Petrucelli, Leonard

Abstract A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation—suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43–depleted human iPSC–derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43–depleted human iPSC–derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43–depleted human iPSC–derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

Genetic variation at the transmembrane protein 106B gene ( TMEM106B) has been linked to risk of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) through an unknown mechanism. We found that presence of the TMEM106B rs3173615 protective genotype was associated with longer survival after symptom onset in a postmortem FTLD-TDP cohort, suggesting a slower disease course. The seminal discovery that filaments derived from TMEM106B is a common feature in aging and, across a range of neurodegenerative disorders, suggests that genetic variants in TMEM106B could modulate disease risk and progression through modulating TMEM106B aggregation. To explore this possibility and assess the pathological relevance of TMEM106B accumulation, we generated a new antibody targeting the TMEM106B filament core sequence. Analysis of postmortem samples revealed that the TMEM106B rs3173615 risk allele was associated with higher TMEM106B core accumulation in patients with FTLD-TDP. In contrast, minimal TMEM106B core deposition was detected in carriers of the protective allele. Although the abundance of monomeric full-length TMEM106B was unchanged, carriers of the protective genotype exhibited an increase in dimeric full-length TMEM106B. Increased TMEM106B core deposition was also associated with enhanced TDP-43 dysfunction, and interactome data suggested a role for TMEM106B core filaments in impaired RNA transport, local translation, and endolysosomal function in FTLD-TDP. Overall, these findings suggest that prevention of TMEM106B core accumulation is central to the mechanism by which the TMEM106B protective haplotype reduces disease risk and slows progression.

Abstract Background The ALLFTD (ARTFL‐LEFFTDS Longitudinal Frontemporal Lobar Degeneration) study is an NIH‐funded effort to prepare for clinical trials in sporadic (s‐FTLD) and familial (f‐FTLD) FTLD syndromes by characterizing cohorts, developing new clinical trial outcome measures, and evaluating disease progression. ALLFTD represents the merger and continuation of two prior studies: Advancing Research and Treatment of Frontotemporal Lobar Degeneration (ARTFL) and Longitudinal Evaluation of Familial Frontotemporal Dementia Subjects (LEFFTDS). Methods ALLFTD recruits participants with FTLD spectrum disorders (bvFTD, svPPA, nfvPPA, FTD‐ALS, CBS or PSP), with strong family histories of FTLD, or known FTLD‐associated genetic variants within the family. 26 sites in North America enroll participants for clinical and neuropsychological evaluations, MR Imaging, blood draws, and CSF collection in willing participants; participants are requested to return annually for follow‐up evaluation. All participants are genotyped for dementia‐associated mutations. Participants also enroll in the FTD Disorders Registry for follow‐up surveys. A subset of participants undergo remote assessment via the ALLFTD app. Visits conducted under ARTFL and LEFFTDS are included in the ALLFTD dataset. Results ALLFTD has been successful in developing methods to conduct remote or hybrid visits and in actively evaluating participants. Since ALLFTD began in January 2020, 1034 participants (736 new; 298 previously enrolled in ARTFL or LEFFTDS) have been evaluated in the longitudinal arm; an additional 119 participants have been participated in a shortened, biofluid‐focused visit. 349 have returned for follow‐up visits. Including prior participants, the ALLFTD dataset contains 3871 visits from 2343 individuals. 1277 people have sporadic syndromes; 1045 were enrolled as familial. Among s‐FTLD, bvFTD is the most common phenotype (32.8%) followed by PSP (15.9%). 61.4% of f‐FTLD participants were clinically normal at first visit; bvFTD is the most common syndrome in symptomatic f‐FTLD (40%). 477 are confirmed carriers of FTLD‐associated genetic mutations. MRIs were obtained for 2594 visits; 3492 visits have associated blood biospecimens banked; 978 visits have associated CSF samples. Conclusions The ALLFTD consortium is actively evaluating participants across North America to better characterize FTLD syndromes and support the planning and development of FTLD clinical trials. Longitudinal datasets including clinical, genetic, and imaging data are available by request.

