of the mRNA. This regulation adds an unexpected layer of complexity to the classic "linear" concept of DNA->mRNA->protein. Fast paced progress in the recent years showed the importance of miRNA in regulating neuronal and immune functions. In the CNS, miRNAs have been implicated in synaptic development and memory formation. We have found that constitutive knock out of miR-155 (miR-155-/-) increases the infract area in the mouse middle cerebral artery occlusion/reperfusion (MCAO/R) model by ~50%. However, miR-155 is expressed in the CNS in by at least astrocytes and microglia. The constitutive knock out of miR-155-/- makes it impossible to determine which cell type is responsible for the substantial increase in infarct area. We therefore wish to generate a conditional knock out model using the Cre/loxP approach. Using this well established approach we will be able to cross the floxed miR-155 (miR-155fl/fl) with readily available Cre-driver mice for astrocytes and microglia to generate cell specific knock out mice. The long-term goal of this project is to generate conditional knock outs of miR-155 to investigate the role of cell-specific miR-155 in CNS injury such as ischemic injury. We believe that the molecular mechanisms identified in subsequent experiments using this model may indentify new pathways for therapeutic intervention in CNS injuries such as stroke. PUBLIC HEALTH RELEVANCE: The broad, long-term goal of this project is to understand the role of miR-155 in the activation of astrocytes microglial cells. We believe that the signal transduction mechanisms identified through our experiments may constitute new targets for therapeutic intervention in CNS injuries associated with glial activation such as trauma, multiple sclerosis or stroke.

DESCRIPTION (provided by applicant): The Society for Neuroscience (SfN), the world's leading professional association for the field of neuroscience, seeks a five-year renewal grant for its successful Neurobiology of Disease Workshop (NDW). The next decade is expected to lead to breakthroughs in neuroscience and rapid translation of discoveries to improving human health and will require new generations of disease-focused basic researchers. A cornerstone of SfN's mission is to provide educational resources to neuroscientists at all stages of their careers, and the NDW has had a strong 30-year track record as the most successful national-level workshop specifically aimed at attracting young basic neuroscientists to the study of diseases of the nervous system. The overall goal of the NDW remains to inspire new basic science researchers from multiple disciplines to focus on disease research that will ultimately contribute to advancing the treatment of important neurological disorders. The four specific aims of the program are: 1) to introduce young neuroscientists to important unsolved scientific problems that relate directly to diseases of the nervous system; 2) to show neuroscientists in training the human side of diseases of the nervous system; 3) to promote the exchange of information on state-of-the-art disease-related research among the faculty and students; and 4) to broaden the reach of the workshop by disseminating its content beyond the SfN annual meeting. SfN will achieve these aims by assembling top experts in a specific disease area each year to introduce the latest problems and ideas through a day-long workshop for 250 participants. The workshops, held at the Society's annual meetings, are comprised of a morning of carefully rehearsed and integrated didactic lectures and patient interviews, followed by an afternoon of small interactive discussion groups led by active scientific and clinical experts. New to this grant cycle will be efforts aimed at increasing the impact of the NDW program. Young neuroscientists unable to attend the workshop will benefit from recorded lectures and slides on CD-ROM. In addition, SfN will organize a follow-up webinar and online discussion forum after each workshop, to further encourage and engage NDW attendees and others to pursue studies and discussions on the specific neurological disease or disorder.

