Hui Zheng - US grants

Mutations in Presenilin-1 (PS1) are linked to early onset of familial Alzheimer's disease (FAD) and lead to increased production of Abeta42, a peptide that is accumulated during aging and plays a critical role in AD pathogenesis. PS1 is a multi- pass transmembrane protein that is essential for mouse embryonic development and is required for Abeta peptide generation. PS1 also interacts with beta-catenin and has been implicated in regulating beta-catenin stability in vitro. The in vivo function of PS1 in the adult central nervous system and other tissues is poorly understood, due to the early lethal phenotype of the PS1 null mouse. We have reported that transgenic mouse lines expressing either the wild-type human PS1 protein or PS1 containing the A246E FAD mutation, under the neuronal-specific human Thy-1 promoter, can protect the PS1 null mouse against embryonic lethality and simultaneously restore Abeta expression. This "rescue" system allows us to further define PS1 in vivo activities by introducing specific modifications, such as PS1 mutations affecting Abeta synthesis or beta-catenin interaction respectively. It also offers us a unique opportunity to study the effect of PS1 loss-of-function in adult peripheral tissues of existing mice as they are rescued by a neuronal specific expression of the PS1 transgene. Our preliminary data indicate that lack of PS1 expression in the skin of these mice leads to epidermal hyperplasia and neoplasm, suggesting that PS1 may play an important role in skin tissue, possibly through beta-catenin signaling pathway. Our long-term objective is to use our established rescue system to dissect multiple pathways which PS1 seems to participate in vivo. The specific aims of the proposal are: 1] To explore the molecular mechanism of PS1 activity in adult epidermis; 2] To identify the in vivo significance of PS1- beta-catenin interaction; 3] To determine whether the developmental activity and Abeta generating property of PS1 can be differentiated. We believe that these studies will advance our understanding of the physiological function of PS1 and the pathogenic mechanism of Alzheimer's disease.

DESCRIPTION (provided by applicant): Mutations in presenilin (PS1 and PS2) are linked to early onset of familial Alzheimer's disease (FAD). Presenilin is required for the processing of Notch and the b-amyloid precursor protein, molecules that play critical roles in development and AD pathogenesis respectively. In addition, PS1 associates with b-catenin, a multi-functional protein involved in Wnt signaling and cadherin/catenin-mediated cell adhesion. We reported that PS! down regulates b-catenin through this interaction. As such, loss of PS1 is associated with enhanced b-catenin signaling and tumorigenesis in mice. However, PS1 deficiency leads to simultaneous disruption of all presenilin activities and the definitive role of PS1-catenin pathway cannot be determined. Although PS1 -b-catenin interaction has been established, the role of PS2 in b-catenin regulation remains to be addressed. We recently discovered that PS2 facilitates PS1-mediated skin tumorigenesis. This calls for a need to systematically investigate the involvement of PS2 in b-catenin pathway. Directly relevant to AD, PS1 is localized at synaptic contacts and PS1 FAD mutant protein exhibits impaired b-catenin regulating activities, raising the possibility that an impaired b-catenin homeostasis may contribute to synaptic dysfunction, a hallmark intimately linked to AD dementia. The following hypotheses are formulated based on these findings: a) Presenilin-b-catenin association plays a central role in b-catenin signaling and cadherin/catenin-mediated cell adhesion in vivo. Deregulation of b-catenin is causal for loss-of-PS1-induced neoplasia and alters synaptic activity in the central nervous system; b) PS1FAD mutation leads defective b-catenin regulation and synaptic dysfunction; c) PS1 and PS2 cooperate to negatively regulate b-catenin. This application is aimed at testing these hypotheses using a novel biological tool we recently created: The PS1 knock-in mice with specific deletion of b-catenin interaction. This allele genetically separates the two important activities PS1 mediates, namely Notch and b-catenin, under in vivo physiological conditions, thus allowing us to definitely determine the role of PS1 -b-catenin pathway. The availability of the PS1M146V FAD knock-in mice makes it feasible to evaluate the effect of FAD mutation on b-catenin pathway. Further, the mechanisms of PS2 in b-catenin regulation will be investigated. The proposed study will lead to a comprehensive understanding of the molecular mechanisms of presenilin and the effect of FAD mutation on b-catenin regulation, tumorigenesis and synaptic formation and maintenance.

