Benjamin Wolozin - US grants

DESCRIPTION (Provided by Applicant): Mutations in the gene coding for Parkin cause a rare familial form of Parkinsonism, autosomal recessive juvenile Parkinsonism, that results in death of dopaminergic neurons in the substantia nigra. To understand how parkin causes disease, we need to understand the regulation and function of parkin. Our studies have lead us to investigate ubiquitination, which is a process that regulates protein degradation. We hypothesize that parkin regulates ubiquitination of other proteins in response to cellular contact with matrix proteins (such as collagen and laminin), and thereby controls regulation of the cytoskeleton and signal transduction by matrix proteins and their integrin receptors. Loss of parkin function could cause neurodegeneration by inhibiting matrix signaling and impairing maintenance of processes by neurons. Our preliminary data support this hypothesis by demonstrating that parkin-dependent ubiquitination is activated by cellular binding to matrix proteins. We have also identified parkin binding proteins that are associated with integrins. Conversely, cell lines that have reduced parkin expression (due to anti-sense parkin cDNA) decrease ubiquitination, retract processes upon cellular exposure to matrix proteins, and have abnormal signal transduction. The goal of this proposal is to investigate the regulation of ubiquitination by parkin (Aim 1), determine the role of parkin in regulating signaling in response to exposure of cells to matrix proteins (Aim 2) and identify common functional deficits associated with disease-related mutations in parkin. Interestingly, parkin is also linked to other forms of neurodegeneration. Parkin binds to alpha-synuclein, and in brains from donors with Parkinson's disease parkin accumulates in inclusions that contain alpha-synuclein, and shows 75% less binding of parkin to two proteins, filamin and hCDCrel2a. We intend to investigate the mechanism of parkin dysfunction by determining how parkin function is altered in Lewy body diseases, and whether oxidation or alpha-synuclein aggregation causes the dysfunction of parkin (Aim 3). The research in this proposal will provide insight into the function of parkin, determine how mutations in parkin produce disease, and provide a new window to understand the molecular pathophysiology of Parkinson's disease.

DESCRIPTION (provided by applicant): The causes of Parkinson's disease (PD) are poorly understood, and so treatment is currently limited to palliative options. Studies of PD have identified both environmental and genetic factors that contribute to the pathophysiology of PD. We will investigate how three PD-related proteins, alpha-synuclein, parkin/KO8E3.7 (the C. elegans homologue of parkin) and DJ-1, affect the toxicity due to inhibition of complex I of the mitochondrial electron transport chain. We will mainly use rotenone as the model toxin for complex I disruption. The three PD-related proteins each have very different putative functions, and how these activities integrate with the pathophysiology of PD is poorly understood. Our preliminary data indicate that expressing alpha-synuclein or deleting KO8E3.7 increases oxidative damage and apoptosis induced by rotenone treatment in C. elegans. In addition, we have identified compounds, D-beta-hydroxybutyrate (a mitochondrial complex II stimulant), tauro-ursodeoxycholic acid (an anti-apoptotic bile acid) and probucol (a potent anti-oxidant), that each partially inhibit rotenone toxicity when applied alone, but in combination prevent rotenone toxicity in C. elegans. In this proposal, we will determine the mechanism of by which alpha- synuclein, parkin/KO8E3.7 and DJ-1 increase mitochondrial damage in both C. elegans and mammalian neurons (primary mouse neuronal cultures and the human neuronal BE-M17 line). We will also determine whether the treatment strategies identified in C. elegans protect mammalian neurons in vitro and in vivo. We hypothesize that mutations in PD-related genes destabilize the mitochondrial electron transport chain leading to an increased tendency to produce free radicals and activate apoptosis. We also hypothesize that combined use of agents that enhance electron transport and inhibit apoptosis (or oxidation) will protect neurons against degenerative processes associated with PD. Aim 1 will determine whether manipulation of PD-related genes inhibit mitochondrial or proteasomal function, and whether the sensitivity to inhibition increases with age. Aim 2 will determine whether manipulation of PD-related genes increase apoptosis during rotenone-induced toxicity. Aim 3 will determine whether combined use of a mitochondrial complex II stimulant plus an apoptotic inhibitor or antioxidant protects neurons against degeneration in transgenic mice carrying the A53T alpha-synuclein transgene..