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder caused by inheriting a CAG repeat expansion in the coding region of the ataxin-7 gene, resulting in a polyglutamine (polyQ) expanded mutant protein. SCA7 has features in common with other neurodegenerative disorders caused by polyQ expansion mutations such as Huntington's Disease. One such feature is that degeneration occurs in selectively vulnerable neural populations. In SCA7 the populations that degenerate include Purkinje cells (PCs), Bergmann Glia (BG) and neurons of the inferior olive (IO) which send climbing fiber axons to synapse on PC dendrites. Using animal models of SCA7, we have shown that disease gene expression specifically in BG, PCs and IO neurons influence the SCA7 disease phenotype. Interestingly, using a floxed- polyQ ataxin-7 mouse model of SCA7 we observed that expression of Cre recombinase in all three cell types dramatically delayed symptom onset. Taken together, these findings support the hypothesis that mutant ataxin-7 expression in these three cell types contributes to dysfunctional cellular interactions and is a critical mediator of SCA7 cerebellar degeneration. Unfortunately, the molecular mechanisms responsible for SCA7 pathology in these 3 specific cell types have not yet been determined. We have also observed that suppression of mutant gene expression after symptom onset halts disease progression, but does not reverse pathology in PCs or BG. However, IO inputs to the cerebellum are both decreased and redistributed in SCA7 mice. IO-PC synapse pathology was the only detectable abnormality prevented by suppression of mutant gene expression after symptom onset, suggesting that loss of IO-PC synapses contributes to SCA7 disease progression. Since SCA7 cerebellar pathology involves altered interactions between specific cell types that reside in distinct regions of the CNS, standard approaches to the study of altered gene expression or protein content cannot distinguish between cell type specific changes mechanistically involved in the pathologic process and reactive changes that develop in response to the degeneration of selectively vulnerable cells. To address this issue, we have developed techniques to isolate RNA specifically from the three cellular populations known to impact cerebellar pathology and motor behavior. In this project, we will employ these methods to address the hypothesis that cell type specific changes in both coding and non-coding RNAs lead to the specific pattern of cellular dysfunction observed in the cerebellum of SCA7 mice. We will further identify which RNA changes are reversible following suppression of mutant ataxin-7 expression by inducible Cre-recombinase. These discovery driven experiments are high-risk, but have the potential to yield critical information regarding the molecular mechanisms responsible for selective vulnerability in a polyQ disorder.

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? DESCRIPTION (provided by applicant): The rising global prevalence of Alzheimer disease (AD) has heightened the urgency to develop effective AD therapeutics. Many lines of evidence support the hypothesis that AD pathogenesis involves dysfunctional innate immune effector cells in both the periphery and central nervous system (CNS). Innate immunity promotes tissue repair, clearance of debris, release of cytokines and chemokines which act to signal appropriate interface with adaptive immune responses. However, dysregulation of CNS innate immune cells, microglia, or of peripheral monocytes, contributes to a neurotoxic environment directly injuring synapses, creating a feed forward loop of neural injury. Recent technological advances have produced powerful tools to survey the molecular regulators of the innate immune network contributing to AD pathogenesis. MicroRNAs, a class of small non-coding RNAs, have emerged as powerful modulators of CNS and peripheral innate immunity, regulating pro-inflammatory activity, phagocytosis and contributing to mechanisms of A? clearance. MiRNA profiles are altered in brain tissue, serum and CSF of patients with AD. In particular, miR146 and miR155, proximal inflammatory modulating miRNAs, are dysregulated in AD implicating miRNAs in the inflammatory arm of AD pathogenesis. Work by our group and others have demonstrated that miR146 and miR155 participate in in the development of age-dependent chronic inflammation in vivo. These data underscore putative pathways by which AD pathogenesis is influenced by inflammatory miRNA regulatory networks. In this multi-PI application, we have designed a systems approach to investigate the role of miRNAs in CNS and circulating innate immune cell regulation in the context of AD. MicroRNA profiling, gene co-expression analysis and amyloid-? (A?) clearance studies will be performed in peripheral macrophage and microglia-like cells differentiated from individuals with early symptomatic AD and early or asymptomatic carriers of familial autosomal dominant Alzheimer disease genes (PSEN1, PSEN2 and APP). In parallel, we will employ the murine APP/PS1 and 5XFAD AD models to determine the impact of altered miR146 and miR155 expression in vivo on cognitive deficits, synapse loss, A? clearance and chronic CNS inflammation. By capturing the inflammatory miRNA landscape and inflammatory profile in patients destined to develop dementia of the Alzheimer type and modeling microRNA modulation in vivo, we aim to identify targetable pathways which leverage the bidirectional communication between CNS and peripheral inflammation.

