Dongming Cai, Ph.D. - US grants

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is characterized by the formation of amyloid plaques and cerebrovascular amyloid deposits, the principal component of which is the -4kDa amyloid peptide. Abeta is derived from a large transmembrane precursor, the amyloid precursor protein (betaAPP), physiological functions of which remains to be elucidated. Previous studies suggest that the distribution of betaAPP between the trans-Golgi network (TGN) and cell surface has a direct influence upon the generation of Aa This phenomenon makes delineation of the mechanisms responsible for regulating aAPP trafficking from the TGN relevant to understanding the pathogenesis of AD. Recent evidence from several groups including ours suggests a role for presenilin-1 (PS1) in regulating intracellular trafficking/maturation, within the secretory pathways of select proteins including betaAPP, nicastrin, TrkB and a PSI-binding protein (ICAM-5/telecephalin). Using a cell-free vesicle trafficking reconstitution system derived from neuroblastoma cells, we demonstrated that, PS1 regulates the biogenesis of betaAPP-containing vesicles from the trans-Golgi Network (TGN) and the endoplasmic reticulum (ER). PS1 deficiency or the expression of loss-of-function variants leads to robust vesicle formation, concomitant with increased maturation and or cell surface accumulation of betaAPP. In contrast, release of vesicles containing aAPP is impaired in FAD-linked PS1 mutant cells, resulting in reduced betaAPP delivery to the cell surface. Moreover, diminution of surface betaAPP is profound at axonal terminals in neurons expressing a PS1 FAD variant. These results suggest an alternative mechanism by which FAD-linked PS1 variants modulate aAPP processing along a pathologic, amyloidogenic pathway. Therefore, the objective of this proposal is to address the cellular mechanisms underlying PS1 regulation of aAPP trafficking as well as to assess physiological and pathological implications of this regulated axonal transport of aAPP in neuronal functions. Based on these premise, the specific aims for the project are as follows: 1) To determine whether recruitment and (in)activation of certain cytosolic factors affects PS-1 regulated betaAPP trafficking. 2) To assess the correlation of PSI-regulated axonal transport of betaAPP with its neuronal functions such as neurite outgrowth and synaptic plasticity. 3) To elucidate the effects of identified cytosolic factors on these neuronal functions via modulation of PSI-regulated betaAPP trafficking.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is characterized by the formation of amyloid plaques and cerebrovascular amyloid deposits, the principal component of which is the -4kDa amyloid peptide. Abeta is derived from a large transmembrane precursor, the amyloid precursor protein (betaAPP), physiological functions of which remains to be elucidated. Previous studies suggest that the distribution of betaAPP between the trans-Golgi network (TGN) and cell surface has a direct influence upon the generation of Aa This phenomenon makes delineation of the mechanisms responsible for regulating aAPP trafficking from the TGN relevant to understanding the pathogenesis of AD. Recent evidence from several groups including ours suggests a role for presenilin-1 (PS1) in regulating intracellular trafficking/maturation, within the secretory pathways of select proteins including betaAPP, nicastrin, TrkB and a PSI-binding protein (ICAM-5/telecephalin). Using a cell-free vesicle trafficking reconstitution system derived from neuroblastoma cells, we demonstrated that, PS1 regulates the biogenesis of betaAPP-containing vesicles from the trans-Golgi Network (TGN) and the endoplasmic reticulum (ER). PS1 deficiency or the expression of loss-of-function variants leads to robust vesicle formation, concomitant with increased maturation and or cell surface accumulation of betaAPP. In contrast, release of vesicles containing aAPP is impaired in FAD-linked PS1 mutant cells, resulting in reduced betaAPP delivery to the cell surface. Moreover, diminution of surface betaAPP is profound at axonal terminals in neurons expressing a PS1 FAD variant. These results suggest an alternative mechanism by which FAD-linked PS1 variants modulate aAPP processing along a pathologic, amyloidogenic pathway. Therefore, the objective of this proposal is to address the cellular mechanisms underlying PS1 regulation of aAPP trafficking as well as to assess physiological and pathological implications of this regulated axonal transport of aAPP in neuronal functions. Based on these premise, the specific aims for the project are as follows: 1) To determine whether recruitment and (in)activation of certain cytosolic factors affects PS-1 regulated betaAPP trafficking. 2) To assess the correlation of PSI-regulated axonal transport of betaAPP with its neuronal functions such as neurite outgrowth and synaptic plasticity. 3) To elucidate the effects of identified cytosolic factors on these neuronal functions via modulation of PSI-regulated betaAPP trafficking.