DESCRIPTION (provided by applicant): Mutations in LRRK2 are a common genetic cause of Parkinson's disease (PD). Diseases associated with LRRK2 are associated with both alpha-synuclein pathology and with tau pathology. The association of LRRK2 with multiple types of pathologies suggests that the biology of LRRK2 could provide particular insight to our understanding of mechanisms of neurodegeneration. Our studies suggest that LRRK2 modifies cellular responses to stress. Expressing wild type LRRK2 confers to C. elegans striking sensitization to stresses associated with increased protein misfolding and protection against mitochondrial toxins, such as rotenone and paraquat. Knockdown of lrk-1, the C. elegans homologue of LRRK2, has the opposite effects and renders C. elegans less vulnerable to proteasomal inhibition and more vulnerable to rotenone. C. elegans expressing PD-associated LRRK2 mutants show more toxicity than seen with wild type LRRK2. These observations are supported by protein-binding studies in human cell lines and suggest that the action of LRRK2 requires the protein folding machinery and the stress kinase cascade. The knockdown and binding studies show evidence of functional and physical interactions with identification of several binding proteins including CHIP, MKK3, 6 and 7, and JIP2 & 4, as well as functional links to hsp60 and 70. The interaction between LRRK2 and MKK6 appears to be particularly important in the pathophysiology of PD because the PD-related mutations in LRRK2 show increased binding that is selective for MKK6. Many of these binding proteins share a common function in that they mediate different elements of the cellular stress response. We hypothesize that LRRK2 coordinates the cellular stress response through its interaction with proteins linked to the protein misfolding pathways and the stress kinase cascades. A secondary element of this hypothesis is that mutations in LRRK2 associated with PD enhance cell death and neurodegeneration by increasing signaling through the stress kinase cascades. The first aim will investigate how LRRK2 modifies the response to cell injury. This aim will also examine how knockdown of LRRK2 binding proteins modifies the actions of LRRK2 in C. elegans lines and LRRK2 inducible human cell lines. The second aim will determine the structural mechanisms by which LRRK2 interacts with its binding proteins. The third aim examines how expression of wild type, G2019S and R1441C LRRK2 modify toxicity and inclusion formation caused by genetic changes related to PD (alpha-synuclein and tau) in C. elegans. This aim will elucidate whether wild type LRRK2 normally protects against pathophysiological changes associated with PD, and whether PD-related mutations in LRRK2 enhance these changes.

DESCRIPTION (provided by applicant): TDP-43 was recently identified as one of the major proteins that accumulate in inclusions in sporadic Amyotrophic Lateral Sclerosis (ALS) and in Fronto-temporal dementia with ubiquitin inclusions (FTLD- U). Abnormalities in TDP-43 biology are sufficient to cause neurodegenerative disease because mutations in TDP-43 are linked to familial ALS, which suggests that TDP-43 is linked directly to the disease process. The work in this grant proposal will build on our discovery that TDP-43 regulates the translational response to stress. Neurons respond to stresses, such as occur in neurodegenerative diseases, by entering a state of translational arrest. Translationally silenced mRNA is sequestered into inclusions termed stress granules (SG), which are composed of an mRNA and RNA binding proteins, such as TIA-1. Inclusions composed of SGs are intriguing because of their dynamic nature. The proteins that nucleate SGs have the ability to reversibly aggregate partly because they contain aggregation prone polyglutamine and prion domains. TDP-43 is also a RNA binding protein. Our research demonstrates that TDP-43 co-localizes with SGs, and is essential for SG formation. Disease related mutations in TDP-43 appear to enhance SG formation, and conversely, agents that inhibit SG formation rapidly disaggregate TDP-43 inclusions. Our data also