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1.
Brain ; 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35521889

RESUMO

Intermediate CAG (polyQ) expansions in the gene ataxin-2 (ATXN2) are now recognized as a risk factor for ALS. The threshold for increased risk is not yet firmly established, with reports ranging from 27 to 31 repeats. We investigated the presence of ATXN2 polyQ expansions in 9,268 DNA samples collected from people with ALS, ALS with frontotemporal dementia (ALSFTD), FTD alone, Lewy body dementia (LBD) and age matched controls. This analysis confirmed ATXN2 intermediate polyQ expansions of ≥31 as a risk factor for ALS with an odds ratio of 6.31. Expansions were an even greater risk for ALSFTD (odds ratio 27.59) and a somewhat lesser risk for FTD alone (odds ratio 3.14). There was no increased risk for LBD. In a subset of 1362 ALS patients with complete clinical data, we could not confirm previous reports of earlier onset of ALS or shorter survival in 25 patients with expansions. These new data confirm ≥31 polyQ repeats in ATXN2 increase the risk for ALS and also for the first time show an even greater risk for ALSFTD. The lack of a more aggressive phenotype in ALS patients with expansions has implications for ongoing gene-silencing trials for ALS.

2.
Cell Rep ; 39(1): 110598, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35385738

RESUMO

Understanding the pathogenic mechanisms of disease mutations is critical to advancing treatments. ALS-associated mutations in the gene encoding the microtubule motor KIF5A result in skipping of exon 27 (KIF5AΔExon27) and the encoding of a protein with a novel 39 amino acid residue C-terminal sequence. Here, we report that expression of ALS-linked mutant KIF5A results in dysregulated motor activity, cellular mislocalization, altered axonal transport, and decreased neuronal survival. Single-molecule analysis revealed that the altered C terminus of mutant KIF5A results in a constitutively active state. Furthermore, mutant KIF5A possesses altered protein and RNA interactions and its expression results in altered gene expression/splicing. Taken together, our data support the hypothesis that causative ALS mutations result in a toxic gain of function in the intracellular motor KIF5A that disrupts intracellular trafficking and neuronal homeostasis.


Assuntos
Esclerose Amiotrófica Lateral , Esclerose Amiotrófica Lateral/genética , Transporte Axonal/genética , Mutação com Ganho de Função , Humanos , Mutação/genética
3.
Stem Cell Reports ; 17(4): 993-1008, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35276091

RESUMO

The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.


Assuntos
Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Controle de Qualidade
4.
Mov Disord ; 2022 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-35262230

RESUMO

BACKGROUND: To date, variants in the GBA gene represent the most frequent large-effect genetic factor associated with Parkinson's disease (PD). However, the reason why individuals with the same GBA variant may or may not develop neurodegeneration and PD is still unclear. OBJECTIVES: Therefore, we evaluated the contribution of rare variants in genes responsible for lysosomal storage disorders (LSDs) to GBA-PD risk, comparing the burden of deleterious variants in LSD genes in PD patients versus asymptomatic subjects, all carriers of deleterious variants in GBA. METHODS: We used a custom next-generation sequencing panel, including 50 LSD genes, to screen 305 patients and 207 controls (discovery cohort). Replication and meta-analysis were performed in two replication cohorts of GBA-variant carriers, of 250 patients and 287 controls, for whom exome or genome data were available. RESULTS: Statistical analysis in the discovery cohort revealed a significantly increased burden of deleterious variants in LSD genes in patients (P = 0.0029). Moreover, our analyses evidenced that the two strongest modifiers of GBA penetrance are a second variation in GBA (5.6% vs. 1.4%, P = 0.023) and variants in genes causing mucopolysaccharidoses (6.9% vs. 1%, P = 0.0020). These results were confirmed in the meta-analysis, where we observed pooled odds ratios of 1.42 (95% confidence interval [CI] = 1.10-1.83, P = 0.0063), 4.36 (95% CI = 2.02-9.45, P = 0.00019), and 1.83 (95% CI = 1.04-3.22, P = 0.038) for variants in LSD genes, GBA, and mucopolysaccharidosis genes, respectively. CONCLUSION: The identification of genetic lesions in lysosomal genes increasing PD risk may have important implications in terms of patient stratification for future therapeutic trials. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

