RESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral/inmunología , Interacciones Huésped-Patógeno/inmunología , Neuronas Motoras/inmunología , Proteína FUS de Unión a ARN/inmunología , Virus Sincitiales Respiratorios/inmunología , Médula Espinal/inmunología , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/inmunología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/virología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/inmunología , Línea Celular , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/inmunología , Gránulos Citoplasmáticos/virología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/inmunología , Cuerpos de Inclusión/virología , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Neuronas Motoras/metabolismo , Neuronas Motoras/virología , Neuroglía/inmunología , Neuroglía/metabolismo , Neuroglía/virología , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/inmunología , Poli I-C/farmacología , Cultivo Primario de Células , Agregado de Proteínas/genética , Agregado de Proteínas/inmunología , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/inmunología , Proteína FUS de Unión a ARN/genética , Virus Sincitiales Respiratorios/patogenicidad , Médula Espinal/metabolismo , Médula Espinal/patología , Médula Espinal/virologíaRESUMEN
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.
