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Pathogenic variants in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis and/or frontotemporal dementia characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial amyotrophic lateral sclerosis and/or frontotemporal dementia cases not caused by UBQLN2 pathogenic variants, particularly C9orf72-linked cases. This makes the mechanistic role of mutant ubiquilin 2 protein and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 44 genotypically diverse amyotrophic lateral sclerosis cases with or without frontotemporal dementia, including eight cases with UBQLN2 variants (resulting in p.S222G, p.P497H, p.P506S, p.T487I (two cases), and p.P497L (three cases)). Using multiplexed (5-label) fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43, dipeptide repeat aggregates, and p62, in the hippocampus of controls (n = 6), or amyotrophic lateral sclerosis with or without frontotemporal dementia in sporadic (n = 20), unknown familial (n = 3), SOD1-linked (n = 1), FUS-linked (n = 1), C9orf72-linked (n = 5), and UBQLN2-linked (n = 8) cases. We differentiate between i) ubiquilin 2 aggregation together with phosphorylated TDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation promoted by UBQLN2 pathogenic variants that cause amyotrophic lateral sclerosis/and frontotemporal dementia. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in amyotrophic lateral sclerosis with or without frontotemporal dementia, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene variants and to understand the mechanisms of UBQLN2-linked disease.
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RATIONALE: Dextro-transposition of the great arteries (D-TGA) is a severe congenital heart defect which affects approximately 1 in 4,000 live births. While there are several reports of D-TGA patients with rare variants in individual genes, the majority of D-TGA cases remain genetically elusive. Familial recurrence patterns and the observation that most cases with D-TGA are sporadic suggest a polygenic inheritance for the disorder, yet this remains unexplored. OBJECTIVE: We sought to study the role of common single nucleotide polymorphisms (SNPs) in risk for D-TGA. METHODS AND RESULTS: We conducted a genome-wide association study in an international set of 1,237 patients with D-TGA and identified a genome-wide significant susceptibility locus on chromosome 3p14.3, which was subsequently replicated in an independent case-control set (rs56219800, meta-analysis P=8.6x10-10, OR=0.69 per C allele). SNP-based heritability analysis showed that 25% of variance in susceptibility to D-TGA may be explained by common variants. A genome-wide polygenic risk score derived from the discovery set was significantly associated to D-TGA in the replication set (P=4x10-5). The genome-wide significant locus (3p14.3) co-localizes with a putative regulatory element that interacts with the promoter of WNT5A, which encodes the Wnt Family Member 5A protein known for its role in cardiac development in mice. We show that this element drives reporter gene activity in the developing heart of mice and zebrafish and is bound by the developmental transcription factor TBX20. We further demonstrate that TBX20 attenuates Wnt5a expression levels in the developing mouse heart. CONCLUSIONS: This work provides support for a polygenic architecture in D-TGA and identifies a susceptibility locus on chromosome 3p14.3 near WNT5A. Genomic and functional data support a causal role of WNT5A at the locus.
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Polimorfismo de Nucleótido Simple , Transposición de los Grandes Vasos/genética , Animales , Células Cultivadas , Humanos , Ratones , Herencia Multifactorial , Miocitos Cardíacos/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Transposición de los Grandes Vasos/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Pez CebraRESUMEN
Humans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions. We used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease. We found no evidence for enrichment of positively-selected SNPs in the heritability of Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease, suggesting that common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk. These findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.