5.
NPJ Genom Med ; 7(1): 8, 2022 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-35091648

RESUMO

There is a strong genetic contribution to Amyotrophic lateral sclerosis (ALS) risk, with heritability estimates of up to 60%. Both Mendelian and small effect variants have been identified, but in common with other conditions, such variants only explain a little of the heritability. Genomic structural variation might account for some of this otherwise unexplained heritability. We therefore investigated association between structural variation in a set of 25 ALS genes, and ALS risk and phenotype. As expected, the repeat expansion in the C9orf72 gene was identified as associated with ALS. Two other ALS-associated structural variants were identified: inversion in the VCP gene and insertion in the ERBB4 gene. All three variants were associated both with increased risk of ALS and specific phenotypic patterns of disease expression. More than 70% of people with respiratory onset ALS harboured ERBB4 insertion compared with 25% of the general population, suggesting respiratory onset ALS may be a distinct genetic subtype.

6.
JAMA Neurol ; 78(10): 1236-1248, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34459874

RESUMO

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation. Objective: To identify the genetic variants associated with juvenile ALS. Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism. Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members. Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway. Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.


Assuntos
Esclerose Amiotrófica Lateral/genética , Predisposição Genética para Doença/genética , Serina C-Palmitoiltransferase/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Mutação , Sequenciamento Completo do Exoma , Adulto Jovem
7.
Nat Neurosci ; 24(8): 1077-1088, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34059832

RESUMO

Nucleocytoplasmic transport (NCT) decline occurs with aging and neurodegeneration. Here, we investigated the NCT pathway in models of amyotrophic lateral sclerosis-fused in sarcoma (ALS-FUS). Expression of ALS-FUS led to a reduction in NCT and nucleoporin (Nup) density within the nuclear membrane of human neurons. FUS and Nups were found to interact independently of RNA in cells and to alter the phase-separation properties of each other in vitro. FUS-Nup interactions were not localized to nuclear pores, but were enriched in the nucleus of control neurons versus the cytoplasm of mutant neurons. Our data indicate that the effect of ALS-linked mutations on the cytoplasmic mislocalization of FUS, rather than on the physiochemical properties of the protein itself, underlie our reported NCT defects. An aberrant interaction between mutant FUS and Nups is underscored by studies in Drosophila, whereby reduced Nup expression rescued multiple toxic FUS-induced phenotypes, including abnormal nuclear membrane morphology in neurons.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Neurônios/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila , Humanos , Mutação , Proteína FUS de Ligação a RNA/genética
8.
Neurol Genet ; 7(3): e596, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34169147

RESUMO

OBJECTIVE: Despite the strong genetic component of frontotemporal dementia (FTD), a substantial proportion of patients remain genetically unresolved. We performed an in-depth study of a family with an autosomal dominant form of FTD to investigate the underlying genetic cause. METHODS: Following clinical and pathologic characterization of the family, genetic studies included haplotype sharing analysis and exome sequencing. Subsequently, we performed immunohistochemistry, immunoblotting, and a microtubule repolymerization assay to investigate the potential impact of the candidate variant in tubulin alpha 4a (TUBA4A). RESULTS: The clinical presentation in this family is heterogeneous, including behavioral changes, parkinsonian features, and uncharacterized dementia. Neuropathologic examination of 2 patients revealed TAR DNA binding protein 43 (TDP-43) pathology with abundant dystrophic neurites and neuronal intranuclear inclusions, consistent with frontotemporal lobar degeneration-TDP type A. We identified a likely pathogenic variant in TUBA4A segregating with disease. TUBA4A encodes for α-tubulin, which is a major component of the microtubule network. Variants in TUBA4A have been suggested as a rare genetic cause of amyotrophic lateral sclerosis (ALS) and have sporadically been reported in patients with FTD without supporting genetic segregation. A decreased trend of TUBA4A protein abundance was observed in patients compared with controls, and a microtubule repolymerization assay demonstrated disrupted α-tubulin function. As opposed to variants found in ALS, TUBA4A variants associated with FTD appear more localized to the N-terminus, indicating different pathogenic mechanisms. CONCLUSIONS: Our findings support the role of TUBA4A variants as rare genetic cause of familial FTD.

