C9orf72 Repeat Research Articles

Genetic variability in sporadic amyotrophic lateral sclerosis.

With the advent of gene therapies for amyotrophic lateral sclerosis, there is a surge in gene testing for ALS. Although there is ample experience with gene testing for C9orf72, SOD1, FUS and TARDBP in familial ALS, large studies exploring genetic variation in all ALS-associated genes in sporadic ALS (sALS) are still scarce. Gene testing in a diagnostic setting is challenging given the complex genetic architecture of sALS with genetic variants with large and small effect sizes. Guidelines for interpretation of genetic variants in gene panels and for counselling of patients are lacking. We aimed to provide a thorough characterization of genetic variability in ALS genes by applying the ACMG criteria on whole genome sequencing data from a large cohort of 6013 sporadic ALS patients and 2411 matched controls from Project MinE. We studied genetic variation in 90 ALS-associated genes and applied customized ACMG-criteria to identify pathogenic and likely pathogenic variants. Variants of unknown significance were collected as well. In addition, we determined the length of repeat expansions in C9orf72, ATXN1, ATXN2 and NIPA1 using the ExpansionHunter tool. We found C9orf72 repeat expansions in 5.21% of sALS patients. In 50 ALS-associated genes, we did not identify any pathogenic or likely pathogenic variants. In 5.89%, a pathogenic or likely pathogenic variant was found, most commonly in SOD1, TARDBP, FUS, NEK1, OPTN or TBK1. Significantly more cases carried at least one pathogenic or likely pathogenic variant compared to controls (OR 1.75; p-value 1.64 × 10-5). Isolated risk factors in ATXN1, ATXN2, NIPA1 and/or UNC13A were detected in 17.33% of cases. In 71.83%, we did not find any genetic clues. A combination of variants was found in 2.88%. This study provides an inventory of pathogenic and likely pathogenic genetic variation in a large cohort of sALS. Overall, we identified pathogenic and likely pathogenic variants in 11.13% of ALS patients in 38 known ALS genes. In line with the oligogenic hypothesis, we found significantly more combinations of variants in cases compared to controls. Many variants of unknown significance may contribute to ALS risk, but diagnostic algorithms to reliably identify and weigh them are lacking. This work can serve as a resource for counselling and for the assembly of gene panels for ALS. Further characterization of the genetic architecture of sALS is necessary given the growing interest in gene testing in ALS.

Cortical iron accumulation in MAPT- and C9orf 72-associated frontotemporal lobar degeneration.

Neuroinflammation has been implicated in frontotemporal lobar degeneration (FTLD) pathophysiology, including in genetic forms with microtubule-associated protein tau (MAPT) mutations (FTLD-MAPT) or chromosome 9 open reading frame 72(C9orf72) repeat expansions (FTLD-C9orf72). Iron accumulation as a marker of neuroinflammation has, however, been understudied in genetic FTLD to date. To investigate the occurrence of cortical iron accumulation in FTLD-MAPT and FTLD-C9orf72, iron histopathology was performed on the frontal and temporal cortex of 22 cases (11 FTLD-MAPT and 11 FTLD-C9orf72). We studied patterns of cortical iron accumulation and its colocalization with the corresponding underlying pathologies (tau and TDP-43), brain cells (microglia and astrocytes), and myelination. Further, with ultrahigh field ex vivo MRI on a subset (four FTLD-MAPT and two FTLD-C9orf72), we examined the sensitivity of T2*-weighted MRI for iron in FTLD. Histopathology showed that cortical iron accumulation occurs in both FTLD-MAPT and FTLD-C9orf72 in frontal and temporal cortices, characterized by a diffuse mid-cortical iron-rich band, and by a superficial cortical iron band in some cases. Cortical iron accumulation was associated with the severity of proteinopathy (tau or TDP-43) and neuronal degeneration, in part with clinical severity, and with the presence of activated microglia, reactive astrocytes and myelin loss. Ultra-high field T2*-weighted MRI showed a good correspondence between hypointense changes on MRI and cortical iron observed on histology. We conclude that iron accumulation is a feature of both FTLD-MAPT and FTLD-C9orf72 and is associated with pathological severity. Therefore, in vivo iron imaging using T2*-weighted MRI or quantitative susceptibility mapping may potentially be used as a noninvasive imaging marker to localize pathology in FTLD.