Presenilin (PS) represents a family of multi-pass transmembrane proteins consisting of, among others, Sel-12 and Hop-1 in C. elegans and PS1 and PS2 in mammals. Presenilin is required for the proteolytic activation of Notch and the enzymatic processing of beta-amyloid precursor protein (APP), molecules that play pivotal roles in cell fate determination during development and Alzheimer's disease (AD) pathogenesis through aging respectively. Presenilin-dependent Notch processing and signaling is highly conserved and exhibit strong amino acid sequence preference. In contrary, cleavage of APP by presenilin occurs at multiple sites, generating two major beta- amyloid peptides: Abeta40 and Abeta42. Mutations in human PS cause early onset of familial Alzheimer's disease (FAD) and the pathogenic effect is most likely mediated by their ability to increase the ratio of Abeta42/Abeta40. However, it has been difficult to understand this apparent "gain-of-misfunction" mechanism because the FAD mutations spread throughout the entire molecule and the mutant protein is only partially active compared to wild-type presenilin in C. elegans. The current application is aimed at resolving these unsettling and sometimes conflicting data. We hypothesize here that PS FAD mutation leads to concurrent partial loss of function on Notch and APP. Reduced activity at the preferred Abeta40 site by the PS FAD mutation drives the increase in the Abeta42/Abeta40 ratio. This hypothesis allows interpretation of presenilin function and the effect of PS FAD mutation on Notch and APP pathways across all species under one mechanism. Furthermore, since PS-mediated Notch activity is highly conserved; we propose that the C. elegans presenilin represents a partial active allele in mammalian system exhibiting similar effects as the PS FAD mutant protein. We plan to test the hypothesis in vivo using two powerful transgenic mouse systems: One is the PS1 FAD knock-in system that allows determination of FAD mutation under physiological context and the other is our novel PS1 "rescue" system that makes it possible to test various presenilin alleles for their Notch developmental and AD pathogenic activities. Because of the indispensable role of presenilin in multiple biological processes and the robust pathogenic impact of the PS mutations in Alzheimer's disease pathogenesis, determining the molecular mechanisms underlying these presenilin-mediated pathways and the effect by the FAD mutations in vivo is crucially important. The studies proposed will lead to better evaluation of presenilin-based therapy as well as advanced understanding of AD pathogenesis in general.

DESCRIPTION (provided by applicant): Mammalian presenilins consist of two homologous proteins: PS1 and PS2. They are indispensable for the proteolytic processing of a variety of substrates including Notch and the amyloid precursor protein (APP), molecules that play critical roles in cell fate determination and Alzheimer's disease (AD) pathogenesis, respectively. In addition, PS1 associates with beta-catenin---a multi-functional protein involved in cell adhesion, Wnt signaling and tumorigenesis. The original application was aimed at investigating the role of PS 1-beta-catenin pathway in skin tumorigenesis and dissecting the PSI-mediated activities in vivo. We have achieved all of our objectives within the grant-funding period. We reported that PS 1 facilitates beta-catenin turnover. As such, loss of PS 1 is associated with enhanced beta-catenin signaling, activation of its downstream target cyclin D1, accelerated cell proliferation and skin tumorigenesis in mice. Using our novel human PS1 "rescue" system in which expression of wild-type PSI could rescue the mouse PS1 null lethal phenotype, we established that a) PS 1 in Notch processing and beta-catenin interaction can be genetically and functionally uncoupled; and b) Aspartate 257 of PS 1 is critical for Notch and APP proteolysis, thus providing strong support that these two pathways are mediated through the same mechanisms. Two important findings emerged during the course of the study: a) We discovered a widespread role of presenilins in regulating cell proliferation and tumorigenesis: Presenilin deficiency leads to age-dependent myeloproliferative defect indicative of human chronic myelogenous leukemia (CML). Investigating the nature of the defect and determining the molecular mechanisms of presenilins in hematopoiesis is one of the research topics of the current application; b) We established a potent and specific regulation of cyclin D1 by presenilins in both mitotic cells and developing neurons. This observation, combined with the fact that activation of cyclin D1 is an early marker in degenerating neurons of AD patients, leads to an appealing hypothesis that compromised presenilin function, by genetic or environmental insults could result in deregulation of cyclin D1 and unscheduled cell cycle re-entry, with the outcome of neoplasia in peripheral tissues and neurodegeneration in the central nervous system. This competitive renewal is aimed at experimental approaches to test this hypothesis using our powerful mouse genetic systems. The studies combined will significantly advance our understanding of the molecular mechanisms underlying presenilin activities in tumorigenesis, neurodegeneration and AD pathogenesis.