suggests that translational inhibitors can suppress or even rapidly disperse TDP-43 inclusions. We hypothesize that SGs contribute to formation of TDP-43 inclusions, such as occur in FTLD-U and ALS, and that inhibiting SGs can inhibit formation of TDP-43 inclusions. Aim 1 of this proposal will move our work from cell lines to primary neurons. Our preliminary work has been performed in cell lines such as human BE-M17 neuroblastoma cells, but the regulation of SGs differs in neurons. We will determine the types of conditions that stimulate formation of TDP-43 inclusions in primary cortical and spinal motor neuron cultures. We will also determine how mutations in TDP-43 affect inclusion formation. Aim 2 will identify the structural domains of TDP-43 inclusion formation and suppression/dispersion of TDP-43 inclusions. This work will identify the conditions and domains on TDP-43 that one can target to develop therapeutic approaches that can suppress or reverse TDP-43 inclusions and possibly treat ALS and FTLD-U. PUBLIC HEALTH RELEVANCE: This project investigates TDP-43, which is one of the major proteins that accumulate in Amyotrophic Lateral Sclerosis (ALS) and in Fronto-temporal dementia with ubiquitin inclusions (FTLD-U). We will determine the types of conditions that stimulate aggregation of TDP-43 and novel methods to prevent or reverse TDP-43 aggregation.

DESCRIPTION (provided by applicant): There is currently no therapy for ALS and it is universally fatal. The research in this proposal will identify compounds that can inhibit TDP-43 aggregation using inclusion formation as a primary readout. Protein aggregation has been implicated as a primary driving force in multiple neurodegenerative illnesses. TDP-43 is one of the most promising targets for pharmacotherapy of ALS because it is one of the major proteins that accumulate as inclusions in Amyotrophic Lateral Sclerosis (ALS), it shows a strong tendency to aggregate, mutations in TDP-43 cause familial ALS and we observe that mutations increase the tendency of TDP-43 to inclusions composed of aggregated protein. We performed a chemical screen to identify small molecules that inhibit TDP-43 aggregation using the high-throughput resources of the Laboratory of Drug Discovery in Neurodegeneration (LDDN) at Brigham and Women's Hospital. The work in this proposal describes the secondary screens and medicinal chemistry to test for and optimize inclusion inhibition and neuroprotection. The optimization studies used in the proposal will primarily use primary cultures of cortical and motor neurons expressing human TDP-43 (WT and A315T). Some studies will also use neuronal PC12 cells that inducibly express WT TDP-43. In each case, the develop cellular cytoplasmic TDP-43 inclusions. We will also analyze the efficacy of the lead compounds to prevent motor deficits in vivo, using C. elegans expressing TDP-43. Our screen of 75,000 novel compounds and 1600 FDA approved and bioactive compounds identified 22 lead compounds on 10 different chemical scaffolds that inhibit inclusion formation. The work in this proposal will identify those chemicals that also provide neuroprotection in models of TDP-43 neurotoxicity. The work will be done through an iterative process of biological testing in the laboratory of Dr. Wolozin, at Boston University School of Medicine, and chemical optimization via medicinal chemistry, performed at the LDDN. Aim 1 will validate the lead compounds from high-throughput fluorescence inclusion assay. We will take our 22 lead compounds from the screening campaign, and test the ability of each compound to inhibit degeneration and inclusion formation in neuronal models of TDP-43 mediated toxicity. We will examine primary cultures of cortical and motor neurons. We will also examine lines of C. elegans expressing WT or A343T TDP-43, to investigate behavior and inclusion formation. Aim 2 will focus on chemical optimization. We will use medicinal chemistry to optimize the pharmacological properties of the two best lead compounds. We will use an iterative process, in which the lead compounds are chemically modified, tested for inclusion dispersion.