DESCRIPTION (provided by applicant): The ApoE4 genotype is the strongest genetic risk factor for developing AD. However, the mechanisms that underlie this link between ApoE4 genotype and AD are not well understood. Objective/Hypothesis: the objectives of this proposal are to understand the molecular underpinnings of the association between ApoE4 genotype-specific changes in brain phospholipid homeostasis and ApoE4 increased susceptibility to develop late-onset AD. Our preliminary data indicate that the levels of PI(4,5)P2 are reduced in postmortem human brain tissues of ApoE4 carriers, in the brain of ApoE4 homozygous knock-in (KI) mice, and in primary neurons expressing ApoE4 alleles, if compared to ApoE3 counterparts. The expression of synaptojanin 1 (synj1) that dephosphorylates PI(4,5)P2 reducing its levels, is elevated in ApoE4 brains. Our recent observations demonstrate that synj1 reduction (with subsequent elevation of PI(4,5)P2 levels) can accelerate endosomal/lysosomal degradation of A? and ameliorate cognitive deficits in AD transgenic mice. In this proposal we are testing the hypothesis that ApoE genotype is a critical determinant of brain phospholipid homeostasis and that the ApoE4 isoform is dysfunctional in this process (increased synj1 expression and reduced PIP2 levels). As a consequence, ApoE4 impairs A? clearance through endosomal/lysosomal degradation pathway, accelerates cognitive decline, and disrupts synaptic functions. These ApoE4-induced changes in the cascade of aberrant molecular events lead to long-term neurodegenerative process and AD development. Rationale/Experimental Design: In this application, we will study whether reducing synj1 thus normalizing brain phospholipid metabolism can rescue ApoE4-related neuropathological changes by utilizing mouse models of synj1 haploinsufficiency with human ApoE4 or E3 homozygous KI background in studies that assess: 1) AD-related cognitive dysfunction (aim 1.1); 2) AD-related biochemical changes such as A?learance and ApoE secretion (aim 1.2 and 1.3); 3) AD related morphological changes and synaptic phospholipid homeostasis (aim 2); 4) molecular mechanisms underlying ApoE isoform specific changes in synj1 expression/PIP2 homeostasis (aim 3). Relevance/Impact: The proposed studies in this application will be the first mechanistic studies that link ApoE4 genotype-specific changes in brain phospholipid homeostasis to ApoE4 increased susceptibility to develop AD. These studies may uncover new therapeutic options for the treatment of AD targeting at ApoE4 pathogenic nature.

Alzheimer's disease (AD) is a neurodegenerative disease that affects approximately one-half of the U.S. population greater than 85 years old. The burden of AD at the patient level falls disproportionately on females, as many studies find that age-matched females have a higher proportion of AD cases. Indeed, evidence suggests that the APOE ?4 allele (ApoE4), which is the strongest risk factor for AD, may have an especially large effect in females compared to males. Surprisingly, although many studies have focused on genetic and gene expression risk factors for AD, and many studies have focused on clinical, neuropathologic, and neuroimaging differences in AD between females and males, few if any studies have focused on the genetic and gene expression mechanisms that mediate the apparent gender differences in AD presentation. In order to address this gap in our understanding of AD pathogenesis, we will employ what is to the best of our knowledge the largest gene expression study of postmortem human brains in AD, which includes currently 1400+ postmortem human brain samples, and will grow as more data is generated over the course of this project. We will perform an extensive quality control and covariate correction of the assembled data to ensure that gender status is annotated correctly and that covariates such as age do not confound our analyses. This cleaned and aggregated human postmortem brain gene expression data set will be made available to the research community to improve standards among the field of AD research and accelerate AD research by avoiding duplication of work. Next, we will systematically search for genes and pathways that differentiate AD progression between females and males, as well as ApoE4 carriers and non-carriers, and we will validate the differences in gene and protein expression levels using additional postmortem human brain samples to confirm the biological relevance of the findings. Then, we will generalize the analysis of human postmortem brain gene expression data to the network level, which will allow us to detect higher-order trends and identify target genes that drive major differences in AD progression between females and males. We will validate several key targets that are likely to play a mechanistic role in AD via genetic analysis of AD risk in females and males with and without APOE ?4 carriers. Finally, we will corroborate the findings at the gene expression and network level via targeted validation studies in AD animal models, such as female and male ApoE4 KI mice without and with 3xTg AD background. We expect that this proposed research program will lead to a dramatic improvement in our understanding of AD biology, because instead of attempting to adjust away sex differences in AD progression, we will explicitly study them and work towards a comprehensive understanding of the molecular mechanisms underlying sex differences in AD pathogenesis. It will also pave a path towards distinct targeted drug discovery efforts for AD in females and males, which will be crucial to help decrease the burden of this devastating disease.