C9orf72 expansions are the most common cause of genetic frontotemporal dementia in a Southeast Asian cohort.

Frontotemporal dementia (FTD) encompasses a spectrum of neurodegenerative disorders, including behavioural variant FTD (bvFTD), semantic variant primary progressive aphasia (svPPA) and non-fluent variant PPA (nfvPPA). While a strong genetic component is implicated in FTD, genetic FTD in Asia is less frequently reported. We aimed to investigate the frequency of Southeast Asian FTD patients harbouring known genetic FTD variants. A total of 60 FTD-spectrum patients (25 familial and 35 sporadic) from Singapore and the Philippines were included. All underwent next-generation sequencing and repeat-primed PCR for C9orf72 expansion testing. Neurofilament light chain (NfL) levels were measured in a subset of patients. Overall, 26.6% (16/60 cases) carried pathogenic or likely pathogenic variants in a FTD-related gene, including: MAPT Gln351Arg (n= 1); GRN Cys92Ter (n= 1), Ser301Ter (n= 2), c.462 + 1G > C (n= 1); C9orf72 expansion (35-70 repeats; n= 8); TREM2 Arg47Cys (n= 1); and OPTN frameshift insertion (n= 2). Genetic mutations accounted for 48% (12/25) of patients with familial FTD, and 11.4% (4/35) of patients with sporadic FTD. C9orf72 repeat expansions were the most common genetic mutation (13.3%, 8/60), followed by GRN (6.7%, 4/60) variants. Within mutation carriers, plasma NfL was highest in a C9orf72 expansion carrier, and CSF NfL was highest in a GRN splice variant carrier. In our cohort, genetic mutations are present in one-quarter of FTD-spectrum cases, and up to half of those with family history. Our findings highlight the importance of wider implementation of genetic testing in FTD patients from Southeast Asia.

A G-quadruplex structure within the 3'-untranslated region of hnRNP K affects its translation by controlling the accessibility of miR-1249-3p to its binding site.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in motor neuron loss in the brain and spinal cord. Mutations of the DNA/RNA binding proteins Tar-DNA binding protein 43 (TDP-43) and fused in sarcoma/translocated in liposarcoma protein (FUS/TLS) are causative of ALS, and these two RNA-binding proteins are the major protein components of the pathological inclusions observed in over 90% of ALS cases. Moreover, a hexanucleotide GGGGCC (G4C2) expansion in the C9ORF72 gene is a common genetic cause of ALS, being found in at least 8% of sporadic and more than 40% of familial ALS. Thus, it is proposed that alterations to RNA metabolism play a pathogenic role in ALS. This hypothesis is supported by a genome-wide study of microRNAs expression in serum from ALS patients that revealed a significant deregulation of miR-142-3p and miR-1249-3p. Interestingly, miR-1249-3p has been shown to regulate the translation of the heterogenous nuclear protein K (hnRNP K), a DNA/RNA binding protein implicated in transcription and translation regulation, as well as in several diseases, including multiple cancers. In ALS, hnRNP K has been shown to associate with TDP-43 and to bind to the RNA C9ORF72 (G4C2) repeat expansion. In this study we identified and characterized by biophysical methods a G-quadruplex (GQ) structure in the 3’-untranslated region of hnRNP K mRNA, which encompasses the miR-1249-3p binding site. We show that this GQ is recognized by the Fragile X Messenger Ribonucleoprotein 1 (FMRP), suggesting a possible mechanism by which the GQ structure affects miR-1249-3p to its binding site accessibility, therefore, regulating the levels the hnRNP K protein.

Protein-RNA condensates: Complementary or competing interactions in ALS progression?

Membraneless organelles within cells provide organization of the intracellular environment through the process of liquid liquid phase separation (LLPS). The fused in sarcoma (FUS) protein is an RNA binding protein that undergoes LLPS with RNA as part of normal activity in healthy cells. PolyPR and polyGR, dipeptide repeat proteins (DPRs) caused by C9orf72 repeat expansion disorders, have been shown to promote protein aggregation of RNA binding proteins, including FUS. This aggregation contributes to the progression of ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia), which have ineffective treatments and no cure. The pathway towards protein aggregation, and away from stable condensates, is poorly understood, but has been linked to changes in condensate behavior induced by the addition of DPRs. Here we investigate the effect of DPR and RNA on the stability of FUS condensates through molecular dynamics simulations.

