RESUMO
Valosin-containing protein (VCP) is a highly expressed member of the type II AAA+ ATPase family. VCP mutations are the cause of inclusion body myopathy, Paget's disease of the bone, and frontotemporal dementia (IBMPFD) and they account for 1%-2% of familial amyotrophic lateral sclerosis (ALS). Using fibroblasts from patients carrying three independent pathogenic mutations in the VCP gene, we show that VCP deficiency causes profound mitochondrial uncoupling leading to decreased mitochondrial membrane potential and increased mitochondrial oxygen consumption. This mitochondrial uncoupling results in a significant reduction of cellular ATP production. Decreased ATP levels in VCP-deficient cells lower their energy capacity, making them more vulnerable to high energy-demanding processes such as ischemia. Our findings propose a mechanism by which pathogenic VCP mutations lead to cell death.
Assuntos
Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação/genética , Neurônios/ultraestrutura , Adenosina Trifosfatases/deficiência , Adulto , Idoso , Análise de Variância , Animais , Animais Recém-Nascidos , Estudos de Casos e Controles , Proteínas de Ciclo Celular/deficiência , Células Cultivadas , Córtex Cerebral/citologia , Saúde da Família , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Peroxidação de Lipídeos/genética , Proteínas Luminescentes/genética , Magnésio/metabolismo , Masculino , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mitocôndrias/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Miosite de Corpos de Inclusão/genética , Miosite de Corpos de Inclusão/metabolismo , Miosite de Corpos de Inclusão/patologia , NAD/metabolismo , Neuroblastoma/patologia , Osteíte Deformante/genética , Osteíte Deformante/metabolismo , Osteíte Deformante/patologia , Consumo de Oxigênio/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transfecção , Proteína com ValosinaRESUMO
Hexanucleotide repeat expansions in C9orf72 are a major cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Understanding the disease mechanisms and a method for clinical diagnostic genotyping have been hindered because of the difficulty in estimating the expansion size. We found 96 repeat-primed PCR expansions: 85/2,974 in six neurodegenerative diseases cohorts (FTLD, ALS, Alzheimer disease, sporadic Creutzfeldt-Jakob disease, Huntington disease-like syndrome, and other nonspecific neurodegenerative disease syndromes) and 11/7,579 (0.15%) in UK 1958 birth cohort (58BC) controls. With the use of a modified Southern blot method, the estimated expansion range (smear maxima) in cases was 800-4,400. Similarly, large expansions were detected in the population controls. Differences in expansion size and morphology were detected between DNA samples from tissue and cell lines. Of those in whom repeat-primed PCR detected expansions, 68/69 were confirmed by blotting, which was specific for greater than 275 repeats. We found that morphology in the expansion smear varied among different individuals and among different brain regions in the same individual. Expansion size correlated with age at clinical onset but did not differ between diagnostic groups. Evidence of instability of repeat size in control families, as well as neighboring SNP and microsatellite analyses, support multiple expansion events on the same haplotype background. Our method of estimating the size of large expansions has potential clinical utility. C9orf72-related disease might mimic several neurodegenerative disorders and, with potentially 90,000 carriers in the United Kingdom, is more common than previously realized.
Assuntos
Esclerose Lateral Amiotrófica/genética , Expansão das Repetições de DNA , Degeneração Lobar Frontotemporal/genética , Degeneração Neural/genética , Proteínas/genética , Esclerose Lateral Amiotrófica/patologia , Encéfalo/patologia , Proteína C9orf72 , Estudos de Coortes , DNA/genética , Degeneração Lobar Frontotemporal/patologia , Predisposição Genética para Doença , Haplótipos , Humanos , Repetições de Microssatélites , Degeneração Neural/patologia , Polimorfismo de Nucleotídeo Único , Reino UnidoRESUMO
Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.