[unreadable] DESCRIPTION (provided by applicant): Mutations in presenilins (PS1 and PS2) are causal for early-onset familial Alzheimer's disease (FAD). These mutations are known to alter the gamma-secretase processing of the amyloid precursor protein (APR), resulting in an elevated ratio of Abeta42/Abeta40, peptides that constitute the principal components of the amyloid plaque pathology characteristic of Alzheimer's disease. However, this increased ratio of Abeta42/Abeta40 does not provide mechanistic insights as it can result from elevated production of Abeta42, a reduction of Abeta40 or both combined. In the original grant application, we hypothesized that the PS FAD mutations lead to partial loss of activity on APP cleavage at the predominant Abeta40 site. Reduced production of Abeta40 is the primary cause for the increase in the ratio of Abeta42/Abeta40. Using our novel mouse genetic approach, we provided strong experimental support for this "partial loss-of-function" hypothesis for the PS FAD mutations, not only in gamma-secretase processing of APP, but also in other PS-mediated physiological pathways. Building on the partial loss-of-function hypothesis and our established mouse genetic systems, in particular the PS conditional knockout, PS1 FAD knock-in and our novel PS1 "rescue" system, in this competitive renewal, we propose to decipher the gamma-secretase dependent and gamma-secretase independent activities of PS and determine the effects of PS FAD mutations on tau phosphorylation, and neuronal and synaptic function in vivo. Furthermore, we will test our new hypothesis that NFkappaB and GSK are critical downstream effectors of PS by pharmacological and genetic manipulations combined with biochemical, immunohistochemical, electrophysiological and neuroimaging analyses. Presenilin plays a pivotal role in AD pathogenesis. Mutations in PS have been shown to affect both Abeta production and tau hyperphosphorylation, which are the determinants of the amyloid plaque and neurofibrillary tangle pathologies respectively. Therefore, delineating these PS-mediated pathways and investigating the mechanisms of PS mutations as proposed are critically important for our understanding of AD pathogenesis and for developing effective therapeutics that target the specific activities of presenilin. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Genetic and biochemical studies establish a central role of the amyloid precursor protein (APP) in Alzheimer's disease (AD): APP processing generates b-amyloid (Ab) peptides, which are the principal components of the amyloid plaque pathology; mutations in APP are causal for a subset of early onset of familial Alzheimer's disease (FAD). Although b-amyloid plaques are the hallmark of AD, synaptic dysfunction is closely associated with cognitive impairment, and cholinergic neurons undergo profound changes in AD. The mechanisms underlying these pathogenic events are not clearly defined. We reasoned that understanding the physiological function of APP, which thus far remains elusive and controversial, would provide pathogenic insights. To this end, we have created mice deficient in APP and revealed that APP is important in hippocampal synaptic plasticity and cholinergic synapse function. Our recent investigation of the molecular and cellular mechanity and learning and memory. Our current proposal is aimed at deciphering the biochemical and functional mechanisms of these pathways in various neuronal circuitry and investigate the effects of Ab and APP FAD mutations. We are equipped with the novel APP conditional knockout mice and humanized APP/Ab FAD knock-in mice to address these critical questions concerning the pathophysiology of APP in vivo. PUBLIC HEALTH RELEVANCE: APP plays a pivotal role in AD pathogenesis. Deciphering the in vivo function of APP in neurons and synapses and evaluating the effects of b-amyloid peptides and the disease-causing mutations as proposed represents a critical area of AD research. Our studies will provide a comprehensive understanding of the role of APP in various neuronal circuitry and reveal novel pathogenic insights into Alzheimer's disease.