Alzheimer's disease (AD) is a neurodegenerative disease that affects approximately one-half of the U.S. population greater than 85 years of age. APOE2 is associated with a lower risk and delayed onset for AD. Numerous studies suggest a role of APOE2 in human longevity as shown by a strong association between the APOE2 allele and reduced aging-related cognitive decline. However, while most research effort in APOE and AD has been focused on detrimental effects of APOE4, very limited studies have addressed the protective effects of APOE2. Cellular mechanisms pertaining to A? clearance, neurofibrillary tangle burden, synaptogenesis and synaptic plasticity, glial activation, and neuro-inflammation have been proposed, but an in- depth molecular characterization of these effects in aging and AD has not been established, partially due to a low allele frequency of APOE2. For example, APOE2 has a much lower allele frequency than APOE3 and APOE4 alleles in pre-existing large-scale gene expression data sets of human postmortem brains (N?30 of APOE2 carriers in both non-demented controls and AD cohorts). This low allele frequency poses a significant challenge to identify molecular networks and drivers mediating APOE2 effects. To overcome this challenge, we propose to leverage pre-existing human brain data sets and integrate them with new data sets to be generated from additional human brain samples, ?2/?3 and ?2/?4 iPSC-derived brain cells (Aim 1) and EFAD animal models (Aim 2). After extensive quality control and covariate correction of the assembled data sets, we will perform integrative high-resolution network modeling analysis to identify multiscale molecular networks and key drivers mediating APOE2-specific protective effects against aging and AD (Aim 3). In this application, we propose to define how interactions with APOE3 and APOE4 alter the APOE2-MNs and functional read-outs characteristic of aging and AD-related pathology. We will also determine how sex affects these processes. We will also validate the key differences in APOE2-MNs using postmortem human brain samples, iPSC-derived brain cells and EFAD mouse brain tissue to confirm the biological relevance of the findings. We will then characterize the functional relevance of top key drivers of the most informative subnetworks of APOE2 using gene perturbation techniques in iPSC-derived brain cells and EFAD mice (Aim 4). All the data and models developed through this study will be shared with the community. Our proposed studies will systematically construct, characterize, and validate APOE2-specific MNs in context of interacting with other APOE alleles and sex that potentially impact longevity, learning and memory, as well as AD development and progression. We expect this work will facilitate identification and development of targeted therapeutic approaches to AD incorporating APOE genotype and sex, the basis of personalized medicine. Therefore, we expect the proposed research will help decrease the burden of this devastating disease and have a large impact on AD research.

Project Summary Alzheimer?s disease (AD) is a multifactorial disorder with complex etiologies and the impact of sex on AD varies over the course of clinical and neuropathological development. Basic and clinical research studies support sex- specific contributions to AD pathogenesis and progression. Apolipoprotein E4 (APOE4) allele has been identified as a primary genetic risk factor. The interplay between sex and APOE4 allele in AD risks, clinical manifestation, pathological processes as well as treatment responsiveness in various clinical trials have been explored. However, the molecular mechanisms underlying sex dimorphism in AD and how APOE4 stratifies sex divergence in AD remain elusive. Multi-omics data in tandem with systems biology approaches offer a new avenue to not only dissect sex- and APOE-stratified molecular mechanisms of AD but also develop sex-specific diagnostic and therapeutic interventions for AD. Single-cell transcriptomic datasets as well as cell deconvolution of bulk tissue transcriptomes provide in-depth insights into brain region-specific and cell-type specific impact on sex dimorphism in AD. In this application, we propose to develop sex- and APOE-specific network models of AD by performing integrative multiscale network analysis of large-scale bulk and single multi-omics data. In particular, we will develop the first cohort of single nucleus multi-omics data in AD (simultaneous RNA-sequencing and ATAC-sequencing of each single cell) that can meaningfully be stratified by sex and APOE genotype. We will also curate existing single nucleus RNA-seq datasets in AD and combine with our own single cell multi-omics dataset to identify sex-specific genetic variants and molecular signatures of AD (Aim 1). We will perform integrative network analysis to investigate the interplay between sex and APOE genotypes in AD at brain-region and single-cell levels and identify from the network models sex- and APOE-specific, network drivers for AD (Aim 2). We will then identify potential therapeutics of selected key drivers with drug candidate prediction through virtual clinical trials of large electronic medical record (EMR) databases (Aim 3). Finally, we will functionally validate the top predicted sex- and APOE-specific molecular network drivers by genetic manipulations (up- or down-regulation of gene expression), as well as pharmacological perturbation with drug candidates identified from virtual drug screening using relevant human iPSC culture systems and AD mouse models (Aim 4). The findings from this project will guide future development of efficacious sex- and APOE-stratified interventions for AD.

