Zhenyu Yue - US grants

[unreadable] DESCRIPTION (provided by applicant): A central question in the cell biology relevant to Parkinson's disease (PD) is the roles for normal and pathogenic forms of alpha-synuclein protein in neurons and neurodegeneration. Recent evidence showed that alpha-synuclein is phosphorylated in Lewy body, a hallmark of pathogenesis of PD, and phosphorylation of alpha-synuclein is critical in mediating neurotoxicity. However, the mechanisms or cellular pathways that lead to alpha-synuclein phosphorylation, cytotoxicity, and nigral degeneration in PD are not clear. Recent discovery of mutations of LRRK2 which cause familial PD presents an opportunity to identify such cellular mechanisms or pathways. The genetic studies indicated that the mutations of LRRK2 cause the most common autosomal dominant PD to date. Although the patients with LRRK2 mutations all exhibited typical PD, the postmortem studies revealed neuro-pathological heterogeneity as shown in neurodegeneration, formation of Lewy bodies, or neurofibrillary tangles. LRRK2 encodes a large complex protein (280kD) containing several conserved domains such as sequence for Ras GTPase and MAPKKK kinase. Mutations of LRRK2 (such as R1441G) that are associated with PD are located in those domains, and likely cause altered biochemical activities of LRRK2 with deregulation of related cellular pathways. This proposal aims to 1) detect LRRK2-mediated cellular functions or pathways and to test the hypothesis of the phosphorylation of alpha-synuclein by LRRK2 kinase activity; 2) and to investigate the pathogenic mechanisms of familial PD mutation R1441G of LRRK2. To accomplish these goals, we generated transgenic mice producing epitope-tagged LRRK2 wild type or mutant which allows us to isolate LRRK2-associated protein complexes under normal and pathological conditions. These mice provide unique tools to assess the phosphorylation of alpha-synuclein (or other substrates) by LRRK2 kinase activity and to study pathogenesis of PD in vivo. In addition, we developed a specific anti-LRRK2 antibody which allows us to detect the expression and localization of endogenous LRRK2. These important reagents will help us uncover the cellular functions or pathways implicated in neuropathogenesis of PD. It will also reveal roles of LRRK2 in regulating or interacting with other molecular pathways. This proposal will provide valuable information for PD therapeutic design directed towards enzymatic activity of LRRK2. [unreadable]

DESCRIPTION (provided by applicant): The goal of this proposal is to evaluate potential motor function deficits and cognitive impairments in a series of BAC-mediated transgenic mice, which over-express mutant LRRK2 carrying Parkinson's disease (PD)-linked familial mutation. PD is characterized primarily by the deficits in motor function in the CNS. Recently, non-motor symptoms including cognitive impairments have been increasingly recognized as important manifestations of neuropathology in PD. However, no animal models are currently available that recapitulate PD symptoms including motor and non- motor impairments. Missense mutations in LRRK2 (Leucine-Rich Repeat Kinase 2) or PARK8 have recently been linked to the most common familial forms as well as some sporadic forms of PD. We recently established transgenic mice over-expressing LRRK2 wild-type (WT), PD-linked mutant G2019S or R1441G by using bacterial artificial chromosome (BAC)-mediated transgenic approach, which utilizes endogenous promoter and necessary regulatory elements for the proper expression of LRRK2 in the mouse brains. These BAC-transgenic mice provide us with valuable tools to specifically characterize potential hyper-activated pathways mediated by LRRK2 in the pathogenesis of PD. In our proteomic study of LRRK2-binding proteins in the brain, we found that LRRK2 interacts with proteins that are associated with synaptic membrane or regulation of synaptic plasticity. Recent study also indicates that LRRK2 is expressed at high levels in the cortex, hippocampus and amygdala that serve cognitive functions. Since the impaired synaptic functions in these areas are frequently linked to the cognitive deficits, it raises the possibility that over-expression of PD-linked mutants of LRRK2 may alter the synaptic activity and plasticity, which in turn may perturb the cognitive execution. Thus, we propose to assess the motor functions as well as to investigate non-motor complications in LRRK2 transgenic mice, It will be of critical importance to know (1) Whether LRRK2 transgenic mice display motor function deficits, (2) whether non-motor manifestations including cognitive deficits develop in the LRRK2 transgenic mice, and (3) whether the onset of the non-motor symptoms precede the motor dysfunction. Hence this study will explore these models to determine the role of LRRK2 in motor functions and its potential interaction or coordination with cognitive functions. PUBLIC HEALTH RELEVANCE: The pathological symptoms of Parkinson's disease include motor deficits as well as non-motor impairments. This project is to investigate motor function and non- motor complications (e.g. cognitive dysfunctions) in Parkinson's disease by using novel transgenic approach. We will conduct behavioral and electrophysiological characterizations of the established transgenic animals expressing gene mutations which are linked to the Parkinson's disease. This study is expected to provide insight into the neurobiology for Parkinson's disease.