9.
Nat Commun ; 12(1): 2558, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33963192

RESUMO

GEMIN5, an RNA-binding protein is essential for assembly of the survival motor neuron (SMN) protein complex and facilitates the formation of small nuclear ribonucleoproteins (snRNPs), the building blocks of spliceosomes. Here, we have identified 30 affected individuals from 22 unrelated families presenting with developmental delay, hypotonia, and cerebellar ataxia harboring biallelic variants in the GEMIN5 gene. Mutations in GEMIN5 perturb the subcellular distribution, stability, and expression of GEMIN5 protein and its interacting partners in patient iPSC-derived neurons, suggesting a potential loss-of-function mechanism. GEMIN5 mutations result in disruption of snRNP complex assembly formation in patient iPSC neurons. Furthermore, knock down of rigor mortis, the fly homolog of human GEMIN5, leads to developmental defects, motor dysfunction, and a reduced lifespan. Interestingly, we observed that GEMIN5 variants disrupt a distinct set of transcripts and pathways as compared to SMA patient neurons, suggesting different molecular pathomechanisms. These findings collectively provide evidence that pathogenic variants in GEMIN5 perturb physiological functions and result in a neurodevelopmental delay and ataxia syndrome.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Transtornos do Neurodesenvolvimento/metabolismo , Neurônios/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN/genética , Alelos , Sequência de Aminoácidos , Animais , Pré-Escolar , Deficiências do Desenvolvimento/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Feminino , Técnicas de Silenciamento de Genes , Ontologia Genética , Células HEK293 , Humanos , Mutação com Perda de Função , Masculino , Hipotonia Muscular/genética , Dissinergia Cerebelar Mioclônica/genética , Transtornos do Neurodesenvolvimento/diagnóstico por imagem , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/fisiopatologia , Linhagem , Polimorfismo de Nucleotídeo Único , RNA-Seq , Ribonucleoproteínas Nucleares Pequenas/genética , Rigor Mortis/genética , Proteínas do Complexo SMN/metabolismo
10.
Sci Adv ; 7(3)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33523907

RESUMO

Despite the considerable progress in unraveling the genetic causes of amyotrophic lateral sclerosis (ALS), we do not fully understand the molecular mechanisms underlying the disease. We analyzed genome-wide data involving 78,500 individuals using a polygenic risk score approach to identify the biological pathways and cell types involved in ALS. This data-driven approach identified multiple aspects of the biology underlying the disease that resolved into broader themes, namely, neuron projection morphogenesis, membrane trafficking, and signal transduction mediated by ribonucleotides. We also found that genomic risk in ALS maps consistently to GABAergic interneurons and oligodendrocytes, as confirmed in human single-nucleus RNA-seq data. Using two-sample Mendelian randomization, we nominated six differentially expressed genes (ATG16L2, ACSL5, MAP1LC3A, MAPKAPK3, PLXNB2, and SCFD1) within the significant pathways as relevant to ALS. We conclude that the disparate genetic etiologies of this fatal neurological disease converge on a smaller number of final common pathways and cell types.


Assuntos
Esclerose Amiotrófica Lateral , Esclerose Amiotrófica Lateral/genética , Testes Genéticos , Estudo de Associação Genômica Ampla , Humanos , Polimorfismo de Nucleotídeo Único
11.
Ann Neurol ; 89(4): 686-697, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33389754