DESCRIPTION (provided by applicant): Proteolytic cleavages of the amyloid precursor protein (APP) generate beta-amyloid peptides (A?). Although beta-amyloid pathology is the hallmark of Alzheimer's disease (AD), synaptic dysfunction is believed to play a primary role in AD pathogenesis. Since A? is produced as part of APP processing, we reasoned that understanding the mechanisms of APP and its processing products in synaptic function, and investigating the effects of A? in the context of APP are of crucial importance. Whereas various neuronal and synaptic activities of APP have been proposed, their physiological relevance remains largely unestablished. To this end, we generated mice deficient in APP and reported that APP plays a functional role in hippocampal synaptic plasticity and learning and memory. We recently created a strain of APP conditional knockout mice. Analysis of these animals demonstrates an essential role for the APP family of proteins in neuronal survival and synaptic structure and function. Intriguingly, APP-mediated synaptogenic activity requires its expression in both pre- and postsynaptic compartments, supporting a functional interaction of APP across synapse. Our proposal is aimed at testing this trans-synaptic APP interaction model, deciphering the activities of APP processing products, including A?, in APP-mediated synaptic property, and identifying the APP downstream targets using a combination of state-of-the-art in vitro technologies and physiological and disease-relevant mouse models. In particular, we are equipped with the novel APP conditional knockout mice and humanized APP/A? knock-in mice and are uniquely positioned to address these fundamental questions concerning the pathophysiology of APP and A? in central synapses.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is an age-related neurodegenerative disorder defined by the deposition of ¿- amyloid (A¿) plaques and neurofibrillary tangles (NFT), of which the principal components are A¿ peptides derived from the amyloid precursor protein (APP) and hyper-phosphorylated tau, respectively. The relationship between the extracellular A¿ and intracellular NFT pathologies and how they mediate synaptic dysfunction, which is regarded as a causal event in AD, remain poorly understood. Mutations in APP and presenilin (PSEN) genes lead to early onset of familial AD (FAD), establishing their critical roles in AD pathogenesis. Based on the genetic evidence, we have established APP and PS1 knockin mouse models (APP/hA¿/PS1) in which the disease-causing mutations and human A¿ sequence are introduced into the endogenous APP or PSEN1 loci such that the pathogenic effects of the FAD mutations and A¿ can be investigated under their physiological context. Analysis of the knockin mice show that they exhibit profound anxiety phenotypes, likely mediated by impaired inhibitory neuronal function. We have now developed a second generation of the knockin mice which allow us to express the APP/PS1 mutations and A¿ in specific neurons and brain regions. Equipped with these powerful mouse models, the application seeks to gain mechanistic and functional understanding of AD pathogenesis by addressing the following fundamental questions: 1) What is the relationship between anxiety/stress and dementia? 2) How does the specific impairment of excitatory or inhibitory neurons contribute to synaptic dysregulation and whether they differentially mediate cognitive and stress-related behaviors? 3) What are the molecular and cellular mechanisms mediating mutant APP/hA¿/PS1-induced NFT pathology? Our proposal will reveal unprecedented insights into AD pathogenesis and we are equipped with innovative and sophisticated mouse models to address these questions.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common form of age-related neurodegenerative disorder defined by the deposition of ¿-amyloid (A¿) peptides which are generated from processing of the amyloid precursor protein (APP). Mutations or gene duplication of APP are causal for a subset of early onset AD, establishing its central role in AD pathogenesis. However, the vast majority of AD cases are late onset in which advanced age and the ¿4 allele of the apolipoprotein E (ApoE) gene are established risk factors. Age is associated with many changes, one of them is adult neurogenesis, a process that generates functional neurons throughout life but its ability declines with age. Impaired adult neurogenesis in the hippocampus has been implicated in learning and memory decline associated with aging and in AD. Interestingly, ApoE has been shown to play multiple roles in adult hippocampal neurogenesis through both cell-autonomous and non cell-autonomous mechanisms the latter requires GABAergic interneurons and is differentially influenced by the ApoE3 and ApoE4 isoforms. Using various APP genetic mutants we created during the current funding cycle and as part of our long-standing research in understanding the APP pathophysiology, we uncovered a potent role of APP in mediating adult hippocampal neurogenesis and, remarkably, many of the phenotypes observed in APP mutant mice resemble those of ApoE mutants. The overarching goals of the renewal application are to decipher the cellular mechanisms of APP in adult hippocampal neurogenesis, to investigate the genetic and functional interactions of APP and ApoE, and to evaluate the effects of APP gene dosage and A¿ on APP/ApoE-mediated adult neurogenesis using the APP YAC genomic transgenic mice. These mice will allow us to elucidate the pathogenic mechanisms since both APP gene duplication and mutation are causal for AD. This proposal is highly innovative and significant in that it brings APP and ApoE, molecules that play critical roles in familial early-onset and sporadic late-onset of AD, togethe and addresses a question that's directly relevant to aging and Alzheimer's disease.