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Enfermedad de Alzheimer , Esclerosis Amiotrófica Lateral , Hombre de Neandertal , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Animales , Humanos , Hombre de Neandertal/genética , Enfermedades Neurodegenerativas/genética , Selección GenéticaRESUMEN
Transactive response DNA binding protein 43 (TDP-43) is an RNA processing protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Nuclear TDP-43 mislocalizes in patients to the cytoplasm, where it forms ubiquitin-positive inclusions in affected neurons and glia. Physiologically, cytoplasmic TDP-43 is associated with stress granules (SGs). Here, we explored TDP-43 cytoplasmic accumulation and stress granule formation following osmotic and oxidative stress. We show that sorbitol drives TDP-43 redistribution to the cytoplasm, while arsenite induces the recruitment of cytoplasmic TDP-43 to TIA-1 positive SGs. We demonstrate that inducing acute oxidative stress after TDP-43 cytoplasmic relocalization by osmotic shock induces poly (ADP-ribose) polymerase (PARP) cleavage, which triggers cellular toxicity. Recruitment of cytoplasmic TDP-43 to polyribosomes occurs in an SH-SY5Y cellular stress model and is observed in FTD brain lysate. Moreover, the processing body (P-body) marker DCP1a is detected in TDP-43 granules during recovery from stress. Overall, this study supports a central role for cytoplasmic TDP-43 in controlling protein translation in stressed cells.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Esclerosis Amiotrófica Lateral/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/patología , HumanosRESUMEN
Aberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro and in transgenic animal models of amyotrophic lateral sclerosis. Detailed examination of the protein in disease-affected tissues from amyotrophic lateral sclerosis patients, however, remains scarce. We used histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from amyotrophic lateral sclerosis cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration. We provide evidence of the mislocalization and accumulation of structurally disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial amyotrophic lateral sclerosis cases, and sporadic amyotrophic lateral sclerosis cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in amyotrophic lateral sclerosis cases, and clearly differentiated all forms of amyotrophic lateral sclerosis from controls. Substantial heterogeneity in the presence of these changes was also observed between amyotrophic lateral sclerosis cases. Our data demonstrate that varying forms of SOD1 proteinopathy are a common feature of all forms of amyotrophic lateral sclerosis, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in amyotrophic lateral sclerosis. Most of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development.
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Esclerosis Amiotrófica Lateral , Procesamiento Proteico-Postraduccional , Superóxido Dismutasa-1 , Esclerosis Amiotrófica Lateral/genética , Humanos , Mutación , Médula Espinal/patología , Superóxido Dismutasa-1/genéticaRESUMEN
Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Esclerosis Amiotrófica Lateral/genética , Autofagosomas/fisiología , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia/genética , Demencia/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Proteínas Relacionadas con la Autofagia/genética , Biomarcadores/metabolismo , Línea Celular , Demencia/metabolismo , Demencia/patología , Predisposición Genética a la Enfermedad , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Unión Proteica , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Regulación hacia Arriba , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
INTRODUCTION/AIMS: Fasciculations are an early clinical hallmark of amyotrophic lateral sclerosis (ALS), amenable to detection by high-density surface electromyography (HDSEMG). In conjunction with the Surface Potential Quantification Engine (SPiQE), HDSEMG offers improved spatial resolution for the analysis of fasciculations. This study aims to establish an optimal recording duration to enable longitudinal remote monitoring in the home. METHODS: Twenty patients with ALS and five patients with benign fasciculation syndrome (BFS) underwent serial 30 min HDSEMG recordings from biceps brachii and gastrocnemii. SPiQE was independently applied to abbreviated epochs within each 30-min recording (0-5, 0-10, 0-15, 0-20, and 0-25 min), outputting fasciculation frequency, amplitude median and amplitude interquartile range. Bland-Altman plots and intraclass correlation coefficients (ICC) were used to assess agreement with the validated 30-min recording. RESULTS: In total, 506 full recordings were included. The 5 min recordings demonstrated diverse and relatively poor agreement with the 30 min baselines across all parameters, muscles and patient groups (ICC = 0.32-0.86). The 15-min recordings provided more acceptable and stable agreement (ICC = 0.78-0.98), which did not substantially improve in longer recordings. DISCUSSION: For the detection and quantification of fasciculations in patients with ALS and BFS, HDSEMG recordings can be halved from 30 to 15 min without significantly compromising the primary outputs. Reliance on a shorter recording duration should lead to improved tolerability and repeatability among patients, facilitating longitudinal remote monitoring in patients' homes.