Our long-term goal is to understand neuroprotective mechanisms of autophagy and identify therapeutic targets of autophagy to treat neurodegenerative diseases associated with intraneuronal protein aggregates. The physiological function of autophagy in neuron is to maintain metabolic homeostasis and serve as quality control through constant degradation. Importantly, the constitutive autophagy in neurons shows high selectivity, targeting specific protein and organelle cargo to the lysosomal degradation. However, the molecular mechanism for the selective autophagy remains poorly characterized in neurons. Increasing evidence shows that selective autophagy is mediated through a family of proteins called autophagy receptors, which are characterized by the ability to recognize degradation signals on cargo proteins and also bind LC3/GABARAP proteins on the forming autophagosome. Our current goal is to understand the physiological function and selective nature of autophagy in neurons and dissect the molecular mechanism whereby selective autophagy clears disease related proteins particularly related to Alzheimer?s disease (AD). AD is characterized pathologically by the extracellular amyloid plaques and intraneuronal neurofibrillary tau tangles. Recent failures of AD clinical trials show the urgency to have deeper understanding of the pathogenic pathways and develop novel therapeutic strategies of AD. Indeed, multiple lines of evidence suggest that basal autophagy prevents the accumulation of phosphorylated tau (p-tau). Furthermore, our lab and others suggests that autophagy selectively degrades amyloid ? precursor protein (APP) and its metabolites (e.g. C- terminal fragments or CTFs and A?). We hypothesize that autophagy selectively removes toxic tau species and APP/APP metabolites through specific autophagy receptors. Given increasing evidence implicating autophagy in controlling the levels of p-Tau, APP and its metabolites, we propose that targeting selective autophagy pathway offers a novel disease-modifying strategy for the treatment of AD. We propose the following Aims to test above hypothesis: Aim 1. Determine the physiological function and the selective nature of autophagy in neurons. Aim 2. Examine the role for selective autophagy in the regulation of tau homeostasis and tauopathies. Aim 3. Determine the mechanism for selective autophagy in the clearance of APP and its metabolites. We seek to establish molecular basis for how selective autophagy regulates the homeostasis of the two most important AD related proteins, phospho-tau and APP (and its metabolites) in CNS; our study is expected to provides insight into the pathogenesis of AD and assist in the development of novel disease-modifying strategy for AD treatment.