? DESCRIPTION (provided by applicant): Tauopathies consist of a group of diseases, including frontotemporal dementias and the most common form Alzheimer's disease, and are characterized by the accumulation of intracellular neurofibrillary tangles (NFTs) composed of aggregates of hyperphosphorylated Tau protein and extensive neurodegeneration. Tau is normally localized to the neuronal axons where it binds and stabilizes the microtubules. Aberrant Tau phosphorylation leads to its dissociation from the microtubules followed by aggregation and redistribution to cell bodies and dendrites. In addition, Tau has been shown to be secreted, and this mechanism has been implicated in the prion-like transfer of Tau pathology. Accumulating evidence has implicated impaired autophagy-lysosome pathway in neurodegenerative diseases including Alzheimer's disease. Recently, the Transcription Factor EB (TFEB) was discovered as a master regulator of cellular clearance through coordinated expression of autophagy and lysosomal target genes. We found that TFEB is highly efficacious in ameliorating phospho-Tau/NFT pathology, neurodegeneration, and behavioral deficits in rTg4510 Tau transgenic mice while exhibiting no adverse effect on wild-type mice; TFEB is effective when introduced both before and after the onset of neuropathology. Besides the autophagy-lysosome pathway, substantial evidence also supports a non-cell-autonomous role of TFEB in cellular clearance through neuronal exocytosis and astroglial-mediated endocytosis. Therefore, we hypothesize in this proposal that TFEB promotes lysosomal exocytosis and subsequent astroglial uptake of Tau, and that TFEB-mediated glial uptake of extracellular Tau prevents cell-to-cell transfer of the NFT-like pathology. We are equipped with sophisticated mouse models and innovative approaches that allow us to address these questions in vitro and in vivo at the subcellular, cellular, and network levels. At the completion of the proposal, we wil have gained mechanistic and functional insights into the poorly defined role of astrocytes in Tau clearance and the interplay between neurons and astrocytes in mediating Tau pathogenesis.

Astrocytes are the most abundant cell type in the CNS that play vital roles in all facets of brain physiology. Activation of astrocytes, or reactive astrogliosis, is associated with morphological, gene expression, and functional alterations. Although changes in astrocyte dynamics a common feature in Alzheimer's disease (AD), its underlying mechanisms and functional consequences remains poorly understood. Our recent studies identified five astrocyte subpopulations that display diverse molecular and functional characteristics in the brain. Significantly, one of the subpopulations, the synaptogenic astrocytes, strongly correlated with human AD expression datasets. This is exciting because synaptic dysfunction is widely accepted as an early and causal event in AD pathogenesis. Our long term goal is to decode the astrocyte types and determine the impact of the diverse astrocyte populations in aging and AD. The objectives of this proposal are to define the cellular and molecular heterogeneity of astrocytes in the cortex and hippocampus and to ascertain the functional role of synaptogenic astrocytes during aging and in AD mouse models. We hypothesize that diverse astrocyte subpopulations in the adult brain differentially contribute to AD pathogenesis and that reduced function of synaptogenic astrocytes plays a crucial role in driving synaptic dysfunction in early AD. To test the hypotheses we will profile changes of astrocyte subpopulations and define their molecular signatures in wild-type mice and AD mouse models as a function of age and AD pathology and cross-validate these results in postmortem human samples and associated expression datasets. We will decipher the role of synaptogenic astrocytes in AD pathogenesis by genetic targeting and functional testing of selected candidates. These studies will be led by two investigators with exceptional track-record in astrocyte biology (Deneen) and AD pathophysiology (Zheng) and assisted by outstanding bioinformatics support. Overall the proposal will significantly advance our understanding of astrocyte heterogeneity in brain regions critical to AD and how early and late astrocyte dysfunction contributes to AD pathogenesis. It will also lead to the identification of novel biomarkers and therapeutic targets.