Traumatic Brain Injury Associates with an Earlier Onset in Sporadic Frontotemporal Dementia.

Currently, there are few studies considering possible modifiable risk factors of frontotemporal dementia (FTD). In this retrospective case-control study, we evaluated whether a history of traumatic brain injury (TBI) associates with a diagnosis of FTD or modulates the clinical phenotype or onset age in FTD patients. We compared the prevalence of prior TBI between individuals with FTD (N = 218) and age and sex-matched AD patients (N = 214) or healthy controls (HC; N = 100). Based on the patient records, an individual was categorized to the TBI+ group if they were reported to have suffered from TBI during lifetime. The possible associations of TBI with age of onset and disease duration were also evaluated in the whole FTD patient group or separately in the sporadic and genetic FTD groups. The prevalence of previous TBI was the highest in the FTD group (19.3%) when compared to the AD group (13.1%, p = 0.050) or HC group (12%, p = 0.108, not significant). Preceding TBI was more often associated with the sporadic FTD cases than the C9orf72 repeat expansion-carrying FTD cases (p = 0.003). Furthermore, comparison of the TBI+ and TBI- FTD groups indicated that previous TBI was associated with an earlier onset age in the FTD patients (B = 3.066, p = 0.010). A preceding TBI associates especially with sporadic FTD and with earlier onset of symptoms. The results of this study suggest that TBI may be a triggering factor for the neurodegenerative processes in FTD. However, understanding the precise underlying mechanisms still needs further studies.

Longitudinal Effects of Asymptomatic C9orf72 Carriership on Brain Morphology.

We investigated effects of C9orf72 repeat expansion and gene expression on longitudinal cerebral changes before symptom onset. We enrolled 79 asymptomatic family members (AFMs) from 9 families with C9orf72 repeat expansion. Twenty-eight AFMs carried the mutation (C9+). Participants had up to 3 magnetic resonance imaging (MRI) scans, after which we compared motor cortex and motor tracts between C9+ and C9- AFMs using mixed effects models, incorporating kinship to correct for familial relations and lessen effects of other genetic factors. We also compared cortical, subcortical, cerebellar, and connectome structural measurements in a hypothesis-free analysis. We correlated regional C9orf72 expression in donor brains with the pattern of cortical thinning in C9+ AFMs using meta-regression. For comparison, we included 42 C9+ and 439 C9- patients with amyotrophic lateral sclerosis (ALS) in this analysis. C9+ AFM motor cortex had less gyrification and was thinner than in C9- AFMs, without differences in motor tracts. Whole brain analysis revealed thinner cortex and less gyrification in parietal, occipital, and temporal regions, smaller thalami and right hippocampus, and affected frontotemporal connections. Thinning of bilateral precentral, precuneus, and left superior parietal cortex was faster in C9+ than in C9- AFMs. Higher C9orf72 expression correlated with thinner cortex in both C9+ AFMs and C9+ ALS patients. In asymptomatic C9orf72 repeat expansion carriers, brain MRI reveals widespread features suggestive of impaired neurodevelopment, along with faster decline of motor and parietal cortex than found in normal aging. C9orf72 expression might play a role in cortical development, and consequently explain the specific brain abnormalities of mutation carriers. ANN NEUROL 2023;93:668-680.

Telomere length analysis in amyotrophic lateral sclerosis using large-scale whole genome sequence data.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of upper and lower motor neurons, leading to progressive weakness of voluntary muscles, with death following from neuromuscular respiratory failure, typically within 3 to 5 years. There is a strong genetic contribution to ALS risk. In 10% or more, a family history of ALS or frontotemporal dementia is obtained, and the Mendelian genes responsible for ALS in such families have now been identified in about 50% of cases. Only about 14% of apparently sporadic ALS is explained by known genetic variation, suggesting that other forms of genetic variation are important. Telomeres maintain DNA integrity during cellular replication, differ between sexes, and shorten naturally with age. Sex and age are risk factors for ALS and we therefore investigated telomere length in ALS. Samples were from Project MinE, an international ALS whole genome sequencing consortium that includes phenotype data. For validation we used donated brain samples from motor cortex from people with ALS and controls. Ancestry and relatedness were evaluated by principal components analysis and relationship matrices of DNA microarray data. Whole genome sequence data were from Illumina HiSeq platforms and aligned using the Isaac pipeline. TelSeq was used to quantify telomere length using whole genome sequence data. We tested the association of telomere length with ALS and ALS survival using Cox regression. There were 6,580 whole genome sequences, reducing to 6,195 samples (4,315 from people with ALS and 1,880 controls) after quality control, and 159 brain samples (106 ALS, 53 controls). Accounting for age and sex, there was a 20% (95% CI 14%, 25%) increase of telomere length in people with ALS compared to controls (p = 1.1 × 10-12), validated in the brain samples (p = 0.03). Those with shorter telomeres had a 10% increase in median survival (p = 5.0×10-7). Although there was no difference in telomere length between sporadic ALS and familial ALS (p=0.64), telomere length in 334 people with ALS due to expanded C9orf72 repeats was shorter than in those without expanded C9orf72 repeats (p = 5.0×10-4). Although telomeres shorten with age, longer telomeres are a risk factor for ALS and worsen prognosis. Longer telomeres are associated with ALS.