The disease mechanism underlying Parkinson's disease (PD) is poorly understood. Mutations in LRRK2 (PARK8) have recently been linked to the most common familial forms (autosomal dominant) as well as some sporadic forms of Parkinson's disease (PD). LRRK2 protein contains multiple conserved domains including a kinase and a GTPase domain. Recent characterization of LRRK2 suggests that PD-associated mutations of LRRK2 cause enhanced kinase activity, which is linked to the neurotoxicity in neuron cultures. Our long-term goal is to elucidate the structure/function of LRRK2 in the CNS, to define the mutant LRRK2-mediated pathogenic pathways in PD, and to provide information for ultimate understanding of the pathogenesis of PD. The evidence that the pathology caused by familial PD gene mutations is restricted to the brain underscores the importance of brain-specific context in the onset and development of PD. Previously we have used an integrated system combining BAC (bacterial artificial chromosome)-mediated transgenic mice, proteomics and biochemical study to investigate the cellular function of LRRK2 in the context of brain. We have purified LRRK2 protein from the transgenic brain and found that the brain LRRK2 is associated with robust kinase and GTPase activity as compared to that from other tissues or cell cultures. Thus, we hypothesize that the kinase/GTPase activities of LRRK2 are specifically regulated by co- factors (e.g. proteins and lipids) in the brain. We will specifically test this hypothesis using purified LRRK2 from the brain (Aim 1). To further understand the regulation of LRRK2 enzymatic activity, we have identified phosphorylation sites in LRRK2 expressed in mouse brain. We have also developed an antibody against specific phosphorylation of LRRK2 to assist in the functional analysis of phospho- LRRK2. Since the levels of autophosphorylation of LRRK2 kinase are correlated with neurotoxicity, we propose to investigate the relationship between the identified phosphorylation and autophosphorylation in LRRK2, and to assess the functional significance of the identified phosphorylation of LRRK2 in LRRK2-mediated pathogenesis. In addition, we will test the hypothesis that specific phosphorylation of LRRK2 regulates LRRK2 kinase/GTPase activity and potentially modifies LRRK2-mediated pathological process in the brain (Aim 2). Finally, despite the recent evidence linking increased kinase activity and GTP binding of LRRK2 to neurotoxicity, the studies were performed mostly in cell cultures. In this proposal, we will use our BAC-mediated LRRK2 transgenic mice to validate these in vitro studies and further test the hypothesis of hyperactivity of LRRK2 kinase in the pathogenesis of PD in vivo using animal models (Aim 3). By integrating biochemistry, cell biology and novel mouse transgenic approach, our study is expected to provide mechanistic insight into the biology and pathology of LRRK2 protein, which is considered a promising drug target for the treatment of PD. Parkinson's disease (PD) is a major human neurodegenerative disease, but the pathogenic mechanism is not clear. This proposal will investigate the central function of a PD-related gene and define the pathogenic pathway mediated by PD-mutations of the gene in the central nervous system. It is expected to provide information for ultimate understanding of the etiology of PD and validation of drug targets for treatment of PD.

DESCRIPTION (provided by applicant): Our long-term goal is to understand the cellular and molecular basis for the initial pathogenic events of Parkinson's disease (PD), such as dopamine (DA) deficiency and aberrant synaptic activity that precedes and contributes to abnormalities in movement, learning and emotion. Using a BAC transgenic approach, we have previously investigated normal and pathophysiological functions of Leucine-rich-repeat-kinase 2 (LRRK2), a newly identified causative gene for familial PD, in mouse models. We have recently reported that LRRK2 is involved in regulating striatal DA transmission and consequent control of motor function. The LRRK2 mutation G2019S, which is the single most common genetic cause of PD, exerts pathogenic effects by impairing these functions of LRRK2. The emerging evidence thus suggests that this LRRK2 PD-linked mutation can initiate a series of pathological events (including the impairment of striatal DA transmission) at an early phase