AbstractBackgroundPathogenic mutations in the MAPT (tau) gene cause familial frontotemporal lobar degeneration (f‐FTLD). No disease‐modifying treatments exist. Tau therapeutics being tested in other tauopathies may be suitable candidates, but the small number of identified mutation carriers prohibits traditional clinical trial designs from achieving adequate power. Platform trial designs permit multiple therapies to be tested in parallel and improve power by sharing placebo groups across treatment arms. We evaluate whether a platform design could improve the likelihood of identifying an effective treatment for MAPT mutation carriers.MethodALLFTD and GENFI observational data were used to simulate MAPT prevention and early symptomatic clinical trials (CDR®+NACC‐FTLD≤1). Only presymptomatic participants (CDR®+NACC‐FTLD = 0) within 2.5 years of expected symptom onset based on disease progression models (DPM) were included. First, we evaluated the time and sample size required to test five treatments using a platform design compared to sequential randomized control trials. Platform designs assumed a max of three treatment arms at any time, with new arms enrolling upon completion of prior arms. Each arm randomized 60 mutations carriers 3:1 treatment to placebo, with a placebo group shared across arms. Minimum follow up was 1.5 years with common close. Sequential trials assumed 90 participants allocated 1:1 treatment to placebo, with three‐month intervals between trials. Using a platform design, we compared a novel method for testing therapeutic effects using DPMs to account for differential rates of expected progression compared to traditional mixed models for repeated measures (MMRM) tests controlling for baseline CDR®+NACC‐FTLD.ResultFive treatments could be tested in a platform design using 300 participants (75% treated) over five years, compared to 450 participants (50% treated) over 13 years using traditional, sequential trials. Platform trial simulations indicated that with 80% power, DPM‐based analytic approach could detect a 25% treatment effect, whereas traditional MMRM could only detect a 40% effect.ConclusionPlatform designs combined with DPM‐based analyses provide a path to testing more treatments with fewer participants. Adequately powered MAPT platform trials are feasible but require a global clinical trial infrastructure.

AbstractBackgroundTMEM106B, a lysosomal trafficking protein, is an important genetic susceptibility risk factor for frontotemporal lobar degeneration (FTLD). In GRN mutation carriers, minor allele homozygosity in the TMEM106B rs1990622 SNP modulates the risk of FTLD. TMEM106B is often the main component of amyloid fibrils in FTLD with TDP43 aggregates. TMEM106B has been quantified in human brain specimens, but its role as a fluid biomarker is unknown. Here, we investigate the clinical value of CSF TMEM106B in FTLD.MethodsCSF TMEM106B was quantified with SOMAmer proteomics (v3.0, SomaLogic®) in an original cohort of C9orf72, GRN and MAPT symptomatic and asymptomatic mutation carriers and family non‐carrier controls, recruited through ALLFTD (n = 182), and a validation cohort of sporadic neuropathology‐confirmed FTLD‐tau and FTLD‐TDP43, clinically‐diagnosed progressive supranuclear palsy – Richardson syndrome (PSP‐RS) and age‐matched controls (n = 96). CSF TMEM106B was correlated with disease severity, measured by the Frontotemporal Lobar Degeneration module global scores (CDR+NACC/FTLD); brain volume, measured with Bayesian linear mixed‐effect modeling, using age, sex and total‐intracranial volume‐corrected permutations with threshold‐free cluster enhancement; and CSF neurofilament light chain (NfL) measured with Simoa. TMEM106B SOMAmer specificity was validated in cell culture overexpression systems and human brain specimens with high TMEM106B burden.ResultsIn both cohorts, there were no differences in CSF TMEM106B by sex, phenotype or neuropathological diagnosis, except for lower levels in PSP‐RS compared to controls (‐0.31 Log2‐fold change, p < 0.001). CSF TMEM106B did not correlate with age. Regardless of disease‐causing mutation, lower CSF TMEM106B was associated with worse disease severity (Figure 1 and 2). CSF TMEM106B was lower in homozygous TMEM106B rs1990622 protective allelein C9orf2 and MAPT, but not in GRN mutation carriers (Figures 3). CSF TMEM106B showed a trend for inverse correlation with NfL in the whole sample (r = ‐0.135, p = 0.052), and correlated positively with left frontal volume in symptomatic FTLD mutation gene carriers (p < 0.05).ConclusionCSF TMEM106B is quantifiable in CSF in vivo. CSF TMEM106B levels are affected by an interaction of disease severity and FTLD and TMEM106B genotypes. Further work is needed to determine its value as a clinical biomarker.