The Neurobiology of Disease Workshop (NDW) provides basic scientists and trainees with an in-depth introduction to neurological disorders. It is organized as a day-long workshop held the Friday before the Society for Neuroscience (SfN) Annual Meeting. The overall goal of the Workshop is to introduce the world of neurological disease to the basic scientist, with the expectation that whatever their interest or expertise in the nervous system, they may bring extraordinary new questions, ideas and even solutions to problems in the field. Reflecting the dynamic and rapidly advancing state of neuroscience research, the topic of each NDW is identified only one year in advance. Although the specific topics change each year, the Workshop?s agenda format remains consistent. The core of the Workshop begins with a patient presentation. Growing less common in clinical departments, and certainly unique to a basic science workshop, is the patient presentation that begins the day-long NDW program. This is a live interview performed by an experienced clinician in front of the entire audience. Not only are patients presented, but in applicable cases, family members of the patient also attend the Workshop. Often the family members are asked to describe how the disease has affected their own lives, helping neuroscientists in training see the human side of diseases of the nervous system. The patient presentation is followed by multiple lectures that cover the essential clinical and basic science information necessary to understand the disease or disorder. The set of lectures includes neuropathology (where relevant), the anatomy of the systems involved, neurochemical and physiological correlates, current treatments, and other related information. The faculty members, chosen for their expertise in the field, present the core information, which lasts approximately three to four hours. As detailed above, a patient is presented by a senior clinician early in the Workshop schedule, and time is allotted for the audience to ask questions of the patient or the clinician. Following the patient and core lecture presentations, Workshop participants form breakout groups, each led by a team of two faculty members, for a period of extended discussions. The total time for each discussion group is 1.5 hours and these sessions are designed to stimulate participant thinking and speculation about the disease and to inform them of the research already underway.

PROJECT SUMMARY/ABSTRACT Neuroinflammation plays a critical role in injury and degeneration in the central nervous system (CNS). MG are specialized resident myeloid cells in the CNS that play essential roles the innate immune response. CNS ischemia and traumatic brain injury (TBI) cause recruitment of circulating immune cells and activation of resident MG. Activated MG perform dynamic functions that can be both supportive and destructive to neuronal health. MG also have essential roles in CNS development, plasticity and immune surveillance. However the ontogeny of MG in the adult CNS is still not fully understood. While MG progenitors that colonize the developing brain are born in the embryonic yolk sac, recent reports demonstrate that MG progenitor cells are also present in the adult CNS. When adult MG are depleted, these progenitor cells devide and differentiate into mature adult MG. These findings suggest that MG plasticity not only refers to the molecular and morphological changes of existing cells, but also the generation of a new MG populations. Currently, little is known regarding the potential role for MG progenitors in the normal, aging or injured brain. We are interested in understanding the regulation of MG behavior, including the generation and differentiation of newly born MG during acute and chronic CNS injury and neurodegeneration. We have developed methods to isolate and culture MG progenitor cells from adult mouse brain. This progenitor population is present in the uninjured adult CNS, can be isolated by cell sorting or positive selection and will differentiate into mature MG in vitro. The goals of this proposal are to determine how newly born MG contribute to the mature MG population in the setting of advanced age or disease and to identify molecular pathways that distinguish MG progenitors from mature MG. We propose to study the process of MG repopulation in aging mice and to determine whether age influences the rate of cellular proliferation in adult CNS resident erythromeyloid precursor cells, MG progenitor cells and mature MG. We will employ this approach to determine if the APP/PS1 mouse model of Alzheimer's disease (AD) influences the size of the progenitor and mature MG populations. In addition, to determine the specific molecular signature of MG progenitors cells, we will isolate progenitors and mature MG by ex-vivo flow cytometry and compare the global gene expression profiles of the three populations using RNA sequencing. In summary, the accomplishment of these aims will help to further understand the molecular signals that regulate MG and their progenitors within the CNS.