of PD preceding nigrostriatal degeneration. Our preliminary study has shown that LRRK2-G2019S causes aberrant synaptic plasticity in the striatum and hippocampus of our LRRK2 BAC transgenics. Therefore, these results suggest that LRRK2-G2019S triggers the deregulation of multiple neural pathogenic pathways that are consistent with abnormalities in both motor and cognitive deficits in PD. Furthermore, we found that brain LRRK2-G2019S has enhanced kinase activity, and in mouse brain LRRK2 kinase is responsible for the phosphorylation of Erzin/Radixin/Moesin (ERM), an event that is associated with spine morphogenesis. We hypothesize that the pathogenic role of LRRK2 is at both presynaptic and postsynaptic sites: (1) LRRK2 regulates DA homeostasis/transmission, whereas the G2019S mutation impairs DA transmission and causes DA deficiency; (2) LRRK2-G2019S triggers deregulation of multiple neural circuits implicated in clinical manifestations of motor, cognitive and psychiatric symptoms of PD; (3) Some pathological consequences of PD are caused by a combination of DA transmission deficits and postsynaptic abnormalities. Hence, we plan to test these hypotheses in our established BAC transgenic mice expressing LRRK2 variants: Aim 1: Determine cellular and molecular mechanisms through which LRRK2 regulates dopamine transmission that is impaired the PD-linked mutation G2019S; Aim 2: Analyze the pathogenic effects of LRRK2 in the electrophysiology of striatum and hippocampus; Aim 3: Analyze the pathogenic effects of LRRK2 in dendritic morphology and its plasticity; Aim 4: Determine whether LRRK2 transgenic mice develop cognitive deficits and dopamine-related motor abnormalities. The outcome of this study is expected to shed light on the pathogenic mechanism underlying the motor and non-motor symptoms of PD, and reveal causative molecular events at an initial stage of PD that are critical for biomarker identification, early diagnostics and therapeutic intervention.

DESCRIPTION (provided by applicant): The overarching goal of this renewal R01 is to advance our understanding of the biology and pathology of Leucine-Rich Repeat Kinase 2 (LRRK2), whose mutations are the most common genetic cause of Parkinson's disease (PD). LRRK2 encodes a large and complex protein (285kD) including a kinase and a GTPase domain. Previously multiple lines of evidence have led to a gain-of-function hypothesis that LRRK2 pathogenic mutations cause increased kinase activity that is attributable to the neurotoxicity. Recent studies in cell cultures implicate LRRK2 in vesicle trafficking, neurite outgrowth, cytoskeletal dynamics, protein translation and degradation, mitochondria dynamics and inflammatory response. Importantly, the study of genetic animal models show that the pathogenic LRRK2 mutations impair dopamine transmission without causing neurodegeneration, suggesting a pathophysiological role of LRRK2 in neurotransmission at early disease stage prior to neurodegeneration. Emerging evidence has also linked LRRK2 to neuroinflammation that is a contributing factor to neurodegeneration in PD. But the precise mechanisms whereby LRRK2 mutant mediate the neural dysfunction and neurotoxicity remain unclear. Therefore, we hypothesize that (1) LRRK2 regulates SV protein functions and neurotransmission that is impaired by LRRK2 pathogenic mutations; 2) LRRK2 plays a critical role in neuroinflammatory response in glial cells; LRRK2 mutants deregulate glial inflammatory pathway and cause neurotoxicity in PD. Our specific aims are to (1) determine the pathogenic role of LRRK2 in SV traffic and neurotransmission; (2) examine dysfunctional LRRK2 in glial neuroinflammatory response. A major challenge of LRRK2 research is the lack of well-defined neurodegeneration models of LRRK2 that are relevant to the PD pathogenesis. In fact, the incomplete disease penetrance of the common mutation G2019S suggests a significant contribution of environmental factors to PD pathogenesis. Our third aim is then to test the hypothesis in animal models that LRRK2 causes neurodegeneration in PD through genetic lesion and environmental toxin-linked neuroinflammation. Successful completion of the study not only will gain insight into LRRK2 biology and pathophysiology, but also will deliver valuable cell and animal models for interrogating neurodegenerative pathways in PD and developing platforms for LRRK2 inhibitor screening.