Project Summary Alzheimer's disease (AD), in particular la?te onset AD, is the most common form of dementia, accounting for about two thirds of all the dementia cases, and its pathogenesis may start decades early before its actual clinic manifestation. The search for disease modifying treatments is the primary objective of most rigorous therapeutic research efforts on AD. However, AD is currently incurable and available therapies are only effective in partially alleviating selected AD clinical symptoms, but not its onset and/or progression. The unmet need for the timely development of potent therapies for AD has been constantly growing with the heavy burden on our healthcare system reaching a critical level. There is an urgent need to reinvigorate AD drug development by utilizing systems biology, especially network biology approaches which have the potential to present not only global landscape of pathway-pathway interactions but also detailed molecular interaction/regulation circuits underlying AD. Network biology approaches to integrate large-scale multi-omics data in AD have demonstrated that differentially regulated subnetworks in AD, which regulate diverse AD pathogenic phenotypes, often include a large number of key regulators. Therefore, drugs and drug combinations that can modulate such subnetworks as a whole are the most pertinent for therapeutic intervention and have better chance to be successful. In this application, we propose to develop novel molecular network based drug repositioning approaches to identify individual FDA approved drugs as well as their combinations that can potentially reverse molecular signatures and network states of AD. A large number of predicted drugs and drug combinations will be tested in multiple model systems including mouse brain primary cells, human iPSC derived brain cells and AD mouse models. This project will establish an integrative platform comprised of highly innovative systems and experimental biology components for rapid drug discovery for AD.

PROJECT SUMMARY Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder of aging, affecting about 44 million people worldwide with 5.5 million in the U.S. Amyloid plaques in the brain, one of the pathological hallmarks of AD, consist of fibrillary forms of amyloid ? peptide-40 (A?-40) and amyloid ? peptide-42 (A?-42) produced from amyloid precursor proteins by sequential cleavage, and are crucial for the neuro-pathogenesis of AD. Despite major drug development efforts targeting A? peptide cleavage and processing, nearly all experimental drugs tested for AD thus far have failed to show significant efficacy. New therapeutic strategies are urgently needed to offer new prevention and treatment opinions for AD that represents a major unmet medical need. A? aggregates induce oxidative stress and inflammation leading to microglia activation and neurodegeneration in the brain. This process is fueled by pro-inflammatory cytokines such as IL-17, IL-21, IL-22, and IL-23, secreted by CD4+ T-helper 17 (Th17) cells, which are found to be elevated in the peripheral blood of individuals with AD dementia and mild cognitive impairment (MCIAD) over normal aging control subjects. Notably, we discovered that expression of Rorc, a major transcription factor of microglia, and target genes Il18, Nos2, and Casp4 are markedly increased in the brain of AD and MCIAD patients over healthy aging controls, and Rorc up-regulation is more profound in female than male AD patients. We further found that IL-17 and TNFa, produced by Th17 cells, induce transcriptional expression of Rorc, Il6, Il18, Il23, and Tnfa in mouse microglia. Since IL-23 can induce pathogenic Th17 cell development, we postulate that Th17 and microglial cells likely act in a positive feedback loop to promote inflammation contributing to AD pathogenesis. Importantly, our new bromodomain inhibitor that selectively targets major transcription regulator BRD4 effectively inhibits transcription of Il17, Il21, Il22, Rorc and Il6 in mouse Th17 cells, and Il6, Tnfa, Il18, Il23, Nos2, and Casp4 in mouse primary microglia. Furthermore, MS402 blocks over-production of Th17 cells in experimental autoimmune encephalomyelitis in mice, a model mimicking the neuroinflammatory disorders in humans. Our results strongly suggest a promise of our Th17/microglia immunomodulators as a new treatment for AD. Motivated by our favorable findings, in this study, we will (1) investigate the mechanisms of transcriptional regulation of Th17 and microglial cells in AD pathogenesis; (2) develop and characterize Th17 and microglial immunomodulators for AD treatment; and (3) investigate in vivo therapeutic efficacy of Th17 and microglial immunomodulators in AD mouse models.