Genotype–phenotype characterisation of long survivors with motor neuron disease in Scotland

BackgroundWe investigated the phenotypes and genotypes of a cohort of ‘long-surviving’ individuals with motor neuron disease (MND) to identify potential targets for prognostication.MethodsPatients were recruited via the Clinical Audit Research and Evaluation for MND (CARE-MND) platform, which hosts the Scottish MND Register. Long survival was defined as > 8 years from diagnosis. 11 phenotypic variables were analysed. Whole genome sequencing (WGS) was performed and variants within 49 MND-associated genes examined. Each individual was screened for C9orf72 repeat expansions. Data from ancestry-matched Scottish populations (the Lothian Birth Cohorts) were used as controls.Results58 long survivors were identified. Median survival from diagnosis was 15.5 years. Long survivors were significantly younger at onset and diagnosis than incident patients and had a significantly longer diagnostic delay. 42% had the MND subtype of primary lateral sclerosis (PLS). WGS was performed in 46 individuals: 14 (30.4%) had a potentially pathogenic variant. 4 carried the known SOD1 p.(Ile114Thr) variant. Significant variants in FIG4, hnRNPA2B1, SETX, SQSTM1, TAF15, and VAPB were detected. 2 individuals had a variant in the SPAST gene suggesting phenotypic overlap with hereditary spastic paraplegia (HSP). No long survivors had pathogenic C9orf72 repeat expansions.ConclusionsLong survivors are characterised by younger age at onset, increased prevalence of PLS and longer diagnostic delay. Genetic analysis in this cohort has improved our understanding of the phenotypes associated with the SOD1 variant p.(Ile114Thr). Our findings confirm that pathogenic expansion of C9orf72 is likely a poor prognostic marker. Genetic screening using targeted MND and/or HSP panels should be considered in those with long survival, or early-onset slowly progressive disease, to improve diagnostic accuracy and aid prognostication.

Altered Blood-Brain Barrier Dynamics in the C9orf72 Hexanucleotide Repeat Expansion Mouse Model of Amyotrophic Lateral Sclerosis.

For peripherally administered drugs to reach the central nervous system (CNS) and treat amyotrophic lateral sclerosis (ALS), they must cross the blood-brain barrier (BBB). As mounting evidence suggests that the ultrastructure of the BBB is altered in individuals with ALS and in animal models of ALS (e.g., SOD1G93A mice), we characterized BBB transporter expression and function in transgenic C9orf72 BAC (C9-BAC) mice expressing a hexanucleotide repeat expansion, the most common genetic cause of ALS. Using an in situ transcardiac brain perfusion technique, we identified a 1.4-fold increase in 3H-2-deoxy-D-glucose transport across the BBB in C9-BAC transgenic (C9) mice, relative to wild-type (WT) mice, which was associated with a 1.3-fold increase in brain microvascular glucose transporter 1 expression, while other general BBB permeability processes (passive diffusion, efflux transporter function) remained unaffected. We also performed proteomic analysis on isolated brain microvascular endothelial cells, in which we noted a mild (14.3%) reduction in zonula occludens-1 abundance in C9 relative to WT mice. Functional enrichment analysis highlighted trends in changes to various BBB transporters and cellular metabolism. To our knowledge, this is the first study to demonstrate altered BBB function in a C9orf72 repeat expansion model of ALS, which has implications on how therapeutics may access the brain in this mouse model.

Molecular basis of C9orf72 poly-PR interference with the β-karyopherin family of nuclear transport receptors.