DESCRIPTION (provided by applicant): TDP-43 is the principle component of inclusions in amyotrophic lateral sclerosis (ALS) and in some frontotemporal dementia (FTLD-U). TDP-43 is a nuclear RNA binding protein, which translocates to the cytoplasm during stress where it forms cytoplasmic granules. Our research indicates that these cytoplasmic TDP-43 inclusions co-localize with RNA granules termed stress granules (SGs) in cell models and in the human brain. Disease-linked mutations in TDP-43 also increase formation of inclusions associated with SGs. These data point to a strong biological connection between SGs and TDP-43. This proposal will address the role of SG-dependent and independent processes in the pathophysiology of ALS. We hypothesize that SG biology stimulates formation of TDP-43 inclusions, and that pathogenic factors linked to ALS increase TDP-43 inclusion formation through a process mediated by SG pathways. Aim 1 will use induced pluripotent stem cells (IPSCs, generated from control and TDP-43 mutant human cell lines) and hippocampal neurons to characterize the regulation of TDP-43 inclusion formation. We will use imaging to determine how disease-linked mutations in TDP-43 modify formation and dispersion of RNA granules under basal or stressed conditions, including genotoxic stress (e.g., effects of ataxin-2 ? expanded polyglutamine regions), excitatory stress (K+) or growth factor stimulation. In each case we evaluate the role of RPCs (including SGs) in particular locations, such as the soma or dendritic arbor, by genetically restricting RPC formation to the nucleus, soma or soma/dendrite, and examining toxicity. Neurodegeneration will be monitored by measuring dendritic length under the different conditions, and putative changes in dendritic structure will be validated in human tissues. In Aim 2 we will identify molecular factors associated with TDP-43 inclusions. We will determine how pathological mutations in TDP-43 or other SG associated proteins modify the proteins and mRNA that associate with TDP-43 under conditions ? inclusions. In Aim 3 we will determine whether TDP-43 forms inclusion through a SG-augmented mechanism in vivo. This aim will apply the work of Aims 1 & 2 to the in vivo setting, using transgenic mice expressing WT TDP-43. We will identify proteins associated with inclusions in inducible TDP-43 WT transgenic mice. We will investigate examine transgenic mouse lines expressing mutant TDP-43 to determine whether expression of ataxin-2 Q21, 31 or 58 increases TDP-43 motor dysfunction, pathology. Finally we will determine whether ataxin-2 knockout inhibits TDP-43 pathology. Investigating the particular elements of the SG pathway that regulate TDP-43 inclusion formation will identify selective approaches for therapeutic intervention to delay or hal the progression of ALS. PUBLIC HEALTH RELEVANCE: TDP-43 is a RNA binding protein that associates with RNA inclusions, termed stress granules. This proposal will focus on RNA protein complexes and the stress granule pathway to identify molecular factors associated with TDP-43 inclusions and determine how these factors regulate neurodegeneration related to TDP-43.

? DESCRIPTION (provided by applicant): This proposal investigates a novel mechanism for the misfolding and aggregation of microtubule associated protein tau (MAPT), which we hypothesize provides an unrecognized but major aspect of the pathophysiology of tauopathies, such as Alzheimer's disease (AD). We have recently identified a new type of molecular pathology in AD that derives from the aggregation of RNA binding proteins, forming RNA-protein complexes that include stress granules. These stress granules progressively accumulate in the brains of transgenic models of tauopathy, as well as massively accumulate in subjects with AD and FTDP-17. The genesis of this research comes from the simple observations that 1) RNA binding proteins form RNA granules through a striking property of reversible aggregation, which is under physiological regulation, and 2) MAPT binds to some RNA binding proteins, including TIA-1. RNA granules consolidate transcripts for transport, storage and/or degradation. Our results suggest that TIA-1 stimulates phosphorylation and misfolding of MAPT, and that MAPT stimulates formation of TIA-1 positive stress granules; the association of MAPT with stress granules reduces its degradation and appears to stabilize insoluble MAPT. The chronic nature of AD might lead to excessive formation of stress granules and aggregation of MAPT, contributing to neurodegeneration. We hypothesize that MAPT and RNA binding proteins exhibit bidirectional regulation. MAPT promotes the formation and stability of RNA granules, including stress granules. Conversely, RNA binding proteins and the translational signaling cascade stimulate the phosphorylation, and misfolding of MAPT. This hypothesis will be studied in the context of three aims: Aim 1 will determine the mechanisms by which TIA-1 interacts with MAPT. We will use structural studies, imaging and mass spectroscopy to highlight key changes in discover key binding proteins. Aim 2 will determine the role of MAPT in neuronal RNA granule biology, including stress granules. This aim will explore this biology using live cell imaging to explore RNA granule dynamics under basal and stress conditions and will examine whether MAPT modulates the types of transcripts associated with particular RNA granules/ stress granules (using iCLIP). Finally, Aim 3 will determine whether stress granule/RNA translation pathways regulate MAPT-mediated neurodegeneration in vivo. This aim will focus on the RNA binding proteins used in our preliminary studies (e.g., TIA-1) as well as the novel MAPT/ stress granule components identified by mass spectroscopy.