Project Summary/Abstract Our goal is to elucidate autophagy-lysosomal mechanism in the pathogenesis of Parkinson's disease (PD). Early pathological analysis of PD and recent studies of PD-linked genes, such as SNCA, LRRK2, Parkin, Pink1, GBA and ATP13A2, implicates dysfunctional autophagy-lysosomal pathway in the pathogenesis of PD. Recent evidence shows that inhibition of LRRK2 or expression of LRRK2 PD mutants causes aberrant autophagic activity, but the detailed mechanism is unclear. Our preliminary study suggests a link of LRRK2 to an autophagy kinase, which is essential for autophagy and required for axon/neurite outgrowth. Here we propose to investigate the molecular mechanism whereby LRRK2 regulates autophagy through directly targeting autophagy machinery and dysfunctional autophagy as a potential PD pathogenic pathway. We previously reported that disruption of autophagy in mice leads to age-dependent accumulation of endogenous ?-Syn in dystrophic axons and altered dopamine transmission, consist with impaired autophagy as one of the failing cellular mechanisms involved in the pathogenesis of PD. Emerging evidence suggests that autophagy not only participates in degradation of ?-Syn, but also is involved in the secretion pathway of ?-Syn, which underlies the spreading of synucleiopathy. Fibrillar ?-Syn can be secreted through exosomes and/or autophagosome-related structures. Furthermore, increasing autophagy gene beclin1 or transcription factor TFEB expression facilitates the autophagic clearance of ?-Syn and offers neuroprotection in rodent models. However, the precise mechanism for the autophagic clearance of intracellular ?-Syn remains poorly characterized. Finally, whether LRRK2 also contributes to the control of ?-Syn homeostasis through regulation of autophagy is unclear. Given the emerging role of autophagy in the exocytosis of ?-Syn, we hypothesize that autophagy regulates ?-Syn clearance through both degradative and secretory pathways; LRRK2 modulates ?- Syn homeostasis by targeting autophagy. In Project 3 we propose (1) to determine molecular and genetic basis for LRRK2-mediated autophagy regulation. We will use biochemical, cellular and animal models to dissect the role for LRRK2 wildtype and PD mutation G2019S in autophagy control through modulating the autophagy kinase and SNARE-related protein trafficking; (2) to determine the mechanism that autophagy and LRRK2 modulate ?-Syn homeostasis by targeting multiple trafficking pathways. We will apply multidisciplinary approaches to test that LRRK2 and autophagy pathway converge to regulate ?-Syn homeostasis by targeting secretion and degradation in cell and animal models of PD. Our project 3 will be conducted in close collaboration with Drs. Jie Shen and Tom Südhof, who have extensive experience in LRRK2 and ?-Syn research, respectively. Completion of our project 3 is expected to provide timely knowledge for understanding LRRK2 pathogenic mechanism and developing potential therapeutic strategies by targeting autophagy-lysosome pathways for the clearance of ?-synuclein.

? DESCRIPTION (provided by applicant): The goal of our project is to investigate pathogenic mechanism underlying Parkinson's disease (PD) based on inherited forms of PD. We and two other groups have identified the same mutation in SYNJ1 gene on chromosome 21q22 that is associated with early onset Parkinsonism. As one of the most important inositol phosphatases in the brain, synaptojanin1 (encoded by SYNJ1) is involved in many aspects of membrane trafficking and has been implicated in a number of brain disorders including Alzheimer's disease and Down's syndrome. However, its pathogenic mechanism in PD has not been studied. Emerging evidence reveals the convergence of pathogenic pathways for ?-synuclein, LRRK2 and other PD-related genes in deregulating synaptic vesicle (SV) cycling in the early stage of PD. Our preliminary study shows shared phenotypes in SV endocytosis and dopaminergic transmission in SYNJ1 heterozygous mice and LRRK2-G2019S mice. SYNJ1 heterozygous mice develop abnormal dendritic morphology and lipid composition selectively in substantia nigra. Given that synaptojanin1 forms a protein complex with endophilin A, which was identified as a putative LRRK2 substrate, we propose a LRRK2-endophilinA-synaptojanin1 signaling axis for the regulation of SV cycling, which is deregulated in PD. We will test the hypothesis using state-of-art optical tools and genetic animal models. Completion of our project is expected to provide insight into the pathogenic mechanism for PD and open new avenues for identification of novel therapeutics.