Nucleocytoplasmic transport (NCT) is affected in several neurodegenerative diseases including C9orf72-ALS. It has recently been found that arginine-containing dipeptide repeat proteins (R-DPRs), translated from C9orf72 repeat expansions, directly bind to several importins. To gain insight into how this can affect nucleocytoplasmic transport, we use coarse-grained molecular dynamics simulations to study the molecular interaction of poly-PR, the most toxic DPR, with several Kapβs (importins and exportins). We show that poly-PR-Kapβ binding depends on the net charge per residue (NCPR) of the Kapβ, salt concentration of the solvent, and poly-PR length. Poly-PR makes contact with the inner surface of most importins, which strongly interferes with Kapβ binding to cargo-NLS, IBB, and RanGTP in a poly-PR length-dependent manner. Longer poly-PRs at higher concentrations are also able to make contact with the outer surface of importins that contain several binding sites to FG-Nups. We also show that poly-PR binds to exportins, especially at lower salt concentrations, interacting with several RanGTP and FG-Nup binding sites. Overall, our results suggest that poly-PR might cause length-dependent defects in cargo loading, cargo release, Kapβ transport and Ran gradient across the nuclear envelope.

Generation of five induced pluripotent stem cells lines from four members of the same family carrying a C9orf72 repeat expansion and one wild-type member

The most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) is the expansion of a G4C2 hexanucleotide repeat in the C9orf72 gene. The size of the repeat expansion is highly variable and a cut-off of 30 repeats has been suggested as the lower pathological limit. Repeat size variability has been observed intergenerationally and intraindividually in tissues from different organs and within the same tissue, suggesting instability of the pathological repeat expansion. In order to study this genomic instability, we established iPSCs from five members of the same family of which four carried a C9orf72 repeat expansion and one was wild-type.

Neurodevelopmental effects of genetic frontotemporal dementia in young adult mutation carriers.

While frontotemporal dementia (frontotemporal dementia) has been considered a neurodegenerative disease that starts in mid-life or later, it is now clearly established that cortical and subcortical volume loss is observed more than a decade prior to symptom onset and progresses with aging. To test the hypothesis that genetic mutations causing frontotemporal dementia have neurodevelopmental consequences, we have examined the youngest adults in the GENFI cohort of pre-symptomatic frontotemporal dementia mutation carriers who are between the ages of 19 and 30y. Structural brain differences and improved performance on some cognitive tests was found for MAPT and GRN mutation carriers relative to familial non-carriers, while smaller volumes were observed in C9orf72 repeat expansion carriers at a mean age of 26y. The detection of such early differences supports potential advantageous neurodevelopmental consequences of some frontotemporal dementia causing genetic mutations. These results have implications for design of therapeutic interventions for frontotemporal dementia. Future studies at younger ages are needed to identify specific early pathophysiologic or compensatory processes in the neurodevelopmental period.

Characterization of a diverse Frontotemporal Dementia cohort, enriched for Caribbean Hispanic patients.

AbstractBackgroundThe vast majority of biomedical data currently available for any disease is derived from studies in non‐Hispanic white (NHW) populations. Specifically, clinical information, genetic factors as well as biomarkers for frontotemporal dementia (FTD) have been studied predominantly in those NHW populations. To increase representation in biomedical research, we set out to enroll and characterize a diverse FTD patient cohort enriched for Caribbean Hispanic patients.MethodOur current cohort consists of 89 FTD patients (30% NHW, 67% Hispanic), with continuing enrollment from the University of Miami Hospital Neurology Department in Miami, FL and the Caribbean Center for the Study of Memory and Cognition in San Juan, PR. All patients were evaluated using NACC approved Uniform DataSet (UDS) or equivalent in their preferred language. For ∼65% of the cohort we also completed the NACC FTD module forms. We generated genotyping data (Illumina GDA+Neurobooster array) as well as whole genome sequencing and plasma biomarker data (Quanterix Simoa Neuroplex‐3; Ab40, Ab42, and total tau) for a subset of the cohort.ResultInitial genetic analyses showed none of the Hispanic patients are carriers of known FTD mutations originally identified in NHW patients, including the C9orf72 repeat expansion and reported pathogenic variants in MAPT or GRN. We did not identify a significant difference in age‐at‐onset or Clinical Dementia Rating scores at time of enrollment between NHW and Hispanic patients. Additionally, biomarker data on Aβ40/Aβ42 ratio and total tau levels in a subset of 22 FTD patients (∼12/10 Hispanic/NHW) did not show significantly different levels between patients of both ethnicities.ConclusionGenetic analyses of FTD in underrepresented population groups is necessary as genetic information from research in NHW is not always generalizable across race/ethnicity. We are currently working to expand our efforts to include identification of novel genetic risk factors for FTD in the Hispanic patients using whole genome sequencing, full evaluation of the Neuroplex as well as p‐tau181 and NfL biomarkers in the complete cohort and comparison of clinical presentations between ethnicities. The biomedical characterization of FTD across race/ethnicity will help the understanding of disease mechanisms in all patients ultimately preventing further health disparities.