Neuritic plaques and neurofibrillary tangles are the hallmark pathologies of Alzheimer's disease (AD), but the presence individual tangles or plaques is not sufficient to predict degeneration; 45% of the elderly with plaques and tangles lack cognitive loss or dementia. We have recently identified a new type of molecular pathology in AD that derives from the aggregation of RNA binding proteins (RBPs), forming RNA-protein complexes, which are termed RNA granules. Microtubule associated protein tau (MAPT) binds to RBPs, co-localizes with RBPs in RNA granules, and RBPs increase MAPT misfolding /aggregation. Importantly, reducing the RBP TIA1 delays progression of tauopathy, despite increased MAPT aggregation. We hypothesize that variation in the composition of MAPT complexes (soluble or insoluble) and RNA granule complexes represent critical determinants of the molecular heterogeneity of AD and other tauopathies, and identify particular pathways that uniquely contribute to each type of disease. We will apply systems biology approaches that integrate information from proteomics and RNA metabolism to identify key proteins in each complex that are associated with neurodegeneration, and then test the roles of these proteins/genes experimentally. Throughout this proposal we will use unbiased studies (e.g., proteomic and RNAseq) combined with the systems biology algorithms to model context-dependent information flows to identify key molecular interactions and pathways regulating pathology, neurodegeneration and neuroprotection. Aim 1 will determine whether the RBP TIA1 directs the biochemical and functional properties of MAPT aggregation. We have discovered that reducing the RBP TIA1 delays disease progression in PS19 P301S MAPT mice despite producing more aggregation. We will elucidate the mechanisms by which TIA1 reduction produces neuroprotection using both in vitro molecular studies, and use unbiased ?omic? studies (mass spectrometry and RNAseq). We will apply the systems biology algorithms to quantify key gene-gene interactions and pathways, and identify those pathways that parallel the human condition. Aim 2 will determine how MAPT and RBP complexes vary with cognitive decline in humans. We will use mass spectrometry and RNAseq to determine how the composition of complexes of MAPT, TIA1 and other key RBPs varies among human cases exhibiting neuritic plaques and neurofibrillary tangles with or without cognitive decline. Aim 3 will determine whether RBPs direct the strain of MAPT and resulting pathologies that are propagated in vitro and in vivo. We will characterize propagation of tauopathy for MAPT aggregates from PS19, PS19xTIA1+/--mice, as well as human cases exhibiting MAPT pathology with and without cognitive decline. The resulting mice will be analyzed as described in Aim 1. 