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Esclerosis Amiotrófica Lateral , Fasciculación , Humanos , Fasciculación/diagnóstico , Electromiografía , Esclerosis Amiotrófica Lateral/diagnóstico , Músculo Esquelético/fisiología , SíndromeRESUMEN
BACKGROUND: Long QT syndrome (LQTS) is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. A causal rare genetic variant with large effect size is identified in up to 80% of probands (genotype positive) and cascade family screening shows incomplete penetrance of genetic variants. Furthermore, a proportion of cases meeting diagnostic criteria for LQTS remain genetically elusive despite genetic testing of established genes (genotype negative). These observations raise the possibility that common genetic variants with small effect size contribute to the clinical picture of LQTS. This study aimed to characterize and quantify the contribution of common genetic variation to LQTS disease susceptibility. METHODS: We conducted genome-wide association studies followed by transethnic meta-analysis in 1656 unrelated patients with LQTS of European or Japanese ancestry and 9890 controls to identify susceptibility single nucleotide polymorphisms. We estimated the common variant heritability of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 single nucleotide polymorphisms previously associated with the QT-interval in the general population using a polygenic risk score. RESULTS: Genome-wide association analysis identified 3 loci associated with LQTS at genome-wide statistical significance (P<5×10-8) near NOS1AP, KCNQ1, and KLF12, and 1 missense variant in KCNE1(p.Asp85Asn) at the suggestive threshold (P<10-6). Heritability analyses showed that ≈15% of variance in overall LQTS susceptibility was attributable to common genetic variation (h2SNP 0.148; standard error 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT-interval in the general population (rg=0.40; P=3.2×10-3). The polygenic risk score comprising common variants previously associated with the QT-interval in the general population was greater in LQTS cases compared with controls (P<10-13), and it is notable that, among patients with LQTS, this polygenic risk score was greater in patients who were genotype negative compared with those who were genotype positive (P<0.005). CONCLUSIONS: This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT-interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT-interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS.
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Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Síndrome de QT Prolongado/genética , Adolescente , Adulto , Edad de Inicio , Alelos , Estudios de Casos y Controles , Electrocardiografía , Estudios de Asociación Genética , Estudio de Asociación del Genoma Completo/métodos , Genotipo , Humanos , Síndrome de QT Prolongado/diagnóstico , Síndrome de QT Prolongado/mortalidad , Síndrome de QT Prolongado/terapia , Herencia Multifactorial , Fenotipo , Polimorfismo de Nucleótido Simple , Pronóstico , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
The GGGGCC (G4C2) repeat expansion mutation in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (G4C2) n repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA hLinc-p21. As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (G4C2) n RNA foci. Overexpression of (G4C2)72 RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (G4C2)72 RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA NEAT1 Moreover, the knockdown of SFPQ protein in C9ORF72 expansion mutation-positive fibroblasts significantly reduces the number of (G4C2) n RNA foci. In conclusion, (G4C2) n RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation.
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Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Demencia Frontotemporal/genética , Neuronas Motoras/fisiología , Complejos Multiproteicos/metabolismo , Mutación/genética , ARN Nuclear/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proteína C9orf72/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Espacio Intranuclear , Ratones , Factor de Empalme Asociado a PTB/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Nuclear/genética , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , RatasRESUMEN
Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.
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Esclerosis Amiotrófica Lateral/genética , Enzima Desubiquitinante CYLD/genética , Enzima Desubiquitinante CYLD/fisiología , Demencia Frontotemporal/genética , Predisposición Genética a la Enfermedad/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Autofagosomas/metabolismo , Autofagosomas/fisiología , Axones/patología , Encéfalo/metabolismo , Proteínas de Unión al ADN , Enzima Desubiquitinante CYLD/metabolismo , Enzimas Desubicuitinizantes/metabolismo , Demencia Frontotemporal/metabolismo , Ratones , Mutación Missense/genética , FN-kappa B/antagonistas & inhibidores , Cultivo Primario de Células , TransfecciónRESUMEN
Mutations in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS). Accumulating evidence implicates astrocytes as important non-cell autonomous contributors to ALS pathogenesis, although the potential deleterious effects of astrocytes on the function of motor neurons remains to be determined in a completely humanized model of C9orf72-mediated ALS. Here, we use a human iPSC-based model to study the cell autonomous and non-autonomous consequences of mutant C9orf72 expression by astrocytes. We show that mutant astrocytes both recapitulate key aspects of C9orf72-related ALS pathology and, upon co-culture, cause motor neurons to undergo a progressive loss of action potential output due to decreases in the magnitude of voltage-activated Na+ and K+ currents. Importantly, CRISPR/Cas-9 mediated excision of the C9orf72 repeat expansion reverses these phenotypes, confirming that the C9orf72 mutation is responsible for both cell-autonomous astrocyte pathology and non-cell autonomous motor neuron pathophysiology.