Alzheimer disease (AD) is a complex phenotype likely influenced by the combinatorial impact of many genetic elements. While environmental factors and age certainly contribute to phenotypic expression, we hypothesize that the underlying biological process of AD is driven by the burden of genetic variants influencing risk. It is therefore critical to understand the biological implication of AD risk alleles if we hope to realize the goal of more precise and effective AD therapeutics. We are leveraging our interdisciplinary Alzheimer Disease (AD) research programs at the University of Washington and partners in the Seattle area to create a cell-type specific systems biology program in AD. The cellular heterogeneity of human brain confounds interpretation of transcriptomic and epigenomic data from bulk tissue. We have developed an iterative process to functionally annotate the known AD GWAS SNPs in a cell type specific unbiased manner with transcriptomic and epigenomic network analysis of single cell CNS neural tissue. Through innovative combinatorial ATAC-seq methods to map the open chromatin profile from isolated single cells in human brain we will generate chromatin accessibility and transcriptomic profiles from single cells isolated from autopsy fresh brain. In parallel we are deriving iPSC differentiated neuronal and microglial lines generated from the same cohort of sporadic AD. Through development of these datasets and identifying shared and distinct features, we can begin to identify not only the epigenomic and transcriptomic impact of GWAS variants, but that of aging and stem cell reprogramming. We hypothesize that non-coding variants confer AD risk through cellular pathways which can be assessed in vitro. We will pilot this approach to assess the impact of GWAS variants on cellular phenotypes well established as relevant to AD pathogenesis. GWAS AD SNPs and novel genomic variants will tested for association with a subset of AD relevant in vitro cell biology phenotypes in iPSC derived neurons and microglia and transdifferentiated neurons. Through our integrated molecular and cell biology phenotyping we aim to identify candidate pathways by which non-coding variants may confer AD risk, and identify novel pathways associated with AD pathogenesis for further in vitro study.

PROJECT ABSTRACT: Neuroinflammation plays a critical role in injury and degeneration in the central nervous system (CNS). Microglia (MG) are specialized resident myeloid cells in the CNS that play essential roles the innate immune response. MG also have essential roles in CNS development, plasticity and immune surveillance. CNS injury and neurodegeneration lead to inflammatory activation of MG. Activated MG perform dynamic functions that can be both supportive and destructive to neuronal health. In the adult CNS MG turnover occurs very slowly but new MG can be rapidly generated after depopulation or in response to injury. However the ontogeny of new MG in the adult CNS is still not fully understood. While MG progenitors (MGP) that colonize the developing brain are born in the embryonic yolk sac, recent reports suggest that MGP cells may also exist in the adult CNS. Thus MG plasticity may not only refer to the molecular and morphological changes of existing cells, but also the generation of new MG populations. Currently, little is known regarding the potential role of newly born microglia in Alzheimer's disease (AD). We are interested in understanding the regulation of MG behavior, including the generation and differentiation of newly born MG in the setting of neurodegeneration. We have developed novel methods to fate map and isolate a population of cells expressing both markers of progenitor state (prominin 1) and myeloid commitment (Cd45) from the adult mouse brain. This potential progenitor population is present in the uninjured adult CNS, can be isolated by fluorescence activated cell sorting (FACS) and will differentiate into mature MG in vitro and in vivo. The goals of this proposal are to 1) determine if AD pathology influences the proliferation, differentiation, and survival of newly born MG in the adult CNS, 2) to determine if MGP contribute to the microglia population associated with amyloid plaque, 3) examine whether AD pathology influences the epigenetic profile and transcriptome of adult MGP and 4) determine if the inflammatory activation pattern in MGP derived mature microglia is influenced by cellular age or origin. We will employ mouse models of AD that develop early amyloid plaque to determine if these pathological hallmarks of AD influence the population dynamics of the progenitor and mature MG populations. In addition, we plan to employ state of the art single cell sequencing approaches to study how AD pathology influences chromatin architecture and gene expression in these distinguishable populations of CNS myeloid cells. In summary, the accomplishment of these aims will help to further understand dynamics of MG populations and the influence of those population dynamics on the inflammatory response in AD brain.