AbstractBackgroundAlzheimer’s disease (AD) is characterized by the accumulation of neurofibrillary tangles (NFTs), comprised primarily of microtubule‐associated protein tau. NFTs are defining hallmarks of AD and its accumulation into pathological oligomers and fibrils has been associated with cognitive decline in AD and other tauopathies. Despite its pivotal role in the disease process, we still have a poor understanding of how their formation, toxicity, and spread across brain regions is regulated via tau‐associated proteins that regulate the oligomerization, pathological accumulation, and seeding of tau in affected cell types and vulnerable brain regions.MethodWe have established two proximity‐dependent biotin‐identification (BioID) models as novel approaches to identify the composition and proximal molecular environment of tau aggregates. Using an in vitro and ex vivo model approach coupled with mass spectrometry, we have identified proteins that were primarily associated with mutant tau aggregates vs. soluble wildtype tau and stratified them based on significance, molecular pathway and druggability. Immunoblotting and immunofluorescence were performed to assess the interaction of endogenous and overexpressed top candidates with tau, along with visualization with NFTs in the context of human AD pathology to assess their impact on tau‐aggregate formation and toxicity.ResultTo determine the physiological and aggregate specific interacting partners of tau, we are comparing wild type tau (TauWT) with mutant tau (Tau3xMUT) carrying three tauopathy‐associated mutations (A152T/P301L/S320F). The proteomic analysis from our combined proteomic datasets revealed that significantly changed proteins included candidates involved in RNA processing, as well as protein ubiquitination and proteasome degradation. We have begun validating proteins that are enriched in mutant tau in human tissue of AD, as well as primary tauopathies, that may be druggable targets for tauopathies.ConclusionThis study provides novel insights into cellular pathways and molecular mechanisms of neurodegeneration, by identifying in the context of living neurons and brain tissue different functional classes of tau‐associated proteins with relevance for AD pathophysiology. These are expected to include proteins that may mediate the toxic effect of NFTs, facilitate the formation or degradation of pathological tau aggregates, and catalyze posttranslational modifications of tau oligomers and associated proteins.

Abstract Transactive response DNA-binding protein 43 (TDP-43) pathology is categorized as type A-E in frontotemporal lobar degeneration and as type α-β in Alzheimer disease (AD) based on inclusion type. We screened amygdala slides of 131 cases with varying ages at death, clinical/neuroimaging findings, and AD neuropathologic changes for TDP-43 pathology using anti-phospho-TDP-43 antibodies. Seven cases (5%) only showed atypical TDP-43 inclusions that could not be typed. Immunohistochemistry and immunofluorescence assessed the atypical star-shaped TDP-43 pathology including its distribution, species, cellular localization, and colocalization with tau. All 7 had died at an extremely old age (median: 100 years [IQR: 94–101]) from nonneurological causes and none had dementia (4 cognitively unimpaired, 3 with amnestic mild cognitive impairment). Neuroimaging showed mild medial temporal involvement. Pathologically, the star-shaped TDP-43-positive inclusions were found in medial (subpial) amygdala and, occasionally, in basolateral regions. Hippocampus only showed TDP-43-positive neurites in the fimbria and subiculum while the frontal lobe was free of TDP-43 inclusions. The star-shaped inclusions were better detected with antibodies against N-terminal than C-terminal TDP-43. Double-labeling studies confirmed deposition of TDP-43 within astrocytes and colocalization with tau. We have identified a novel TDP-43 pathology with star-shaped morphology associated with superaging, with a homogeneous clinicopathologic picture, possibly representing a novel, true aging-related TDP-43 pathology.