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Astrocitos/metabolismo , Proteína C9orf72/genética , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Potenciales de Acción/fisiología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Astrocitos/patología , Proteína C9orf72/metabolismo , Técnicas de Cocultivo , Humanos , Células Madre Pluripotentes Inducidas/patología , Neuronas Motoras/patología , MutaciónRESUMEN
FUS (fused in sarcoma) mislocalization and cytoplasmic aggregation are hallmark pathologies in FUS-related amyotrophic lateral sclerosis and frontotemporal dementia. Many of the mechanistic hypotheses have focused on a loss of nuclear function in the FUS-opathies, implicating dysregulated RNA transcription and splicing in driving neurodegeneration. Recent studies describe an additional somato-dendritic localization for FUS in the cerebral cortex implying a regulatory role in mRNA transport and local translation at the synapse. Here, we report that FUS is also abundant at the pre-synaptic terminal of the neuromuscular junction (NMJ), suggesting an important function for this protein at peripheral synapses. We have previously reported dose and age-dependent motor neuron degeneration in transgenic mice overexpressing human wild-type FUS, resulting in a motor phenotype detected by â¼28 days and death by â¼100 days. Now, we report the earliest structural events using electron microscopy and quantitative immunohistochemistry. Mitochondrial abnormalities in the pre-synaptic motor nerve terminals are detected at postnatal day 6, which are more pronounced at P15 and accompanied by a loss of synaptic vesicles and synaptophysin protein coupled with NMJs of a smaller size at a time when there is no detectable motor neuron loss. These changes occur in the presence of abundant FUS and support a peripheral toxic gain of function. This appearance is typical of a 'dying-back' axonopathy, with the earliest manifestation being mitochondrial disruption. These findings support our hypothesis that FUS has an important function at the NMJ, and challenge the 'loss of nuclear function' hypothesis for disease pathogenesis in the FUS-opathies.
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Unión Neuromuscular/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Demencia Frontotemporal/metabolismo , Humanos , Ratones , Ratones Transgénicos , Neuronas Motoras , Proteína FUS de Unión a ARN/genética , Sinapsis/metabolismo , Sinaptofisina/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) presents with focal muscle weakness due to motor neuron degeneration that becomes generalized, leading to death from respiratory failure within 3-5 years from symptom onset. Despite the heterogeneity of aetiology, TDP-43 proteinopathy is a common pathological feature that is observed in >95% of ALS and tau-negative frontotemporal dementia (FTD) cases. TDP-43 is a DNA/RNA-binding protein that in ALS and FTD translocates from being predominantly nuclear to form detergent-resistant, hyperphosphorylated aggregates in the cytoplasm of affected neurons and glia. Mutations in TARDBP account for 1-4% of all ALS cases and almost all arise in the low complexity C-terminal domain that does not affect RNA binding and processing. Here we report an ALS/FTD kindred with a novel K181E TDP-43 mutation that is located in close proximity to the RRM1 domain. To offer predictive gene testing to at-risk family members, we undertook a series of functional studies to characterize the properties of the mutation. Spectroscopy studies of the K181E protein revealed no evidence of significant misfolding. Although it is unable to bind to or splice RNA, it forms abundant aggregates in transfected cells. We extended our study to include other ALS-linked mutations adjacent to the RRM domains that also disrupt RNA binding and greatly enhance TDP-43 aggregation, forming detergent-resistant and hyperphosphorylated inclusions. Lastly, we demonstrate that K181E binds to, and sequesters, wild-type TDP-43 within nuclear and cytoplasmic inclusions. Thus, we demonstrate that TDP-43 mutations that disrupt RNA binding greatly enhance aggregation and are likely to be pathogenic as they promote wild-type TDP-43 to mislocalize and aggregate acting in a dominant-negative manner. This study highlights the importance of RNA binding to maintain TDP-43 solubility and the role of TDP-43 aggregation in disease pathogenesis.
