Celeste M. Karch, Ph.D. - US grants

DESCRIPTION (provided by applicant): The overall goal of this research and training plan is to define the molecular mechanisms underlying tauopathies. Tau aggregates are a hallmark pathological feature of tauopathies, which include Alzheimer's disease, progressive supranuclear palsy, frontotemporal dementia, corticobasal degeneration and Pick's disease. While Parkinson's disease is not usually characterized as a tauopathy, a common tau gene (MAPT) haplotype is an established risk factor for disease. This project aims to define the molecular mechanisms underlying tauopathies, will improve our understanding of how tau genetics influences tau biology, and will inform novel avenues for therapeutic intervention. The investigator, Dr. Celeste Karch, will gain advanced training in stem cell biology, genomics, and axonal imaging in support of an innovative approach that establishes novel cell models that use human induced pluripotent stem cell (iPSC)-derived neurons, zinc finger nucleases, and axonal imaging to study the extent to which genetic changes in MAPT, the gene that encodes the tau protein, disrupts tau metabolism in tauopathies. The mentors, who were selected for this training, Drs. Alison Goate, Marc Diamond, Jeffrey Milbrandt, and Yadong Huang, are internationally recognized experts in the fields of human and molecular genetics, tau aggregation, axonal degeneration, and stem cell biology, respectively. The goal of this proposal is to determine how genomic variants in tau that are associated with risk for tauopathies contribute to the development of these diseases using human iPSC-derived neurons. The overarching hypothesis of this proposal is that common mechanisms exist by which disease mutations and risk haplotypes disrupt tau metabolism and contribute to disease pathogenesis. To define these common mechanisms, I will measure several modalities of tau metabolism in iPSC-derived neurons from disease mutation and risk haplotype carriers. Through this research and mentored training plan, Dr. Karch will begin to define the molecular mechanisms underlying tauopathies and will establish new experimental tools and approaches that will form the foundation for a career as an independent, translational neuroscientist.

Project Description The complex molecular events that underlie the development of Alzheimer's disease (AD) are poorly understood and are the key to developing targeted therapies. Our group and others have shown that rare variants in the triggering receptor expressed on myeloid cells 2 gene (TREM2) are associated with increased susceptibility to AD. TREM2 encodes a membrane protein that is part of a receptor-signaling complex that modulates inflammatory responses, phagocytosis and cell survival in myeloid cells, such as microglia. However, the specific role of TREM2 in AD pathogenesis remains unclear. CSF sTREM2 levels are increased in patients with symptomatic AD compared to cognitively normal controls. We have recently discovered that common variants in the MS4A locus are a major regulator of CSF sTREM2 levels. Importantly, the same allele associated with higher CSF sTREM2 levels is associated lower AD risk and delayed age at onset. We also identified a second, independent signal in the MS4A locus that encodes MS4A4A p.M159V, which has the opposite effect on CSF sTREM2 levels and AD risk. Our findings that two independent signals in the MS4A gene region have opposing effects on CSF sTREM2 levels and AD risk points to the connection between MS4A and TREM2 biology in AD pathogenesis. The goal of this project is to elucidate the mechanisms by which MS4A genes alter TREM2 function and drive AD pathogenesis. We hypothesize that MS4A4A is involved in TREM2 trafficking and cleavage and that disrupted interaction between MS4A4A and TREM2 leads to microglial dysfunction and neurodegeneration. To test this hypothesis, we will use genomic and biochemical approaches in human brains and stem cell models. The results of this project will provide the mechanistic framework needed to develop treatments that restore or enhance TREM2 functions in order to prevent neurodegenerative disease.