18F‐fluorodeoxyglucose‐positron emission tomography Findings in Patients with Genetic Frontotemporal Dementia

AbstractBackgroundMutations in microtubule‐associated protein tau (MAPT), granulin (GRN), and hexanucleotide expansion repeat in the open reading frame of chromosome 9 (C9orf72) are found in 60% of familial frontotemporal dementia (FTD) cases. The current study aims to delineate brain regions with reduced glucose metabolism in patients with genetic FTD in comparison with cognitively normal controls.Methods(18)F‐ fluorodeoxyglucose (FDG)‐positron emission tomography (PET) images were retrospectively obtained from 17 genetic FTD patients. All patients were symptomatic at the time of imaging, and the autopsy was available for 13. Pittsburgh compound B (PiB) amyloid PET scans were done for 13 patients (Table 1). FDG‐PET was collected at Lawrence Berkeley National Laboratory on a PET or PET‐CT scanner as six 5‐min frames acquired 30 min post tracer injection. Standard Uptake Value Ratio (SUVR) images were created for each patient using pons as a reference region. W‐score (age‐adjusted Z‐score) maps were created for each patient’s FDG SUVR using a group of neurologically unimpaired controls (N=74, Mean age 65+16, 41 female) as reference.ResultOn review of single‐subject w‐maps, all patients with MAPT mutation showed hypometabolism in the anteromedial temporal lobes with 66% showing anterolateral temporal hypometabolism and 33% in the frontal and dorsolateral parietal lobes. Hypometabolism in temporal lobes was seen in all GRN patients and 75% had frontal and parietal hypometabolism in an asymmetric pattern. 80% of theC9orf72 patients had hypometabolism in anteromedial temporal and orbitofrontal regions. 60% showed hypometabolism in dorsolateral prefrontal, medial frontal, and anterolateral temporal lobes. Posterior temporal, posterior cingulate, parietal lobe, cerebellum, insula, thalamus, and caudate were hypometabolic in less than 40% of the C9orf72 patients (Figure 1).On group‐level analysis, compared to controls, patients with C9orf72 showed hypometabolism in the frontotemporal regions, cerebellum and thalamus. Hypometabolism in the frontotemporal and parietal regions was seen with GRN mutation. MAPT mutation carriers showed frontal and anteromedial temporal hypometabolism (Figure 2).ConclusionHypometabolism in the posterior regions and cerebellum is more suggestive of C9orf72 repeat expansion. An asymmetric pattern of hypometabolism was seen in all GRN mutation carriers. MAPT mutation resulted in a greater degree of hypometabolism in the anterior medial temporal lobes.

Telomere attrition in induced pluripotent stem cell derived neurons from ALS/FTD related C9ORF72 repeat expansion carriers

AbstractBackgroundThe GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dysregulated DNA damage response and the generation of reactive oxygen species (ROS) have been postulated as major drivers of toxicity in C9ORF72 pathogenesis. Telomeres are tandem‐repeated nucleotide sequences that are at the end of chromosomes and protect them from degradation.MethodWe analyzed telomere length in neurons and neural progenitor cells from several induced pluripotent stem cell (iPSC) lines from control subjects and C9ORF72 repeat expansion carriers. In addition, we analyzed RNA levels and protein levels of sheltering complex members.ResultWe found an age‐dependent decrease in telomere length in two‐month‐old iPSC‐derived motor neurons from C9ORF72 carriers as compared to control subjects. We also found a significant decrease in TRF1 and POT1 protein levels in 2 month old motor neurons.ConclusionWe found an ae dependent decrease in telomere length and dysregulation of sheltering complex members in 2‐month old motor neurons from C9ORF72 carriers.