Abstract Alzheimer?s disease (AD) is an age-related neurodegenerative disorder defined by the deposition of ?-amyloid (A?) plaques and accumulation of neurofibrillary tangles (NFTs), of which the principal components are A? peptides derived from the amyloid precursor protein (APP) and hyper-phosphorylated Tau, respectively. The relationship between the extracellular A? and intracellular NFT pathologies and how aging contributes to the disease pathogenesis are critical questions but remain poorly understood. Besides the pathological hallmarks, AD is associated with profound neuroinflammation marked by reactive astrogliosis and microgliosis. The complement pathway is a well-recognized innate immunity modulator. We uncovered an astroglial C3 and neuronal and microglial C3a receptor (C3aR) mediated neuron-immune signaling network that is prominently elevated in human AD and in AD mouse models where inactivation of C3aR ameliorates AD pathology and cognitive impairment. Since C3aR is widely expressed in the central nervous system, the C3aR blockade could exert its beneficial effects by acting on these cell types individually or by influencing the neuron-immune network. Indeed, crosstalk between microglia, astrocytes, and neurons has long confounded elucidation of their individual roles in neuroinflammatory damage. To tackle this problem, we have created a C3ar1 conditional allele that allows us to inactivate the C3aR in different cell types in the CNS and delineate their cell- type-specific roles. We have also developed a powerful technology that enables us to isolate high-quality RNA and to perform RNA sequencing analysis from aged dissociated and fluorescence-activated cell sorting (FACS) sorted individual neurons, astrocytes, and microglia. This affords unbiased analysis of central immune and inflammatory pathways with unprecedented cellular specificity. Building on these exciting biological and technical developments, we propose to a) decipher the role and cell-type-specific contribution of C3-C3aR pathway in AD pathogenesis using APP knock-in mouse models; b) interrogate cell-type-specific changes as a function of A? pathology and C3aR ablation and identify common signatures in human AD; c) determine the cellular mechanisms underlying complement mediated Tau/NFT pathology. These will provide unprecedented insights into the cell-type-specific targets in AD pathogenesis and we are equipped with innovative technology and sophisticated mouse models to address these questions.

Abnormal accumulation of misfolded Tau protein is tightly linked pathogenically to a group of brain degenerative disorders including Alzheimer's disease (AD) that are collectively called Tauopathies. Currently there is no effective prevention or treatment avenue against these debilitating diseases. Targeted clearance of misfolded, pathogenic Tau species thus represents one potentially vital therapeutic strategy. A flurry of recent studies, corroborating with a long history of clinical observations, have led to the proposal that Huntington's disease (HD), another fatal neurodegenerative disorder caused by an abnormal expansion of a glutamine tract (polyQ) in Huntingtin (HTT) protein, is also a tauopathy disease. Interestingly, while working on the HTT homolog in Drosophila, we found that normal HTT can promote the clearance of certain misfolding-prone Tau species by acting as a scaffold in selective autophagy, a subtype of autophagy-lysosomal pathway that requires cargo receptors such as p62/SQSTM1 to recognize and target specific cytosolic components for lysosomal degradation. Subsequent studies in mammalian cells and in mouse AD models further supported this finding, which, together with the known autophagic phenotypes in HD cells and in mice expressing polyQ-deleted HTT, suggest a scenario that polyQ expansion in HTT disrupts its own activity in promoting selective autophagy and normal Tau turnover, leading to the Tau pathology. Such a hypothesis not only supports the HTT-mediated autophagy pathway as a converging mechanism linking HD and Tauopathy, but also raises the promise of harnessing this conserved innate protective pathway for targeted removal of pathogenic Tau in Tauopathies. In this joint R01 application, we will use our established assays in the complementary Drosophila, cellular, and mouse model systems to rigorously and systematically test this hypothesis at the genetic, biochemical and functional levels. Taking advantage of the conserved HTT and autophagy pathways, as well as the availability of a plethora of Tau models and Tau toxicity assays established in Drosophila, we will use flies as an in vivo tool to evaluate the effect of polyQ lengths on the autophagic function of HTT, and to examine the discrete Tau species to search for the ones that can be degraded by the HTT-mediated selective autophagy and for their common signatures; Using mammalian cell-based assays, we will validate the findings from the flies and also probe the molecular mechanisms underlying the modulatory role of the polyQ stretch on HTT activities. Finally, by manipulating Tau and HTT in the well-characterized mouse HD and AD models, we will directly validate our hypothesis and findings in an in vivo setting that is physiologically closer to humans. Completion of this project will reveal novel mechanistic insight into the crosstalk between Tauopathy and HD, and establish the feasibility of future pharmacological exploitation of the novel selectively autophagy pathway to combat morbidity and mortality arising from Tau-associated AD and other Tauopathies.

ABSTRACT This is an Admin supplement request to purchase a ThermoFisher nanoflow liquid chromatography. This new add-on instrument will advance mechanistic studies and drug development in the parent award, aid the development of a novel chemoproteomics strategy, and enhance the overall goals of the parent grant.