RESUMO

OBJECTIVE: The role of the survival of motor neuron (SMN) gene in amyotrophic lateral sclerosis (ALS) is unclear, with several conflicting reports. A decisive result on this topic is needed, given that treatment options are available now for SMN deficiency. METHODS: In this largest multicenter case control study to evaluate the effect of SMN1 and SMN2 copy numbers in ALS, we used whole genome sequencing data from Project MinE data freeze 2. SMN copy numbers of 6,375 patients with ALS and 2,412 controls were called from whole genome sequencing data, and the reliability of the calls was tested with multiplex ligation-dependent probe amplification data. RESULTS: The copy number distribution of SMN1 and SMN2 between cases and controls did not show any statistical differences (binomial multivariate logistic regression SMN1 p = 0.54 and SMN2 p = 0.49). In addition, the copy number of SMN did not associate with patient survival (Royston-Parmar; SMN1 p = 0.78 and SMN2 p = 0.23) or age at onset (Royston-Parmar; SMN1 p = 0.75 and SMN2 p = 0.63). INTERPRETATION: In our well-powered study, there was no association of SMN1 or SMN2 copy numbers with the risk of ALS or ALS disease severity. This suggests that changing SMN protein levels in the physiological range may not modify ALS disease course. This is an important finding in the light of emerging therapies targeted at SMN deficiencies. ANN NEUROL 2021;89:686-697.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Dosagem de Genes , Humanos , Masculino , Reprodutibilidade dos Testes , Fatores de Risco , Índice de Gravidade de Doença , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Sequenciamento Completo do Genoma
12.
Neuron ; 109(3): 448-460.e4, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33242422

RESUMO

We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40-64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington's disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered.


Assuntos
Esclerose Amiotrófica Lateral/genética , Expansão das Repetições de DNA , Demência Frontotemporal/genética , Proteína Huntingtina/genética , Esclerose Amiotrófica Lateral/patologia , Demência Frontotemporal/patologia , Humanos , Mutação , Sequenciamento Completo do Genoma
13.
Brain Commun ; 2(2): fcaa064, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32954321

RESUMO

Increasingly, repeat expansions are being identified as part of the complex genetic architecture of amyotrophic lateral sclerosis. To date, several repeat expansions have been genetically associated with the disease: intronic repeat expansions in C9orf72, polyglutamine expansions in ATXN2 and polyalanine expansions in NIPA1. Together with previously published data, the identification of an amyotrophic lateral sclerosis patient with a family history of spinocerebellar ataxia type 1, caused by polyglutamine expansions in ATXN1, suggested a similar disease association for the repeat expansion in ATXN1. We, therefore, performed a large-scale international study in 11 700 individuals, in which we showed a significant association between intermediate ATXN1 repeat expansions and amyotrophic lateral sclerosis (P = 3.33 × 10-7). Subsequent functional experiments have shown that ATXN1 reduces the nucleocytoplasmic ratio of TDP-43 and enhances amyotrophic lateral sclerosis phenotypes in Drosophila, further emphasizing the role of polyglutamine repeat expansions in the pathophysiology of amyotrophic lateral sclerosis.

14.
Brain ; 143(3): 783-799, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32185393

RESUMO

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.


Assuntos
Esclerose Amiotrófica Lateral/genética , Enzima Desubiquitinante CYLD/genética , Enzima Desubiquitinante CYLD/fisiologia , Demência Frontotemporal/genética , Predisposição Genética para Doença/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/fisiologia , Axônios/patologia , Encéfalo/metabolismo , Proteínas de Ligação a DNA , Enzima Desubiquitinante CYLD/metabolismo , Enzimas Desubiquitinantes/metabolismo , Demência Frontotemporal/metabolismo , Camundongos , Mutação de Sentido Incorreto/genética , NF-kappa B/antagonistas & inibidores , Cultura Primária de Células , Transfecção
15.
Sci Transl Med ; 11(523)2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852800

RESUMO

Motor neuron-specific microRNA-218 (miR-218) has recently received attention because of its roles in mouse development. However, miR-218 relevance to human motor neuron disease was not yet explored. Here, we demonstrate by neuropathology that miR-218 is abundant in healthy human motor neurons. However, in amyotrophic lateral sclerosis (ALS) motor neurons, miR-218 is down-regulated and its mRNA targets are reciprocally up-regulated (derepressed). We further identify the potassium channel Kv10.1 as a new miR-218 direct target that controls neuronal activity. In addition, we screened thousands of ALS genomes and identified six rare variants in the human miR-218-2 sequence. miR-218 gene variants fail to regulate neuron activity, suggesting the importance of this small endogenous RNA for neuronal robustness. The underlying mechanisms involve inhibition of miR-218 biogenesis and reduced processing by DICER. Therefore, miR-218 activity in motor neurons may be susceptible to failure in human ALS, suggesting that miR-218 may be a potential therapeutic target in motor neuron disease.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , MicroRNAs/metabolismo , Neuropatologia/métodos , Esclerose Amiotrófica Lateral/genética , Animais , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Neurônios Motores/metabolismo , Neurônios/metabolismo
16.
Biochim Biophys Acta Gene Regul Mech ; 1862(9): 194413, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31382054