Transcriptome wide correlations with neuropathological hallmarks in the frontal cortex of FTLD patients

AbstractBackgroundA key pathological event in frontotemporal lobar degeneration (FTLD) is the alteration of the RNA metabolism. Despite this, no study has characterized the diversity of RNA species using high‐throughput sequencing approaches and correlated them with the main neuropathological hallmarks across FTLD subtypes.MethodTotal and small RNA sequencing was performed in the frontal cortex of patients neuropathologically diagnosed with FTLD‐TDP (including non‐mutation carriers [sFTLD‐TDP;n = 9], and carriers of the C9orf72 repeat expansion [FTLD‐C9;n = 11]), FTLD‐tau (n = 13, six carrying the p.P301L mutation in MAPT) and controls without neuropathological alterations in the same brain region (n = 7). Gene and miRNA co‐expression modules were identified using WGCNA. Cell‐type proportions were estimated through cell‐type deconvolution using MuSiC. Gene ontology enrichment analyses were performed using Metascape. We assessed in the frontal cortex the presence of pTDP43 (in sFTLD‐TDP and FTLD‐C9), dipeptide repeats and RNA foci (in FTLD‐C9), and tau aggregates (in FTLD‐tau) through quantitative immunohistochemistry and correlated their density with transcriptome‐wide RNA alterations.ResultOur results indicate statistically significant correlations between gene and miRNA co‐expression modules, neuropathological changes and cell‐type proportions specific for each FTLD subtype. The most significant findings include: in sFTLD‐TDP, the density of pTDP43 positively correlated with a gene co‐expression module (R = 0.9,p = 0.04) enriched with splicing functions (p<1×10−8), which directly correlated with a miRNA co‐expression module (R = 0.77,p = 5×10−4) and the proportion of a microglial subpopulation (R = 0.69,p = 4×10−3). In FTLD‐C9, the density of poly(GP) repeats inversely correlated with the proportion of a neuronal subpopulation (R = ‐0.62,p = 0.041), and negatively correlated with a gene co‐expression module (R = ‐0.67,p = 0.024) enriched with protein phosphorylation functions (p<1×10−4). In FTLD‐tau, the density of tau aggregates negatively correlated with the proportion of a neuronal subpopulation (Fig.1A;R = ‐0.77,p = 0.003) and positively correlated with a miRNA (Fig.1B;R = 0.88,p = 8.8×10−5) and a gene (Fig.1C;R = 0.65,p = 0.016) co‐expression module enriched in genes with neuron ensheathing functions (Fig.1D;p<1×10−20).ConclusionOur data demonstrate selective vulnerability of cell‐subtypes to neuropathological changes. In addition, we describe striking correlations between the main neuropathological hallmarks of each FTLD subtype and specific gene and miRNA co‐expression modules, including their hub genes and miRNAs which might be used as biomarkers to identify the FTLD neuropathological substrate in vivo, and reveal novel molecular mechanisms amenable for therapeutic intervention in FTLD.

C9orf72 repeat length might influence clinical sub-phenotypes in dementia patients

BackgroundC9orf72 repeat expansions have been observed in a wide variety of neurodegenerative disorders. The cut-off between normal and pathogenic alleles is not well established as repeat sizing methods are often semi-quantitative. However, intermediate alleles might influence disease prevalence and phenotype, as seen for other repeat expansion disorders. We aimed to further delineate the prevalence of small, intermediate and expanded C9orf72 alleles and elucidate their potential influence on the disease phenotype. MethodsDNA derived from patients (n = 1804) and healthy individuals (n = 643) was obtained from multiple collectives in Austria. Genotyping was performed using a two-step PCR assay followed by Southern blotting. Results3.4% of clinically diagnosed frontotemporal dementia (FTD; n = 5/147) cases and 0.8% of clinically diagnosed Alzheimer's disease (AD; n = 5/602) cases were carriers of a pathological C9orf72 repeat expansion. A significantly earlier disease onset was detected in expansion carriers compared to non-carriers in the FTD and AD cohorts (median 50 years, range 39–64 vs. median 64 years, range 36–92, p = 0.018 and median 63 years, range 54–71 vs. median 74 years, range 45–92, p = 0.006, respectively). C9orf72 intermediate alleles were significantly associated with cerebellar symptoms (p = 0.0004) and sensory deficits in the dementia cohort (p = 0.01). ConclusionsC9orf72 repeat expansion carriers showed earlier disease onset compared to non-carriers with clinical diagnosis of FTD and AD. Furthermore, C9orf72 intermediate repeats might modify the phenotypic expression in dementia.

