RESUMO
Mutations in the FUS gene cause familial amyotrophic lateral sclerosis (ALS-FUS). In ALS-FUS, FUS-positive inclusions are detected in the cytoplasm of neurons and glia, a condition known as FUS proteinopathy. Mutant FUS incorporates into stress granules (SGs) and can spontaneously form cytoplasmic RNA granules in cultured cells. However, it is unclear what can trigger the persistence of mutant FUS assemblies and lead to inclusion formation. Using CRISPR/Cas9 cell lines and patient fibroblasts, we find that the viral mimic dsRNA poly(I:C) or a SG-inducing virus causes the sustained presence of mutant FUS assemblies. These assemblies sequester the autophagy receptor optineurin and nucleocytoplasmic transport factors. Furthermore, an integral component of the antiviral immune response, type I interferon, promotes FUS protein accumulation by increasing FUS mRNA stability. Finally, mutant FUS-expressing cells are hypersensitive to dsRNA toxicity. Our data suggest that the antiviral immune response is a plausible second hit for FUS proteinopathy.
Assuntos
Esclerose Lateral Amiotrófica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Neurônios Motores/imunologia , Proteína FUS de Ligação a RNA/imunologia , Vírus Sinciciais Respiratórios/imunologia , Medula Espinal/imunologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/virologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Linhagem Celular , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/virologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/imunologia , Corpos de Inclusão/virologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Neurônios Motores/metabolismo , Neurônios Motores/virologia , Neuroglia/imunologia , Neuroglia/metabolismo , Neuroglia/virologia , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/imunologia , Poli I-C/farmacologia , Cultura Primária de Células , Agregados Proteicos/genética , Agregados Proteicos/imunologia , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Proteína FUS de Ligação a RNA/genética , Vírus Sinciciais Respiratórios/patogenicidade , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/virologiaRESUMO
Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside paraspeckles, especially prominent under conditions of paraspeckle-inducing stress. Consistently, paraspeckle-dependent microRNA production, a readout for functionality of paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of paraspeckles accompanied by accumulation of non-paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS.