RESUMO

The RNA-binding protein TDP-43, associated to amyotrophic lateral sclerosis and frontotemporal dementia, regulates the alternative splicing of several genes, including the skipping of TNIK exon 15. TNIK, a genetic risk factor for schizophrenia and causative for intellectual disability, encodes for a Ser/Thr kinase regulating negatively F-actin dynamics. Here we show that in the human adult nervous system TNIK exon 15 is mostly included compared to the other tissues and that, during neuronal differentiation of human induced pluripotent stem cells and of human neuroblastoma cells, TNIK exon 15 inclusion increases independently of TDP-43 protein content. By studying the possible molecular interplay of TDP-43 with brain-specific splicing factors, we found that the neuronal NOVA-1 protein competitively inhibits both TDP-43 and hnRNPA2/B1 skipping activity on TNIK by means of a RNA-dependent interaction and that this competitive mechanism is common to other TDP-43 RNA targets. We also show that the TNIK protein isoforms including/excluding exon 15 differently regulate cell spreading in non-neuronal cells and neuritogenesis in primary cortical neurons. Our data suggest a complex regulation between the ubiquitous TDP-43 and the neuron-specific NOVA-1 splicing factors in the brain that may help better understand the pathobiology of both neurodegenerative diseases and schizophrenia.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a RNA/genética , Esquizofrenia/genética , Processamento Alternativo/genética , Linhagem Celular , Proteínas de Ligação a DNA/química , Éxons/genética , Humanos , Neurônios/metabolismo , Neurônios/patologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Esquizofrenia/patologia
17.
Nat Commun ; 10(1): 3827, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444357

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown etiology. Although defects in nucleocytoplasmic transport (NCT) may be central to the pathogenesis of ALS and other neurodegenerative diseases, the molecular mechanisms modulating the nuclear pore function are still largely unknown. Here we show that genetic and pharmacological modulation of actin polymerization disrupts nuclear pore integrity, nuclear import, and downstream pathways such as mRNA post-transcriptional regulation. Importantly, we demonstrate that modulation of actin homeostasis can rescue nuclear pore instability and dysfunction caused by mutant PFN1 as well as by C9ORF72 repeat expansion, the most common mutation in ALS patients. Collectively, our data link NCT defects to ALS-associated cellular pathology and propose the regulation of actin homeostasis as a novel therapeutic strategy for ALS and other neurodegenerative diseases.


Assuntos
Actinas/metabolismo , Esclerose Amiotrófica Lateral/patologia , Neurônios Motores/patologia , Poro Nuclear/patologia , Profilinas/metabolismo , Acrilamidas/farmacologia , Actinas/ultraestrutura , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Esclerose Amiotrófica Lateral/genética , Biópsia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Linhagem Celular , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Embrião de Mamíferos , Fibroblastos , Humanos , Microscopia Eletrônica de Transmissão , Neurônios Motores/citologia , Mutação , Poro Nuclear/efeitos dos fármacos , Poro Nuclear/ultraestrutura , Cultura Primária de Células , Profilinas/genética , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , Pele/citologia , Pele/patologia , Tiazóis/farmacologia , Tiazolidinas/farmacologia
18.
J Biol Chem ; 294(26): 10194-10210, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31092554

RESUMO

Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease-linked RNA-binding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function, and disease have not been explored. Here, using primary cortical and motor neurons, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity included TAR DNA-binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS). We noted that FUS is translocated through a calcium-dependent mechanism and that its translocation coincides with striking alterations in nucleocytoplasmic transport. Furthermore, glutamate-induced up-regulation of glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type subunit 2 (GRIA2) in neurons depended on FUS expression, consistent with a functional role for FUS in excitotoxic stress. These findings reveal molecular links among prominent factors in neurodegenerative diseases, namely excitotoxicity, disease-associated RBPs, and nucleocytoplasmic transport.