Our laboratory discovered an important role for RNA binding proteins (RBPs) in the pathophysiology of tauopathy. Tau accumulates in the neuronal soma as part of a normal biological pathway involving the translational stress response, and formation of stress granules (SGs). The association of tau with SGs also stimulates tau aggregation, indicating that tau aggregation can occur normally as part of the translational stress response. Our recent studies demonstrate a key role for RNA binding proteins in tauopathy in vivo. We demonstrated that reducing levels of the RNA binding protein TIA1 (which nucleates SGs) significantly delays disease progression in the P301S tau mouse. Reducing the RBP TIA1 by half (TIA1+/-) yielded a 33% increase in lifespan, with rescue of synaptic loss, neuronal loss, reduced inflammation and behavioral rescue at 6 months. This strong neuroprotection occurs with a corresponding dramatic (90%) reduction in tau oligomerization. Analysis of the full TIA1 deletion revealed a surprise. The P301S TIA1-/- mice live longer and show behavioral rescue at 6-months, however, they also exhibit a major (>10-fold) increase in reactive microglia, suggesting a strong neuro- inflammatory response. We hypothesize that TIA1 removal in neurons inhibits neurodegeneration, while TIA1 removal from microglia enhances the neuro-inflammatory response. Two alternate scenarios might explain the presence of neuroprotection in the face of an enhanced neuro- inflammatory response. Scenario 1: The benefit accrued to neurons from TIA1 reduction is stronger than the harm resulting from the increased neuro-inflammation. Scenario 2: Loss of TIA1 in microglia produces a neuro-inflammation that is surprisingly beneficial. This proposal will produce conditional TIA1 knockout mice that lack TIA1 in either neurons or microglia, and test these scenarios. Aim 1: Generate conditional TIA1 knockouts (KO) with CRE driven by promoters selective for cholinergic neurons (ChAT), pyramidal neurons of the frontal cortex and hippocampus (CamKII), and microglia (Cx3cr1). Validate expression by immunohistochemistry. Aim 2: Determine how selective deletion of TIA1 from neurons or microglia modifies the neuroprotective and inflammatory phenotypes: We will use the microglial selective TIA1 KO mice to determine whether the enhanced inflammatory response exacerbates inflammation and neurodegeneration, using the facial nerve axotomy model. We will also examine the neuronal selective TIA1 KO mice to determine if protection is observed against a challenge with the axotomy model, as well as the glutamate analogue, kainic acid.

The goal of this proposal is to comprehensively map and identify the subnetworks of synaptic protein complexes that are central players in the synaptic dysfunction occurring with neurodegeneration. We will use the emerging power of quantitative network proteomics in the Emili laboratory to systematically characterize the major protein assemblies present at normal and diseased synapses on a proteome scale. This research will be propelled by discoveries from the Wolozin laboratory demonstrating that a dynamic network of protein interactions drives tau biology and changes with the course of disease. Interpreting these perturbed assembly networks, though, demands knowledge of the localization and compositional specificity of such complexes. The unbiased interactome screening technology developed by the Emili laboratory is uniquely suited for unbiased interrogations of synaptic protein networks. We hypothesize that selective disruption of specific synaptic protein assemblies mediates the functional degeneration associated with tauopathy. Aim 1 will determine how synaptic protein complexes differ between general cortical and cholinergic neurons. We will isolate and biochemical separate synaptic assemblies from total cortical and cholinergic (ChAT::GFP) synapses, using FACS to further purify ChAT:GFP synapses. Separated assemblies will be characterized by precision mass spectrometry and integrative data mining (machine learning) procedures to determine their composition and post- translational modification states, and to map dynamically changing interactions implicated in altered synaptic function during normal aging. Aim 2 will determine how the macromolecular structures of synaptic protein assemblies change with aging and AD. We will analyze cortical synaptosomal complexes from P301S tau and P301S tau x TIA1+/- mice, the latter which exhibited delayed degeneration. Key drivers in synaptic dysfunction will be identified and verified in AD and control human samples by co-immunoprecipitation. Aim 3 will identify complexes that are critical drivers of synaptic function by disrupting prioritized assemblies using genetic and opticogenetic tools. This work will determine key regulators of synaptic function in health and disease, and will also produce expanded genetic tools and outstanding targets for future approaches using bio-engineered regulation.

