John F. Crary - US grants

? DESCRIPTION (provided by applicant): Recent consensus criteria have been put forth by a large group of prominent neuropathologists and neuroscientists to define a new category of Alzheimer's disease (AD) neuropathologic changes. Individuals with this disorder, now termed primary age-related tauopathy (PART), develop AD-type neurofibrillary tangles (NFT) in the absence of amyloid beta peptide (A?) containing-plaques. Patients with PART may have normal cognition, amnestic mild cognitive impairment (aMCI), or an amnestic dementia. The prevalence of PART dementia is unclear, with estimates ranging from 1-7%, but PART is likely more pervasive. Recent studies have found a common neuroimaging and cerebrospinal fluid biomarker profile, termed suspected non-amyloid pathophysiol- ogy (SNAP), in ~25% of non-demented elderly individuals and patients with aMCI which shows remarkable similarities to PART. The objective of this application is to perform a focused histopathological, molecular and rigorous genetic study of PART. Our central hypothesis is that subjects with PART have distinct characteristics that underlie their NFT+/A?- phenotype. Understanding the molecular changes of, and genetic risk factors for, PART will reveal mechanistic insights into its pathogenesis and in turn advance our understanding of the re- gional vulnerability of the temporal lobe to neurodegeneration and the pathogenesis of tauopathies in general. In aim 1, we will validate the new neuropathologic criteria for PART and lay the groundwork for clinical and mechanistic studies that will elucidate disease burden, pathogenesis and progression. This will be performed in a large dementia autopsy series using stereology-based semiquantitative and quantitative measurements of NFT burden. In aim 2, we will test the hypothesis that PART has a molecular signature consisting of AD-type tau pathology alongside non-amyloidogenic APP metabolites. This will be accomplished by comparing the levels and distribution of toxic tau species and APP metabolites using quantitative immunoblot, ELISA, brightfield immunohistochemistry and immunofluorescence confocal microscopy in autopsy brain tissue from PART, AD, and control cases. Finally, in aim 3 we will test the hypothesis that PART has a genetic risk profile that over laps with some aspects of AD genetics (e.g., MAPT haplotypes) but diverges with respect to others (e.g., APOE genotype). This aim will be accomplished by performing a series of association analyses comparing the frequency of known AD risk alleles in PART, AD and controls. We will also perform a genome-wide quantitative trait analysis of NFT burden using a large cohort of PART subjects to discover new candidate risk alleles. This project will establish that a group of individuals with AD tauopathic changes have a distinct constellation of clinical features, molecular changes and genetic risk factors. The proposed research is innovative because considering PART as an A?-independent process represents a paradigm shift in terms of how we conceptual- ize the tauopathic component of AD neuropathologic changes.

PROJECT 3 SUMMARY Understanding the earliest events in Alzheimer disease (AD) has the potential to lead to therapeutic strategies that slow and/or halt disease progression. Addressing AD at its earliest stages is critical as it might be too late to regain cognitive function once neuronal loss has occurred. Development of neurofibrillary tangles (NFT) in the entorhinal cortex (EC) is among the earliest pathological events in AD, but what underlies this regional vul- nerability remains a mystery. NFT are abnormal aggregates of the microtubule-associated protein tau that be- come hyperphosphorylated and aggregated following abnormal kinase activity. Several post-translational modi- fications have been proposed to contribute to tau toxicity, with hyperphosphorylation thought to play a pivotal role. Recently, it has become widely recognized that late-onset AD is associated with abnormalities in mi- croRNAs (miRNAs), small non-coding RNAs that are powerful regulators of gene expression. MiRNAs bind to and silence target mRNAs on the post-transcriptional level. Each miRNA can bind many target transcripts, providing an additional level of regulation that complements canonical transcriptional pathways. While a con- sensus is beginning to emerge that certain miRNAs are consistently altered in AD, the functional significance if these findings remain unclear. The overall objective of this application is to characterize miRNA regulation of the expression of tau and glycogen synthase kinase 3 b (GSK3b), a kinase implicated in tau phosphorylation and leading candidate drug target in AD. This will be accomplished by performing miRNA profiling studies in human autopsy brain tissue and then modeling candidate pathogenic changes with in vitro mammalian neu- ronal cultures and transgenic Drosophila. Our central hypothesis is that down-regulation of miRNAs leads to de-repression of a genetic program that increases expression of genes that drive tau toxicity. To test our hy- pothesis, we propose the following specific aims: 1) to characterize changes in miRNA expression early in AD progression, with a focus on the lateral entorhinal cortex, a region affected early in AD, 2) to characterize miR- NA regulation of the expression of tau and GSK3b, and 3) to demonstrate that miRNAs modulate tau neurotox- icity. This project is significant because it will lead to a better understanding of a genetic program that regulates the expression of genes that we hypothesize contribute the generation of toxic forms of hyperphosphorylated tau and the progression of AD. This proposal is innovative because looking at miRNA regulation has forced a re-assessment of gene expression that will provide a new perspective on the pathogenesis of AD as well as a new framework and new models to investigate mechanisms of tau regulation and toxicity. Together, these ex- periments will help us address the critical need of understanding the earliest events in AD and help pave the way towards new strategies to address AD with miRNA-based biomarkers and therapeutics.