? DESCRIPTION (provided by applicant): Statement of Work. The research proposed in this multi-PI application with three PIs (Drs. Hoang, Yue and Ubarretxena) combines the efforts of three different laboratories to study the structure and activity regulation of Leucine Rich Repeat Kinase 2 (LRRK2). Mutations in this unique multi- domain enzyme have been linked with Parkinson's disease (PD) pathogenesis, and thus LRRK2 has emerged as a key therapeutic target for the treatment of this neurodegenerative disorder. The results obtained from X-ray crystal structures of the GTPase domain and the COR region carrying PD-linked mutations (Dr. Hoang component) and those obtained from the high- resolution electron microscopy 3-D models of full-length LRRK2 (Dr. Ubarretxena component) will be used to inform the biochemical, cell-based and animal studies (Dr. Yue component). In particular, we will learn how the GTPase and kinase activities in LRRK2 are coordinated and how domain-domain interactions in the enzyme, and PD-linked mutations affect these activities.

Our goal is to investigate the molecular mechanism that SYNJ1/PARK20 contributes to the pathogenesis of Parkinson's disease (PD) and understand if SYNJ1 is involved in the common PD pathogenic pathway. The available evidence suggests that diverse cellular functions are involved in PD pathogenic pathways, including synaptic trafficking and autophagy-lysosome. SYNJ1 encodes synaptojanin1 (synj1), a phosphoinositide phosphatase and a binding partner for endophilinA. The synj1 is enriched in nerve terminals where it regulates synaptic vesicle (SV) recycling and synaptic protein targeting by hydrolyzing membrane phosphoinositides via its two phosphatase domains (SAC1 and the 5-phosphatase) and by binding to endophilinA. At least three recessive point mutations located in the SAC1 or 5-phosphatase domain of the SYNJ1 gene, are linked to the Parkinsonism. We further showed that R258Q mutation abolishes SAC1 phosphatase activity of synj1, suggesting a loss of function of synj1 responsible for the disease. Mice carrying R258Q mutant of synj1 develop dystrophic terminals selectively in the nigral DAergic neurons and impaired SV recycling, confirming the causality of the mutation to the disease and suggesting selective effect of SYNJ1 dysfunction in DAergic neurons. While the pathogenic variants of SYNJ1 and other recessive PD genes are extremely rare, whether they are involved in the more common form of PD (idiopathic) remains unclear. We reported that SYNJ1 heterozygous deletion (SYNJ1+/-) midbrain DAergic neurons display slowed SV endocytosis, and our preliminary study of SYNJ1+/- mice show reduced striatal DA content and reduced density of DAergic nerve terminal in an age dependent manner. The result suggests haploinsufficiency of SYNJ1 leads to dysfunctional DAergic neuron terminal dystrophy. Furthermore, we found down-regulation of human SYNJ1 transcripts in sporadic PD brains, raising a possibility that SYNJ1 deficiency is linked to the pathogenic pathway of more common PD. We hypothesize that reduced expression of SYNJ1 predispose DAergic neuron to dystrophic terminal degeneration and neurotoxicity underlying PD pathogenesis. We will investigate the two aims: Aim 1. To validate the hypothesis that haploinsufficiency of SYNJ1 contributes to progressive DAergic neuron vulnerability and PD related symptoms in animal models; Aim 2. To determine that reduced SYNJ1 expression in human iPSC DAergic neurons causes presynaptic vulnerability and PD related pathogenic process. Therefore, our study not only will provide an insight into the molecular mechanism for SYNJ1 rare mutation related Parkinsonism, but importantly may also reveal the pathogenic mechanism for more common form of PD (idiopathic) and identify novel therapeutic targets in regulating phosphoinositide levels.