The goal of this proposal is to determine how ApoE polymorphisms modify comprehensive maps and subnetworks of protein complexes that are central players in the dysfunction occurring Alzheimer?s disease (AD). We will use the emerging power of quantitative network proteomics in the Emili laboratory combined with proximity profiling to systematically characterize the major protein assemblies that occur in a classic disease model of tauopathy. This research will be propelled by discoveries from the Wolozin laboratory demonstrating that a dynamic network of protein interactions drives tau biology and changes with the course of disease. The work will be further informed by cross-referencing key network members to large genetics and genomics datasets focused on AD risk, neuropathological outcomes and human brain expression data to enable prioritization of network members exhibiting disease-linked gene-gene interactions. These advanced interactome screening technologies are uniquely suited for unbiased interrogations of disease-related protein networks in the brain. We hypothesize that tau and RNA binding protein interactomes exhibit a progressive evolution with disease progression in tauopathy, and that the structure of the interactomes is modified by genetic risk factors for AD. Aim 1 will determine how APOE alleles modify neuronal interactomes with disease progression. We will cross PS19 P301S tau mice with mice carrying humanized ApoE3 or ApoE4, and compare protein-protein interaction networks for tau and the RNA binding proteins TIA1 (which interacts with pathological tau) and G3BP (which does not interact with pathological tau). Aim 2 will determine how APOE alleles modify responses to propagated pathological tau. We will compare how propagation of different tau strains changes the proteins that interact with pathological tau, and how expression of specific ApoE isoforms modify the pattern of tau propagation and the nature of the resulting tau interactomes. Aim 3 will determine how reduction of key protein interactors modifies AD-related networks, and disease progression in tauopathy. This work will examine the how reducing key network components modifies related interactome networks and disease progression in PS19xApoE3 or E4 mice under conditions of normal disease progression or accelerated progression induced by tau propagation.

SUMMARY New variants, especially in non-coding regions, are expected to be discovered through the ongoing AD Sequencing Project (ADSP). This proposal will investigate circular RNAs (circRNAs) and RNA binding proteins (RBPs) that regulate or are regulated by these circRNAs. Recent genomic studies have discovered thousands of circRNAs produced from both protein-coding genes and non-coding regions of the genome via a process known as back-splicing. CircRNAs are more enriched in neuronal tissues and are often derived from genes specific for neuronal and synaptic function. The discovery of these circRNAs demands a coordinated investigation of RBPs that interact with the circRNAs. Mutations in and dysfunction of RBPs are known to be major mechanisms contributing to the pathophysiology in frontotemporal dementia, ALS and AD. However, the contributions of the circRNA:RBP network to these disease mechanisms are largely unknown. The novel biology of circRNAs opens an entirely new window into mechanisms of neurodegeneration in ADRD. CircRNAs could contribute to neurodegeneration by acting as sponges that sequester miRNA/RBPs away from normal mRNA targets, altering splicing or expression. RBPs also regulate circRNA production by binding to the flanking intronic sequences of circRNAs which contain many conserved binding sites of splicing factors/RBPs. Thus, sequestration of RBPs in protein aggregates could cause dysfunctional regulation of circRNAs. The history of genomics indicate that discovery of each new nucleotide species expands our understanding of disease mechanisms. The discovery of circRNA presents a major unexplored avenue of RNA metabolism that demands investigation. We hypothesize that changes in the levels of circRNAs contributes to the pathophysiology of ADRD, and that discovery of key circRNAs or circRNA-RBP interactions in aging human brains could uncover novel biomarkers, disease mechanisms or therapeutic targets. In this proposal, by leveraging large public and our own RNA-seq data (rRNA-depleted), we will apply several methods to detect and characterize AD-related circRNAs from multiple human brain regions, and integrate them with ADSP genetic findings (Aim 1). In Aim 2, aside from discovering AD-related RBPs from human brain RNA-seq, proteomics and ADSP WES/WGS data, we will leverage the ENCODE CLIP-seq data for RBP binding to identify putative RBP-circRNA interactions with AD, i.e. AD-related functional RNA elements. Finally, in Aim 3, we will select the top 10% of the circRNAs (~200) and RBPs (~150) for further high-throughput functional evaluation with a novel, powerful 3D human organoid model of ADRD, termed AstAD that exhibits the full range of tau pathology and neurodegeneration. We anticipate that our integrative analyses of ADSP genetics, circRNA, mRNA, RBP and the high-throughput AstAD functional screen readouts can help generate testable hypothesis for future molecular mechanisms experimental design.