Assuntos
Cálcio/metabolismo , Núcleo Celular/metabolismo , Ácido Glutâmico/efeitos adversos , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Receptores de AMPA/metabolismo , Estresse Fisiológico , Transporte Ativo do Núcleo Celular , Esclerose Amiotrófica Lateral , Citoplasma , Demência Frontotemporal , Humanos , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteína FUS de Ligação a RNA/genética , Receptores de AMPA/genética
19.
Hum Mol Genet ; 28(14): 2309-2318, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30985904

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive muscular atrophy and respiratory failure. The G4C2 repeat expansion in the C9orf72 gene is the most prevalent genetic risk for ALS. Mutation carriers (C9ALS) display variability in phenotypes such as age-at-onset and duration, suggesting the existence of additional genetic factors. Here we introduce a three-step gene discovery strategy to identify genetic factors modifying the risk of both C9ALS and sporadic ALS (sALS) using limited samples. We first identified 135 candidate genetic modifiers of C9ALS using whole-genome sequencing (WGS) of extreme C9ALS cases diagnosed ~30 years apart. We then performed an unbiased genetic screen using a Drosophila model of the G4C2 repeat expansion with the genes identified from WGS analysis. This genetic screen identified the novel genetic interaction between G4C2 repeat-associated toxicity and 18 genetic factors, suggesting their potential association with C9ALS risk. We went on to test if 14 out of the 18 genes, those which were not known to be risk factors for ALS previously, are also associated with ALS risk in sALS cases. Gene-based-statistical analyses of targeted resequencing and WGS were performed. These analyses together reveal that rare variants in MYH15 represent a likely genetic risk factor for ALS. Furthermore, we show that MYH15 could modulate the toxicity of dipeptides produced from expanded G4C2 repeat. Our study presented here demonstrates the power of combining WGS with fly genetics to facilitate the discovery of fundamental genetic components of complex traits with a limited number of samples.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Expansão das Repetições de DNA , Drosophila/genética , Cadeias Pesadas de Miosina/genética , Adulto , Idoso , Animais , Animais Geneticamente Modificados , Proteína C9orf72/metabolismo , Proteína C9orf72/toxicidade , Dipeptídeos/metabolismo , Dipeptídeos/toxicidade , Modelos Animais de Doenças , Drosophila/citologia , Drosophila/crescimento & desenvolvimento , Drosophila/ultraestrutura , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Humanos , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação , Cadeias Pesadas de Miosina/metabolismo , Fenótipo , Fatores de Risco , Sequenciamento Completo do Genoma
20.
Cell Rep ; 26(9): 2298-2306.e5, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30811981

RESUMO

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder without effective neuroprotective therapy. Known genetic variants impair pathways, including RNA processing, axonal transport, and protein homeostasis. We report ALS-causing mutations within the gene encoding the glycosyltransferase GLT8D1. Exome sequencing in an autosomal-dominant ALS pedigree identified p.R92C mutations in GLT8D1, which co-segregate with disease. Sequencing of local and international cohorts demonstrated significant ALS association in the same exon, including additional rare deleterious mutations in conserved amino acids. Mutations are associated with the substrate binding site, and both R92C and G78W changes impair GLT8D1 enzyme activity. Mutated GLT8D1 exhibits in vitro cytotoxicity and induces motor deficits in zebrafish consistent with ALS. Relative toxicity of mutations in model systems mirrors clinical severity. In conclusion, we have linked ALS pathophysiology to inherited mutations that diminish the activity of a glycosyltransferase enzyme.


Assuntos
Esclerose Amiotrófica Lateral/genética , Glicosiltransferases/genética , Mutação , Esclerose Amiotrófica Lateral/diagnóstico , Animais , Linhagem Celular , Sobrevivência Celular , Éxons , Feminino , Técnicas de Silenciamento de Genes , Glicosiltransferases/metabolismo , Complexo de Golgi/enzimologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Neurônios/enzimologia , Domínios Proteicos/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética
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