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Proteínas de Unión al ADN/genética , Mutación , Agregación Patológica de Proteínas/genética , Proteínas de Unión al ARN/genética , Médula Espinal/metabolismo , Proteinopatías TDP-43/genética , Adulto , Proteínas de Unión al ADN/metabolismo , Humanos , Masculino , Neuroglía/metabolismo , Neuronas/metabolismo , Fosforilación , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Proteínas de Unión al ARN/metabolismo , Médula Espinal/patología , Proteinopatías TDP-43/metabolismo , Proteinopatías TDP-43/patologíaRESUMEN
An intronic GGGGCC (G4C2) hexanucleotide repeat expansion inC9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 RNA can result in five different dipeptide repeat proteins (DPR: poly GA, poly GP, poly GR, poly PA, and poly PR), which aggregate into neuronal cytoplasmic and nuclear inclusions in affected patients, however their contribution to disease pathogenesis remains controversial. We show that among the DPR proteins, expression of poly GA in a cell culture model activates programmed cell death and TDP-43 cleavage in a dose-dependent manner. Dual expression of poly GA together with other DPRs revealed that poly GP and poly PA are sequestered by poly GA, whereas poly GR and poly PR are rarely co-localised with poly GA. Dual expression of poly GA and poly PA ameliorated poly GA toxicity by inhibiting poly GA aggregation both in vitro and in vivo in the chick embryonic spinal cord. Expression of alternative codon-derived DPRs in chick embryonic spinal cord confirmed in vitro data, revealing that each of the dipeptides caused toxicity, with poly GA being the most toxic. Further, in vivo expression of G4C2 repeats of varying length caused apoptotic cell death, but failed to generate DPRs. Together, these data demonstrate that C9-related toxicity can be mediated by either RNA or DPRs. Moreover, our findings provide evidence that poly GA is a key mediator of cytotoxicity and that cross-talk between DPR proteins likely modifies their pathogenic status in C9ALS/FTD.
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Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Proteína C9orf72/metabolismo , Células Cultivadas , Embrión de Pollo , Expansión de las Repeticiones de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dipéptidos/genética , Dipéptidos/metabolismo , Lóbulo Frontal/metabolismo , Lóbulo Frontal/fisiología , Células HEK293 , Humanos , Cuerpos de Inclusión Intranucleares/metabolismo , Neuronas/metabolismo , Agregado de ProteínasRESUMEN
OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease of motor neurons with a median survival of 2 years. Familial ALS has a younger age of onset than apparently sporadic ALS. We sought to determine whether this younger age of onset is a result of ascertainment bias or has a genetic basis. METHODS: Samples from people with ALS were sequenced for 13 ALS genes. To determine the effect of genetic variation, age of onset was compared in people with sporadic ALS carrying a pathogenic gene variant and those who do not; to determine the effect of family history, we compared those with genetic sporadic ALS and familial ALS. RESULTS: There were 941 people with a diagnosis of ALS, 100 with familial ALS. Of 841 with apparently sporadic ALS, 95 carried a pathogenic gene variant. The mean age of onset in familial ALS was 5.3 years younger than for apparently sporadic ALS (p=6.0×10-5, 95% CI 2.8 to 7.8 years). The mean age of onset of genetic sporadic ALS was 2.9 years younger than non-genetic sporadic ALS (p=0.011, 95% CI 0.7 to 5.2 years). There was no difference between the mean age of onset in genetic sporadic ALS and familial ALS (p=0.097). CONCLUSIONS: People with familial ALS have an age of onset about 5 years younger than those with apparently sporadic ALS, and we have shown that this is a result of Mendelian gene variants lowering the age of onset, rather than ascertainment bias.