Our goal is to elucidate molecular mechanism for neuroprotective autophagy in Huntington's disease (HD) and determine therapeutic potential for selective autophagy in treating the disease. HD is caused by an aberrant expansion of CAG repeat (polyQ) in the HTT gene, which leads to a toxic gain-of-function in the mutant huntingtin (mHTT) protein. Despite over 20 years' research, disease-modifying therapeutics is unavailable. Thus, elucidation of the disease mechanism and mHTT clearance pathways is pivotal for the success of therapeutic development. Autophagy is a catabolic cellular pathway that clears protein aggregates and injured organelles through lysosomes as a quality control system. PolyQ-expanded protein aggregates including fragments of HTT can be degraded by selective autophagy, and thus selective autophagy is considered a drug target for HD. However, autophagy is a complex process subjected to tight regulation, and how exactly autophagy selectively degrade mHTT remains poorly understood. We previously showed that ULK1 regulates p62-mediated selective autophagy under proteotoxic stress. In the context of mHTT, however, we reported dysregulation of ULK1 kinase activity that connects to reduced VPS34 activity and aberrant p62-selective autophagy in the brains of HD model zQ175. Our current study suggests that ULK1 deficiency accelerates mHTT-mediated toxicity. The data thus provides strong evidence for the role of ULK1-p62 mediated selective autophagy in regulating mHTT toxicity. We hypothesize that ULK1 and p62 are promising modifiers of HD disease progression. We propose (1) to determine the role and mechanism for ULK1-p62 signaling in the degradation of mHTT through selective autophagy; (2) to investigate pathogenic mechanism that mHTT disrupts ULK1 kinase activity and causes ULK1 deficiency-mediated selective autophagy impairment and neurotoxicity; (3) determine ULK1 kinase activity as a therapeutic target to inhibit mHtt-mediated neurotoxicity using animal models through genetic and pharmacological approaches. Our study is expected to reveal molecular mechanism for ULK1 protective function against HD and validate ULK1 kinase activity as a drug target for the clearance of mHTT and offering neuroprotection.

Our central goal is to determine neuroprotective mechanism conferred by microglia and autophagy, and understand how dysfunctional autophagy in microglia contributes to the pathogenesis of Alzheimer's disease (AD). Emerging evidence from human genetic and pathological studies has demonstrated the significance of microglia pathophysiology in the pathogenesis of AD. Microglia are the resident innate immune cells in the brain. The exact role for microglia in AD pathogenesis, however, remains poorly understood. Multiple lines of studies revealed the protective function of microglia that restrain the toxic accumulation of ?-amyloid and prevent disease progression. However, evidence also exists suggesting excessive microglial activation can harm the neurons by releasing inflammatory factors and engulfing neuronal synapses. Microglia may phagocytose A?, the main component of plaques as a hallmark of AD pathology; single-cell RNAseq analysis showed the disease-associated microglia (DAM), which localizes at plaques in AD animal models, consistent with a role of TREM2 as a critical regulator of DAM activation. Autophagy is a lysosome clearance pathway that plays an important role in maintaining homeostasis under metabolic stress and neuroprotection. Little is known about glial autophagy. Previous studies from peripheral immune cells demonstrate a significant role of autophagy in immunity and inflammation. Whether microglial autophagy plays such a role, however, remains poorly understood. We recently analyzed AD mouse model and observed the activation of microglial autophagy. We found that DAM is associated with a robust increase of autophagic activity. We also showed that inactivation of microglial autophagy causes reduced number of microglia associated with A? plagues and enhanced neurotoxicity in AD models, which phenocopied the effect of the loss of Trem2 in AD models. Therefore, our overall hypothesis is that autophagy activation is required for DAM metabolic fitness to degrade A? and protect neurons in the AD brains. We also hypothesize that microglial autophagy controls inflammation by selective degradation of inflammasomes via protein receptors that are neuroprotective in AD. Our specific aims are to (1) determine the role for microglial autophagy in neuroprotection by clearing phagocytosed A? and maintaining metabolic fitness in AD mouse models; (2) dissect the mechanism of microglial autophagy that controls inflammation in AD mouse model; (3) determine that autophagy is an integral part of TREM2-mediated neuroprotection mechanism in microglia of AD mouse model.