Project 1: Amyloid Beta SUMMARY/ABSTRACT Alzheimer's disease (AD) is characterized by the accumulation of amyloid-? (A?) and neurofibrillary tangles composed of the tau protein in the brain. More than 200 mutations have been identified in amyloid-? precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) that cause autosomal dominant forms of AD (ADAD). PSEN1 and PSEN2 form the catalytic domain of the ?-secretase enzyme, which cleaves APP to generate many A? proteoforms which can be modified into variants including posttranslational modifications, truncations and sequence variations. Changes in the relative ratios of A?42/40 isoforms have been used to predict pathogenicity of ADAD variants. However, we know less about the contribution of other A? proteoforms to AD pathogenesis and the utility of the A? proteoform signature as a biomarker of mutation status and/or disease course. For example, what are the A? pathogenic cause(s) of ADAD? Several A? proteoforms support a causal role for AD, including A?42, A?43, A?37, A?39 and modifications including pyroglutamate, oxidation, isomerization, and N- and C-terminal truncation. The objective of this study is to define the effects of ADAD mutations and amyloidosis on A? proteoform and disease pathogenesis. To meet this objective, we will define mutation and gene-specific effects on A? proteoform signatures using novel mass spectrometry approaches in human plasma, CSF, stem cell derived neurons, and brain tissue. We will then determine how A? proteoform signatures relate to histologic amyloid plaque structure in human brains. We hypothesize that ADAD mutations produce a common pathogenic A? proteoform signature. The rationale for this proposal is that defining the effects of ADAD mutations and amyloidosis on A? proteoforms will be critical to define the common pathogenic A? signatures which cause AD. This target validation will guide clinical studies, therapeutic strategies and classify future novel ADAD mutations. This work will be performed in collaboration with the Genetics, Biomarker, Clinical, Neuropathology, Imaging, and Biostatistics Cores.

Tauopathies may occur by familial mechanisms in which mutations in the MAPT gene are dominantly inherited causing frontotemporal lobar degeneration (FTLD-tau) or by sporadic mechanisms in which MAPT haplotypes are associated with increased disease risk (e.g. progressive supranuclear palsy and corticobasal degeneration). MAPT mutations and risk haplotypes have been proposed to drive disease pathogenesis through proteoforms that contain 3-microtubue binding domain repeats (3R tau), 4R tau, or both. However, the mechanisms by which tauopathies occur remains poorly understood. We propose that MAPT mutations drive tau aggregation and neuronal dysfunction through altered proteostasis. In preliminary studies, we have shown that induced pluripotent stem cell derived-neurons expressing MAPT R406W exhibit changes in tau turnover compared to isogenic, control neurons, and we observed differences in the turnover of specific tau proteoforms in mutant neurons. Neurons expressing MAPT R406W exhibit enlarged lysosomal structures and secondary elevation of lysosomal enzymes, markers of lysosomes that are unable to properly degrade their contents. Correction of the mutant allele was sufficient to restore these lysosomal defects. This suggests that altered tau kinetics may be due to defects in the endolysosomal pathway. Thus, a unifying feature by which MAPT mutations drive tauopathy is through disrupted proteostasis. The objective of this study is to extend our preliminary findings to manipulate tau proteoforms using genetic or molecular methods to define the mechanisms by which tau proteoforms disrupt proteostasis in tauopathies. We hypothesize that tau proteoforms are sufficient to destabilize proteostasis and to result in the accumulation of tau in vulnerable brain regions. To test this hypothesis, we will determine the extent to which MAPT mutations cause impaired tau phenotypes and proteostasis characteristic of tauopathy. We will also generate a systematic genetic interaction map to elucidate connections between MAPT mutations, proteostasis, and associated therapeutic targets. Together, this study will reveal novel mechanisms underlying tauopathy that are driven by specific tau proteoforms and whether therapeutics designed to block specific tau proteoforms impact pathologic events.