Project Summary/Abstract Alzheimer's disease (AD) is one of the most persistent and devastating ailment of old age. Besides the deposition of ?-amyloid plaques and accumulation of neurofibrillary tangles, AD brains are marked by prominent neuroinflammatory responses, which plays a crucial role in disease pathogenesis. Although aging is known to profoundly influence tissue homeostasis, how age-induced brain inflammation specifically contributes to the progression of AD remains elusive. The cytokine family type I interferon (IFN) is a major innate immune mediator produced in response to microbial infections. We have detected elevated signals of IFN pathway activation in the brains of normal aging mice as well as APP transgenic and knock-in animals expressing human amyloid beta (A?). Previously by manipulating the generation of amyloid, we established that innate immune cells readily produce IFN upon exposure to certain form of amyloid via activating nucleic acid-sensing innate immune receptors in vitro and in vivo, suggesting that amyloid aggregation may serve as endogenous stimulus to chronically activate IFN pathway in AD. Consistent with these observations, a panel of IFN pathway genes were expressed at increased levels in an archived human AD dataset. At this time, the functional significance of IFN pathway activation in the context of AD-related neuroinflammation is unclear. Separately, aging-associated IFN activation predominantly affect choroid plexus (CP), a membrane structure that interfaces the cerebrospinal space and the blood brain capillaries. How this element of IFN signaling affects AD pathogenesis has not been modeled so far. Based on these intriguing observations, we hypothesize that IFN pathway critically participates in normal brain aging and, further under protein homeostatic stress, promotes AD pathogenesis. This application seeks the answers to the urgent scientific question how a novel arm of neuroinflammation contribute to AD from both brain parenchyma and neurovascular barrier. We propose three specific aims - 1: Map age-dependent activation of IFN pathway in normal aging and in response to A? pathology; 2: Define the functional significance of IFN pathway activation in A? pathology; and 3: Identify IFN-regulated molecular signatures central to AD pathogenesis in both mouse AD models and human patients. These studies are expected to generate unprecedented insights on how IFN pathway affects the pathogenesis of AD and reveal potential crosstalk between all major neuroinflammatory pathways, which may facilitate the identification of novel biomarkers and therapeutic targets.

Adult; Age; Aging; Alzheimer's Disease; Atlases; Autophagocytosis; biological adaptation to stress; Biological Process; Biological Response Modifier Therapy; Biology; Brain; brain health; Cell Nucleus; Cell physiology; Characteristics; Collaborations; combat; Data; Degenerative Disorder; detection of nutrient; Etiology; functional decline; Functional disorder; Genes; genetic risk factor; Genetic Transcription; Goals; Hippocampus (Brain); Histones; Homeostasis; hyperphosphorylated tau; Image; Impairment; in vivo; insight; interest; Knock-in Mouse; Lysosomes; Measures; Metabolism; metabolome; metabolomics; Modeling; Modification; Molecular; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear Translocation; Online Systems; Organelles; Output; Pathway interactions; Pattern; Positioning Attribute; Post-Translational Modification Site; Post-Translational Protein Processing; preservation; programs; Property; protein aggregation; Proteome; Proteomics; Regulation; Regulatory Pathway; Research Support; Risk Factors; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Specificity; Stress; tau Proteins; Tauopathies; Technology; Testing; therapeutic target; Time; tool; Transcriptional Activation; transcriptome; Ursidae Family; vacuolar H+-ATPase;

ABSTRACT Alzheimer?s disease (AD) is the most common cause of dementia and one of the leading causes of death in the United States. AD is the only leading cause of death for which no disease-modifying therapy is currently available. Neuroinflammation plays a major role in AD pathogenesis. Epoxyeicosanoid signaling is a key integrator of cell-cell communication in the central nervous system (CNS), coordinating cellular responses across different cell types. Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 epoxygenase that have potent anti-inflammatory activity. In our preliminary study, we demonstrated that pharmacological inhibition of sEH can attenuate neuroinflammation, enhance reduction of plaque pathology, and eventually reverse spatial learning and memory deficits in preclinical models of AD. Although some sEH inhibitors (sEHIs) have been reported, none of them are optimized for CNS applications. Blood brain barrier (BBB) is the main hurdle for CNS drug development. Taking advantage of high throughput virtual screening and medicinal chemistry optimization, we developed EHI-16 as a highly potent, orally available and brain permeable sEHI. Additionally, EHI-16 reduces LPS-induced neuroinflammation in both primary astrocytes and in vivo. In this project, we will further optimize EHI-16 to develop anti-inflammation therapy for AD treatment. To this end, we assembled a highly motivated and experienced team with complementary expertise. Dr. Wang is an expert on small molecule drug discovery and ADMET profiling. Dr. Zheng is a pioneer on AD pathophysiology and mouse modeling. Our expertise, highly promising preliminary data, and proven collaboration track-record will ensure the success of the proposed project. In Aim 1, we will develop potent, orally available, and CNS-penetrable sEHIs. In Aim 2, we will determine the pharmacokinetics-pharmacodynamics relationship of sEHIs and in vivo efficacy in attenuating neuroinflammation and improving cognitive impairment in AD mouse models. In Aim 3, we will determine the toxicity and PK profile of sEHIs in rats and dogs and perform IND-enabling studies. The successful accomplishment of this project will open a new avenue for treating and preventing AD and will advance our scientific knowledge of multiple mechanisms of AD.

