Jansen‐West, Karen

ABSTRACT Intronic GGGGCC hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Despite its intronic location, this repeat avidly supports synthesis of pathogenic dipeptide repeat (DPR) proteins via repeat-associated non-AUG (RAN) translation. However, the template RNA species that undergoes RAN translation endogenously remains unclear. Using long-read based 5’ RNA ligase-mediated rapid amplification of cDNA ends (5’ Repeat-RLM-RACE), we identified novel C9orf72 transcripts initiating within intron 1 in a C9BAC mouse model, patient-derived iNeurons, and iNeuron-derived polysomes. These cryptic m 7 G-capped mRNAs are at least partially polyadenylated and are more abundant than transcripts derived from intron retention or circular intron lariats. In RAN translation reporter assays, novel intronic template transcripts – even those with short (32 nucleotide) leaders – exhibited robust expression compared to exon-intron and repeat-containing lariat reporters. To assess endogenous lariat repeat RNA contributions to RAN translation, we enhanced endogenous lariat stability by knocking down the lariat debranching enzyme Dbr1. However, this modulation did not impact DPR production in patient-derived iNeurons. These findings identify cryptic, linear, m 7 G-capped intronic-initiating C9orf72 mRNAs as an endogenous template for RAN translation and DPR production, with implications for disease pathogenesis and therapeutic development. SIGNIFICANCE STATEMENT An intronic GGGGCC repeat expansion in C9orf72 supports an unusual translational initiation process known as repeat-associated non-AUG (RAN) translation to produce toxic dipeptide repeat (DPR) proteins that contribute to neurodegeneration in ALS and FTD. How an intronic repeat RNA engages with ribosomes to support such translation is unclear. Here we identify a series of novel mRNA transcripts that initiate within the repeat-containing intron to create linear m 7 G-capped templates for RAN translation from GGGGCC repeats. These cryptic mRNAs are present in patient iNeurons, engage with ribosomes, and robustly support RAN translation. This finding has important implications for both our understanding of the mechanism by which RAN translation occurs and on therapeutic development in this currently untreatable class of neurodegenerative disorders.

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.

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.

Lewy body dementia (LBD) is an often misdiagnosed and mistreated neurodegenerative disorder clinically characterized by the emergence of neuropsychiatric symptoms followed by motor impairment. LBD falls within an undefined range between Alzheimer’s disease (AD) and Parkinson’s disease (PD) due to the potential pathogenic synergistic effects of tau, beta-amyloid (Aβ), and alpha-synuclein (αsyn). A lack of reliable and relevant animal models hinders the elucidation of the molecular characteristics and phenotypic consequences of these interactions. Here, the goal was to evaluate whether the viral-mediated overexpression of αsyn in adult hTau and APP/PS1 mice or the overexpression of tau in Line 61 hThy1-αsyn mice resulted in pathology and behavior resembling LBD. The transgenes were injected intravenously via the tail vein using AAV-PHP.eB in 3-month-old hThy1-αsyn, hTau, or APP/PS1 mice that were then aged to 6-, 9-, and 12-months-old for subsequent phenotypic and histological characterization. Although we achieved the widespread expression of αsyn in hTau and tau in hThy1-αsyn mice, no αsyn pathology in hTau mice and only mild tau pathology in hThy1-αsyn mice was observed. Additionally, cognitive, motor, and limbic behavior phenotypes were not affected by overexpression of the transgenes. Furthermore, our APP/PS1 mice experienced premature deaths starting at 3 months post-injection (MPI), therefore precluding further analyses at later time points. An evaluation of the remaining 3-MPI indicated no αsyn pathology or cognitive and motor behavioral changes. Taken together, we conclude that the overexpression of αsyn in hTau and APP/PS1 mice and tau in hThy1-αsyn mice does not recapitulate the behavioral and neuropathological phenotypes observed in LBD.

Hexanucleotide G 4 C 2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G 4 C 2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72 450 mice. However, chronic administration of anti-GA antibodies in AAV-(G 4 C 2 ) 149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.

Frontotemporal dementia and amyotrophic lateral sclerosis (FTD-ALS) are associated with both a repeat expansion in the C9orf72 gene and mutations in the TANK-binding kinase 1 ( TBK1 ) gene. We found that TBK1 is phosphorylated in response to C9orf72 poly(Gly-Ala) [poly(GA)] aggregation and sequestered into inclusions, which leads to a loss of TBK1 activity and contributes to neurodegeneration. When we reduced TBK1 activity using a TBK1-R228H (Arg 228 →His) mutation in mice, poly(GA)-induced phenotypes were exacerbated. These phenotypes included an increase in TAR DNA binding protein 43 (TDP-43) pathology and the accumulation of defective endosomes in poly(GA)-positive neurons. Inhibiting the endosomal pathway induced TDP-43 aggregation, which highlights the importance of this pathway and TBK1 activity in pathogenesis. This interplay between C9orf72 , TBK1 , and TDP-43 connects three different facets of FTD-ALS into one coherent pathway.

AbstractBackground and purposeClinical trials in spinocerebellar ataxia type 3 (SCA3) will require biomarkers for use as outcome measures.MethodsTo evaluate total tau (t‐tau), glial fibrillary acidic protein (GFAP), ubiquitin carboxy‐terminal hydrolase L1 (UCHL1) and neurofilament light‐chain (NfL) as fluid biomarkers in SCA3, ATXN3 mutation carriers (n = 143) and controls (n = 172) were clinically assessed, and the plasma concentrations of the four proteins were analysed on the Simoa HD‐1 platform. Eleven ATXN3 mutation carrier cerebrospinal fluid samples were analysed for t‐tau and phosphorylated tau (p‐tau181). A transgenic SCA3 mouse model (MJDTg) was used to measure cerebellar t‐tau levels.ResultsPlasma t‐tau levels were higher in mutation carriers below the age of 50 compared to controls, and the Inventory of Non‐Ataxia Signs was associated with t‐tau in ataxic patients (p = 0.004). Pre‐ataxic carriers showed higher cerebrospinal fluid t‐tau and p‐tau181 concentrations compared to ataxic patients (p = 0.025 and p = 0.014, respectively). Cerebellar t‐tau was elevated in MJDTg mice compared to wild‐type (p = 0.033) only in the early stages of the disease. GFAP and UCHL1 did not show higher levels in mutation carriers compared to controls. Plasma NfL concentrations were higher in mutation carriers compared to controls, and differences were greater for younger carriers. The Scale for the Assessment and Rating of Ataxia was the strongest predictor of NfL in ataxic patients (p < 0.001).ConclusionOur results suggest that tau might be a marker of early disease stages in SCA3. NfL can discriminate mutation carriers from controls and is associated with different clinical variables. Longitudinal studies are required to confirm their potential role as biomarkers in clinical trials.

Hospitalized patients with COVID-19 show increased serum concentrations of neurofilament light chain that correlate with worse clinical outcomes.

Plasma neurofilament light is a reliable, uniform marker to estimate neuroaxonal injury and forecast all-cause mortality across major stroke types.