RESUMEN
Mutations disrupting the nuclear localization of the RNA-binding protein FUS characterize a subset of amyotrophic lateral sclerosis patients (ALS-FUS). FUS regulates nuclear RNAs, but its role at the synapse is poorly understood. Using super-resolution imaging we determined that the localization of FUS within synapses occurs predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosomes, we identified synaptic FUS RNA targets, encoding proteins associated with synapse organization and plasticity. Significant increase of synaptic FUS during early disease in a mouse model of ALS was accompanied by alterations in density and size of GABAergic synapses. mRNAs abnormally accumulated at the synapses of 6-month-old ALS-FUS mice were enriched for FUS targets and correlated with those depicting increased short-term mRNA stability via binding primarily on multiple exonic sites. Our study indicates that synaptic FUS accumulation in early disease leads to synaptic impairment, potentially representing an initial trigger of neurodegeneration.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteína FUS de Unión a ARN/metabolismo , ARN/metabolismo , Sinapsis/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Núcleo Celular/metabolismo , Corteza Cerebral , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/genéticaRESUMEN
Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a progressive increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo, associated with altered synaptic gene expression. Synaptic ultrastructural and morphological defects were more pronounced in inhibitory than excitatory synapses and associated with increased synaptosomal levels of FUS and its RNA targets. Thus, cytoplasmic FUS triggers synaptic deficits, which is leading to increased neuronal activity in frontal cortex and causing related behavioral phenotypes. These results indicate that FUS mislocalization may trigger deleterious phenotypes beyond motor neuron impairment in ALS, likely relevant also for other neurodegenerative diseases characterized by FUS mislocalization.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Citoplasma/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Sinapsis/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Femenino , Expresión Génica , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Mutación , Fenotipo , Transmisión Sináptica/fisiologíaRESUMEN
The extensive generation of RNA sequencing (RNA-seq) data in the last decade has resulted in a myriad of specialized software for its analysis. Each software module typically targets a specific step within the analysis pipeline, making it necessary to join several of them to get a single cohesive workflow. Multiple software programs automating this procedure have been proposed, but often lack modularity, transparency or flexibility. We present ARMOR, which performs an end-to-end RNA-seq data analysis, from raw read files, via quality checks, alignment and quantification, to differential expression testing, geneset analysis and browser-based exploration of the data. ARMOR is implemented using the Snakemake workflow management system and leverages conda environments; Bioconductor objects are generated to facilitate downstream analysis, ensuring seamless integration with many R packages. The workflow is easily implemented by cloning the GitHub repository, replacing the supplied input and reference files and editing a configuration file. Although we have selected the tools currently included in ARMOR, the setup is modular and alternative tools can be easily integrated.