Asunto(s)
Esclerosis Amiotrófica Lateral/epidemiología , Esclerosis Amiotrófica Lateral/genética , Variación Genética/genética , Adulto , Edad de Inicio , Anciano , Sesgo , Bases de Datos de Ácidos Nucleicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reino UnidoRESUMEN
Accumulation and aggregation of TDP-43 is a major pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 inclusions also characterize patients with GGGGCC (G4C2) hexanucleotide repeat expansion in C9orf72 that causes the most common genetic form of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Functional studies in cell and animal models have identified pathogenic mechanisms including repeat-induced RNA toxicity and accumulation of G4C2-derived dipeptide-repeat proteins. The role of TDP-43 dysfunction in C9ALS/FTD, however, remains elusive. We found G4C2-derived dipeptide-repeat protein but not G4C2-RNA accumulation caused TDP-43 proteinopathy that triggered onset and progression of disease in Drosophila models of C9ALS/FTD. Timing and extent of TDP-43 dysfunction was dependent on levels and identity of dipeptide-repeat proteins produced, with poly-GR causing early and poly-GA/poly-GP causing late onset of disease. Accumulating cytosolic, but not insoluble aggregated TDP-43 caused karyopherin-α2/4 (KPNA2/4) pathology, increased levels of dipeptide-repeat proteins and enhanced G4C2-related toxicity. Comparable KPNA4 pathology was observed in both sporadic frontotemporal dementia and C9ALS/FTD patient brains characterized by its nuclear depletion and cytosolic accumulation, irrespective of TDP-43 or dipeptide-repeat protein aggregates. These findings identify a vicious feedback cycle for dipeptide-repeat protein-mediated TDP-43 and subsequent KPNA pathology, which becomes self-sufficient of the initiating trigger and causes C9-related neurodegeneration.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Proteína C9orf72/metabolismo , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/patología , Degeneración Nerviosa/metabolismo , alfa Carioferinas/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Expansión de las Repeticiones de ADN , Drosophila , Proteínas de Drosophila/metabolismo , Retroalimentación Fisiológica , Demencia Frontotemporal/metabolismo , Humanos , Degeneración Nerviosa/patologíaRESUMEN
Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Demencia Frontotemporal/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Distrofia Muscular de Cinturas/genética , Proteínas Mutantes/genética , Mutación/genética , Miositis por Cuerpos de Inclusión/genética , Osteítis Deformante/genética , Priones/química , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Masculino , Ratones , Datos de Secuencia Molecular , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Miositis por Cuerpos de Inclusión/metabolismo , Miositis por Cuerpos de Inclusión/patología , Osteítis Deformante/metabolismo , Osteítis Deformante/patología , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Priones/metabolismo , Estructura Terciaria de Proteína/genética , ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB-PTPIP51 interaction and ER-mitochondria associations. These disruptions are accompanied by perturbation of Ca(2+) uptake by mitochondria following its release from ER stores, which is a physiological read-out of ER-mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS-expressing cells; mitochondrial ATP production is linked to Ca(2+) levels. Finally, we demonstrate that the FUS-induced reductions to ER-mitochondria associations and are linked to activation of glycogen synthase kinase-3ß (GSK-3ß), a kinase already strongly associated with ALS/FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Retículo Endoplásmico/metabolismo , Demencia Frontotemporal/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Modelos Animales de Enfermedad , Retículo Endoplásmico/ultraestructura , Activación Enzimática , Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/ultraestructura , Mutación , Unión Proteica , Proteína FUS de Unión a ARN/genéticaRESUMEN
Amyotrophic lateral sclerosis is a progressive neurodegenerative disease of motor neurons. About 25 genes have been verified as relevant to the disease process, with rare and common variation implicated. We used next generation sequencing and repeat sizing to comprehensively assay genetic variation in a panel of known amyotrophic lateral sclerosis genes in 1126 patient samples and 613 controls. About 10% of patients were predicted to carry a pathological expansion of the C9orf72 gene. We found an increased burden of rare variants in patients within the untranslated regions of known disease-causing genes, driven by SOD1, TARDBP, FUS, VCP, OPTN and UBQLN2. We found 11 patients (1%) carried more than one pathogenic variant (P = 0.001) consistent with an oligogenic basis of amyotrophic lateral sclerosis. These findings show that the genetic architecture of amyotrophic lateral sclerosis is complex and that variation in the regulatory regions of associated genes may be important in disease pathogenesis.