Synaptic proteomics reveal distinct molecular signatures of cognitive change and C9ORF72 repeat expansion in the human ALS cortex

Increasing evidence suggests synaptic dysfunction is a central and possibly triggering factor in Amyotrophic Lateral Sclerosis (ALS). Despite this, we still know very little about the molecular profile of an ALS synapse. To address this gap, we designed a synaptic proteomics experiment to perform an unbiased assessment of the synaptic proteome in the ALS brain. We isolated synaptoneurosomes from fresh-frozen post-mortem human cortex (11 controls and 18 ALS) and stratified the ALS group based on cognitive profile (Edinburgh Cognitive and Behavioural ALS Screen (ECAS score)) and presence of a C9ORF72 hexanucleotide repeat expansion (C9ORF72-RE). This allowed us to assess regional differences and the impact of phenotype and genotype on the synaptic proteome, using Tandem Mass Tagging-based proteomics. We identified over 6000 proteins in our synaptoneurosomes and using robust bioinformatics analysis we validated the strong enrichment of synapses. We found more than 30 ALS-associated proteins in synaptoneurosomes, including TDP-43, FUS, SOD1 and C9ORF72. We identified almost 500 proteins with altered expression levels in ALS, with region-specific changes highlighting proteins and pathways with intriguing links to neurophysiology and pathology. Stratifying the ALS cohort by cognitive status revealed almost 150 specific alterations in cognitively impaired ALS synaptic preparations. Stratifying by C9ORF72-RE status revealed 330 protein alterations in the C9ORF72-RE +ve group, with KEGG pathway analysis highlighting strong enrichment for postsynaptic dysfunction, related to glutamatergic receptor signalling. We have validated some of these changes by western blot and at a single synapse level using array tomography imaging. In summary, we have generated the first unbiased map of the human ALS synaptic proteome, revealing novel insight into this key compartment in ALS pathophysiology and highlighting the influence of cognitive decline and C9ORF72-RE on synaptic composition.

Serum total TDP-43 levels are decreased in frontotemporal dementia patients with C9orf72 repeat expansion or concomitant motoneuron disease phenotype

BackgroundFrontotemporal dementia (FTD) covers a spectrum of neurodegenerative disorders with various clinical and neuropathological subtypes. The two major pathological proteins accumulating in the brains of FTD patients, depending on their genetic background, are TDP-43 and tau. We aimed to evaluate whether total TDP-43 levels measured from the serum associate with the genotype or clinical phenotype of the FTD patients and whether serum TDP-43 provides prognostic or diagnostic value in the FTD spectrum disorders.MethodsThe study cohort included 254 participants with a clinical diagnosis of FTD (including all major genotypes and clinical phenotypes) and 105 cognitively healthy controls. Serum total TDP-43 levels measured with a single-molecule array (Simoa) were compared within the FTD group according to the genotype, clinical phenotype, and predicted neuropathological subtype of the patients. We also evaluated the associations between the TDP-43 levels and disease severity or survival in FTD.ResultsTotal TDP-43 levels in the serum were significantly lower in the FTD group as compared to the healthy control group (275.3 pg/mL vs. 361.8 pg/mL, B = 0.181, 95%CI = 0.014–0.348, p = 0.034). The lowest TDP-43 levels were observed in the subgroup of FTD patients harboring predicted TDP-43 brain pathology (FTD-TDP, 241.4 pg/mL). The low levels in the FTD-TDP group were especially driven by C9orf72 repeat expansion carriers (169.2 pg/mL) and FTD patients with concomitant motoneuron disease (FTD-MND, 113.3 pg/mL), whereas GRN mutation carriers did not show decreased TDP-43 levels (328.6 pg/mL). Serum TDP-43 levels showed no correlation with disease severity nor progression in FTD.ConclusionsOur results indicate that the total levels of TDP-43 in the serum are decreased especially in FTD patients with the C9orf72 repeat expansion or FTD-MND phenotype, both subtypes strongly associated with TDP-43 type B brain pathology. Serum-based measurement of TDP-43 could represent a useful tool in indicating C9orf72 repeat expansion and FTD-MND-related TDP-43 neuropathology for future diagnostics and intervention studies.