The 17q21.31 MAPT locus is associated with Alzheimer's disease (AD) and sporadic primary tauopathies in the absence of a tau gene coding region mutation, but the causative variants remain unknown. Our long-term goal is to understand the pathogenesis of tauopathies and pave the way towards novel diagnostics and therapeutics. The objective here is to use new diagnostic criteria to better stratify subjects for a molecular genetic study of tauopathy. Using high-resolution fine mapping and long-read single molecule real time (SMRT) DNA sequencing that employs phospholinked nucleotides to produce very long reads (>70,000 bp) to overcome structural complexity at the locus and pinpoint functional alleles. Further, we will use long-read mRNA isoform sequencing (IsoSeq) to assess mRNA levels, splicing and alternative 3' untranslated regions (UTRs) that might influence post-transcriptional regulation, including RNA binding proteins and microRNAs. We will validate our findings using biochemical, histopathological and cellular techniques. Our central hypothesis is that distinct subhaplotypes confer risk for different MAPT-associated diseases through dysregulation of the expression of toxic tau species leading to unique higher order degenerative phenotypes. Our rationale is that understanding these changes will be crucial for diagnostic stratification and further clinical and mechanistic studies. We will test our hypothesis by pursuing the following specific aims: Aim 1. To identify functional risk alleles associated with tauopathy, we will generate a high density genetic map of the 17q21.31 locus using high-coverage genotyping and targeted SMRT sequencing to characterize the locus and correlate novel variation with AD, primary age-related tauopathy, progressive supranuclear palsy, corticobasal degeneration, Parkinson disease and other tauopathies to pinpoint functional risk alleles; Aim 2. To discover and validate novel mRNA splicing events in tauopathy, we will perform a genome wide computational analysis using brains from subjects with AD and other tauopathies to discover novel splicing events in tau and other genes as well as changes in splicing factors then validate these findings using biochemical, histopathological and cellular approaches; and Aim 3. To uncover novel post-transcriptional regulators of tauopathy, we will discover and validate both protein and RNA post-transcriptional regulators of tau and tauopathy and characterize their networks and validate these changes using biochemical, histopathological and cellular approaches. This contribution will be significant because it will provide a foundation for further mechanistic studies that will elucidate the drivers of disease and pave the way for future clinical studies. This proposal is innovative because by studying AD in the context of the tauopathies there is the possibility that it will lead to a harmonized classification system.

Establishing an accurate neuropathological diagnosis is a critical function of the Alzheimer's Disease Center (ADC) Neuropathology Cores and is a prerequisite to all tissue-based research. Although chronic traumatic encephalopathy (CTE), primary age-related tauopathy (PART), argyrophilic grain disease (AGD) and aging- related tau astrogliopathy (ARTAG) are all age-related tauopathies, each has distinctive patterns of abnormal tau deposition. Additionally, each has specific, but overlapping, effects on cognitive and neuropsychiatric function. Neuropathological criteria to diagnose CTE, PART, AGD and ARTAG have been recently proposed, yet no modules exist to capture these disorders in the National Alzheimer's Coordinating Center (NACC) database. Without rigorous, reproducible protocols for the diagnosis of age-related tauopathies, their disease- specific contributors to late life cognitive and neuropsychiatric impairment may be overlooked and obscured. This proposal will draw on the neuropathological expertise of Drs. Ann McKee, Dennis Dickson, and John Crary, who have pioneered investigations of CTE, AGD and PART. We will use 250 cases of AD and age- related tauopathies from the Boston University, Mayo Clinic Jacksonville and Mount Sinai ADCs to identiify novel tau and inflammatory markers that accurately detect and distinguish the age-related tauopathies. After re-examining tissue from the NACC-Neuropathology Data Set (NDS), we will establish the frequency of CTE, PART, AGD and ARTAG among individuals who had ante-mortem clinical diagnoses of normal cognition, mild cognitive impairment, or dementia. We will determine the unique contribution of these disorders to clinical diagnosis and to cognitive and neuropsychiatric impairment. We will also determine whether examining under- studied brain regions, namely the prefrontal cortex, uncal gyrus and amygdala identifies new cases of age- related tauopathies and whether pathology in these regions contributes to neuropsychiatric impairment. Lastly, we will apply knowledge gained from this project to develop CTE, PART, AGD and ARTAG assessment and diagnostic modules that we will use to organize an international NIH consensus conference to harmonize the neuropathological diagnoses of the age-related tauopathies for general use. Overall, this proposal will address critical, yet still unknown issues related to CTE, PART, AGD and ARTAG: 1) Identification of novel tau and neuroinflammatory biomarkers to aid in detection and discrimination of the age-related tauopathies, 2) Determination of age-related tauopathy frequency among memory disorder clinic patients; 3) Determination of age-related tauopathy contribution to cognitive and neuropsychiatric impairment; and 4) Development of harmonized criteria for age-related tauopathy neuropathological diagnosis. This project will also enrich the existing NACC-NDS, making data on age-related tauopathies available to other researchers. Overall, the successful completion of this proposal will transform the way we conceptualize the tauopathies and will facilitate future research on the diagnosis, treatment, and prevention of AD and related disorders.