Core F: Biomarker Project Summary Neurodegenerative diseases result from multifactorial processes that cause pleiotropic changes in the molecular networks that link a host of biological processes, leading to protein aggregation in the brain, and ultimately to the relentless decline in cognition that characterizes most age-related dementing illnesses. Neurodegenerative diseases, such as Alzheimer disease (AD), idiopathic Parkinson's disease (PD), and frontotemporal lobar degeneration (FTLD) occur by mechanisms in which common or rare variants associated with disease risk are directly inherited or arise sporadically. Human somatic and stem cell models have emerged as a powerful system for modeling the complexities of pathological gene expression, particularly in the early phase of disease, in the context of a non-neoplastic human genome. Further, human stem cells can be differentiated into individual cell types affected in disease, such as neurons, astrocytes, microglia, and oligodendrocytes, as well as 3D ?mini-brain? organoids. To this end, we have established a biorepository of stem cell models of AD and related dementias. The collection includes more than 200 human fibroblasts from the Knight ADRC and the Dominantly Inherited Alzheimer Network (DIAN) and induced pluripotent stem cell (iPSC) lines from more than 30 individuals carrying mutations in APP, PSEN1, PSEN2, GRN, MAPT, and risk variants in MAPT, APOE, TREM2, RAB10 and PLD3. Our long-term goal is to develop a set of tools and biomarkers for AD and related dementias. To do this, we will continue to build a biorepository of human somatic and stem cell models. These cells will facilitate the study of basic disease mechanisms, allow for discovery of novel biomarkers, and facilitate drug discovery platforms. We will focus on cell collection that builds on three major areas of strength in the Knight ADRC: (1) contributions of diverse ethnic backgrounds to molecular and cellular biomarkers of AD; (2) genetic and molecular modifiers of age at onset in large families with a dense family history of late onset AD; and (3) comparison of the molecular and cellular biomarkers that are common and unique between autososomal dominant AD and sporadic, late onset AD. Finally, we will make available the human somatic and stem cell lines to the broader research community to revolutionize our understanding of AD and related dementias.

Abstract Alzheimer?s disease (AD) is a complex and heterogenous condition in which multiple molecular pathways are disrupted in different cell-types and lead to disease. Genetic findings indicate that amyloid-beta protein clearance and degradation pathways, cholesterol metabolism and the immune system are associated with AD etiology. However, the specific mechanism, genes and molecular networks have not yet been completely identified. Single-nuclei transcriptomic (snRNA-seq) data from human brains provides a detailed molecular atlas to study the pathways dysregulated in AD. We propose to deepen our understanding of the genes, network and molecular pathways associated with AD by sequencing a high-number of neuronal and glial cells (approximately 3.3 million cells) from human brain carriers of key genetic mutations and high risk variants, non-carrier sporadic AD cases and neuropath-free controls. We will leverage a unique collection of human tissue from the Dominantly Inherited Alzheimer Network and Knight-ADRC brain banks, and select +220 brains to perform systematic cell- type specific transcriptomic analyses. This is a unique and innovative study designed to analyze cell-specific transcriptomic dysregulation in carriers of high effect risk variants (TREM2 and APOE) and fully penetrant pathogenic mutations in APP/PSEN1/PSEN2 and by comparing them to sporadic AD cases and neuropath-free controls. This is a powerful approach to address disease heterogeneity, and will provide highly informative insights into the biology and pathology of neurodegeneration. Replication of these findings will be performed in snRNA-seq data from induced pluripotent stem cell derived neurons, astrocytes, and microglia-like cells that will be genome edited to add/remove genetic variants, as well as datasets that are being publicly released. Finally, we will create a knowledge portal in which all of the processed snRNA-seq data from our study will be harmonized with that of other research groups to provide a comprehensive molecular atlas that will provide additional insights into the biology and pathology of AD for the entire research community.