The objectives of the Administration and Data Integration Core (Core A) are three-fold: first, the Core will provide administrative support and financial management of the Program Project Grant (PPG); second, the Core will support cross-platform bioinformatics integration and sharing of multi-omic datasets generated by the Cores and Projects; third, the Core will facilitate interactions between the Cores and Projects, as well as between Baylor College of Medicine (BCM) and Washington University School of Medicine (WU), and will also provide overall scientific leadership of the PPG. The Core will be led by Dr. Hui Zheng, Director of the Huffington Center on Aging (HCOA) at BCM, and housed within the HCOA administrative unit. The Core will be co-led by Dr. Cristian Coarfa, Associate Professor of Molecular and Cellular Biology and an expert in integrative analysis and visualization of multi-omic data, who will oversee the bioinformatics and data sharing aspects of the PPG. To ensure effective management and successful execution of the objectives, a Scientific Advisory Board (SAB) will be formed upon notification of award that will be composed of three members with established leadership and scientific expertise in AD, tauopathy, aging, lysosomal biology, proteomics, and metabolomics. The Core will consult with the SAB for overall program management and scientific direction. The Core has also recruited individuals from BCM and WU who will offer support on institutional resources and advice on conflict resolution if needed. The specific aims of the Administration and Data Integration Core are as follows: 1) Provide overall scientific leadership; 2) Provide financial, administrative, and organizational support; 3) Perform multi-omics data analysis and integration and ensure rapid public access to the datasets; 4) Generate a web-based first-in-class Aging- and Tauopathy-associated Lysosomal atlas (ATLas) of the lysosomal proteome and metabolome. Overall, the core is ultimately responsible for the PPG?s success, and will work with NIH and SAB closely to ensure that the team as a whole achieves its milestones and deliverables.

Abstract Tauopathies consist of a group of diseases, including frontotemporal dementias and the most common form Alzheimer?s disease, and are characterized by the accumulation of intracellular neurofibrillary tangles (NFTs) composed of aggregates of hyperphosphorylated Tau protein and extensive neurodegeneration. Accumulating evidence has implicated impaired autophagy-lysosome pathway in neurodegenerative diseases including Alzheimer?s disease. The Transcription Factor EB (TFEB) was discovered as a master regulator of cellular clearance through coordinated expression of autophagy and lysosomal target genes. We have found that TFEB is highly efficacious in ameliorating Tau/NFT pathology and behavioral deficits in Tau transgenic mice while exhibiting no adverse effect on wild-type mice, supporting the premise that TFEB may serve as potential therapeutic target. The overarching goal of this project is to investigate a Tau-induced TFEB lysosome-to- nucleus signaling pathway regulating lysosomal homeostasis and cellular clearance in physiological and tauopathy conditions and to identify strategies to augment this pathway for enhanced cellular clearance. Specifically, through proteomics analysis of tauopathy mouse models, we will identify how Tau pathology induces unique TFEB post-translational modifications and nuclear signaling. By leveraging the powerful lysosomal purification and profiling system made available by the Program Project Grant investigators, we will test how Tau pathology alters the lysosomal proteome, metabolome, and pH, the latter is essential for lysosomal function and critically controlled by the vacuolar ATPase (V-ATPase). Accordingly, we will test the specific TFEB/V-ATPase signaling in Tau pathogenesis and downstream glia and immune response. This project is an integral component of the Program Project Grant aimed at understanding how lysosomal function is regulated through lysosome-to-nucleus signaling pathways, how these pathways are changed in aging and Alzheimer?s disease, and how to harness these regulatory pathways to promote brain health, combat age- associated functional decline, and delay neurodegenerative diseases. This project, together with the collaborative efforts of the Program Project Grant, will create a first-in-class Aging- and Tauopathy-associated Lysosomal atlas (ATLas) of the lysosomal proteome and metabolome for mouse cortex and hippocampus which will be made broadly available to the research community.