Alzheimer disease (AD) is a complex phenotype influenced by the cumulative impact of many genetic elements. While environmental factors and age certainly contribute to phenotypic expression, we hypothesize that the underlying biological process of AD is driven by the burden of genetic variants influencing risk. Understanding how genetic burden causes disease can inform efforts to develop more effective AD therapeutics. Furthermore, the spectrum of known AD genetic risk variants implicates numerous cell types and thus knowing how variants impact biological networks and in which cell type is critical to directing therapeutic target design. In this proposal we leverage our collaborative and interdisciplinary AD research programs at the University of Washington and SAGE Bionetworks to create a cell-type specific systems biology program in AD. We hypothesize that non-coding variants confer AD risk through disruption of cellular pathways which can be identified by molecular phenotyping of AD patient brain tissue and assayed in vitro. Specifically, we focus on endosome biology given the link between endosome pathways and neural cell function and the known association with endosomal genetic variant risk and AD. Through the use of an endosome pathway specific polygenic risk score we can enrich our AD cohort for those more likely to manifest AD driven by endosomal dysfunction. We will employ single nuclei transcriptomics and functional studies in reprogrammed neural cells derived from the same cohort to investigate the impact of genetic risk in endosomal pathways on AD pathophysiology. We will 1) determine if a high endosome pathway polygenic risk score predicts endosome dysfunction in neurons and 2) determine how high endosome polygenic risk influences microglia function. Through development of these datasets, which will be available as open-source through SAGE Bionetworks, we can begin to identify the cell type specific transcriptomic changes in AD associated with endosomal variant load as well as the concomitant functional cell alterations using induced pluripotent stem cell derived neurons, microglia and transdifferentiated neurons. Our integrated molecular and cell biology phenotyping approach seeks to identify biological pathways by which aggregate endosomal genetic risk may contribute to AD pathogenesis and elucidate the cellular subtypes most directly impacted by endosomal dysfunction. Understanding candidate biological pathways and the cell types in which they are disrupted will provide valuable information for more effective therapeutic targeting in AD.

ABSTRACT - OVERALL Before it kills, Alzheimer?s disease (AD) exacts a devastating toll on patients, families, caregivers, and communities. Moreover, even in 2020, there is still no prevention or cure for AD. To meet this challenge, the new Duke/University of North Carolina ADRC is poised to transform AD-related research and services across the Research Triangle and Eastern North Carolina. The new Duke/University of North Carolina Alzheimer?s Disease Research Center (Duke/UNC ADRC)?s theme is identifying age-related changes across the lifespan that mediate development, progression, and experience of Alzheimer?s disease (AD). In support of this theme, the Duke/UNC ADRC clinical cohort design and biomarker collection equips investigators with data and resources to discover new opportunities to intervene in the years before AD symptoms manifest. A related Center goal is to identify how factors that arise in early and mid-life contribute to racial and urban/rural disparities in dementia. The Center plans to provide infrastructure and resources aligned with the following specific aims: 1) Stimulate and support research on AD and Alzheimer?s disease-related dementias (AD+ADRD) for investigators from many fields by providing access to well-characterized subjects, curated data and biospecimens from diverse individuals across a wide age-spectrum with and without dementia, with emphasis on pre-clinical and early disease; 2) Attract and prepare diverse, creative, well-trained investigators to conduct high caliber research on AD+ADRD and 3) Improve lives impacted by age-related cognitive decline and increase the inclusiveness of this research. The Center?s Cores (Clinical, Biomarker, Neuropathology, Data Management and Statistics (DMS), Outreach, Recruitment, and Engagement (ORE), Administrative) and Research Education Component (REC) work collectively to pursue these aims, aided by our team?s decades of dementia outreach across North Carolina, strong existing ties to minority and rural communities, and institutional resources and experts in disparities research. The Center?s Clinical, Biomarker, and Neuropathology Cores, in conjunction with the ORE Core will support collection of images, biospecimens, and fluids from over 500 well-characterized participants across a broad age range. The DMS Core will support data curation and analysis by providing integrated data management and statistical/bioinformatics collaborative expertise. The REC will develop a robust and diverse pipeline of future leaders in AD+ADRD research through an innovative combination of widely disseminated curricular elements and more personalized mentoring experiences. The REC?s educational platforms extend to three additional Universities in our catchment area, including two majority minority institutions. The ADRC?s Cores and the REC, supported by the Administrative Core, will work in a coordinated manner to generate and store data and resources from people with and without dementia or AD risk, share data and expertise as widely as possible, and ultimately contribute back to the AD community through dissemination of results, education, and health services.