PROJECT SUMMARY/ABSTRACT Cerebrovascular pathology is present throughout stages of Alzheimer?s Disease and is correlated with cognitive changes. There is strong evidence that vascular dysfunction is a significant driver of neuropathology. Our long- term objective is to understand the function of Alzheimer?s Disease-associated risk genes in vascular cells, their contribution to the development of cerebrovascular pathology and the opportunities to use this information in therapeutic development. There are over 27 Alzheimer?s Disease-associated risk (AD-risk) loci encompassing numerous genetic variants in non-coding and coding regions and hundreds of linked genes. Our overarching hypothesis is that a subset of AD-risk genes impairs vascular function, causing release of inflammatory factors, blood brain barrier (BBB) impairment, and reduced perfusion, thus contributing to neurodegeneration. To address this, we have assembled a multi-disciplinary team with a proven track record of collaboration, including with ADSP and ADGP members, who bring expertise in vascular pathology in dementia, endothelial cell (EC) signaling and EC functional testing, Alzheimer?s Disease genomics, single cell and nuclear transcriptomics, bioinformatics, CRISPR-based gene editing for large scale screening and AD mouse models for in-depth functional assessment in vivo. Notably, we will address differences in gene effects related to the important biological variables, sex and metabolic disease. Men and women differ in their genetic risk for Alzheimer?s Disease, with sex-specific polygenic risk scores providing better prediction of onset, progression, and pathology than pooled-sex scores. Over 80% of individuals with Alzheimer?s Disease have co-morbid metabolic disease, which exacerbates vascular pathology. We have identified the top 50 AD-risk SNPs and 600 AD-associated genes, and these will be targeted for induced pluripotent stem cell (iPSC)-derived endothelial cell (EC) screens by prime editing and CRISPR-based gene inhibition and activation approaches respectively. iPSC-based production of human ECs and mural cells in 2D and 3D models has been optimized and scaled to enable efficient functional testing of the impact of gene changes, including on neuro-vascular interactions in cerebral organoids. Discoveries made in these human cell systems will be validated by an in-depth investigation of gene expression changes in individual ECs and mural cells across a large collection of Alzheimer?s Disease brain samples using single nuclear sequencing. The EC translatome will also be obtained from mouse Alzheimer?s Disease models that incorporate sex and metabolic disease. These diverse datasets will be harmonized and integrated in order to map vascular phenotypes of AD-risk genes and identify critical molecular pathways that are targetable drivers of AD cerebrovascular pathology. These data will add to the breadth of knowledge being gathered by other groups to further elucidate underlying neuronal, glial, microglial, endothelial and mural cell-cell interactions that contribute in a substantial way to the complex architecture of Alzheimer Disease pathology. 1

PROJECT SUMMARY Pathological tau deposition occurs in a subset of neurodegenerative disorders including frontotemporal lobar degeneration with tau inclusions (FTLD-tau). Much of what is known about FTLD-tau is derived from mutant tau models or overexpression. However, a unified view of overall tau metabolism?from its initial production, interactions with molecular chaperones, post-translational modifications, targeting to lysosomes/autophagy, and resultant degradation, to our knowledge has not been generated The long- term goal of this proposed FTD Center without Walls (CWOW) is to improve our understanding of the pathobiological mechanisms underlying FTD-tau. Its overall objective is to elucidate the genes, molecules and pathways that regulate tau metabolism and to determine the impact of disease- associated mutations and variants. Our central hypothesis is that proper tau metabolism requires the precise, coordinated action of molecular chaperones, co-chaperones, PTMs and degradation machinery that each represent regulatory nodes. Genetic mutations in tau and other pathway members can disrupt tau metabolism, leading to tau accumulation, secretion and neurodegeneration. The Center will be led by Dr. Aimee Kao, who will also lead Core A: Administration and Data Core (with Co-lead Dr. Yokoyama) and Project 1: Tau Molecular Chaperones, targeting and proteolysis (with Co-I Dr. Agard). Dr. David Agard will oversee Core B: Macromolecular and Cellular Structure Core. Dr. Jennifer Yokoyama will lead Core C: Genomics and Transcriptomics. Finally, Dr. Celeste Karch will lead Project 2: Tau Half Life and Secretion. We will achieve these objectives through four Specific Aims. Aim 1: Understand the normal process of tau metabolism as a series of decisions that are made at regulatory nodes. Aim 2: Identify and test the functional relevance of genetic variants in MAPT and other tau metabolism genes, in in vitro, cell and iNeuron models, on each of the tau metabolism regulatory nodes. Aim 3: Integrate findings from Projects and Cores to produce a Tau Metabolism and Variant Database (TMVdb), that will serve as a reference point for the field. Aim 4: Integrate findings from Projects and Cores to produce a Tau Polygenic Risk Score (TPRS), which will stratify genetic risk for tauopathy. Upon successful completion of these Aims, the proposed FTD CWOW will have provided fundamental information about tau metabolism, defined mechanistic nodes predisposing to tauopathy and generated the TMVdb and TPRS, new resources for the fields of tauopathy and neurodegeneration research. It will generate critically important information about tau homeostasis and a foundational basis from which to build and frame subsequent investigations into tau pathobiology and toxicity.