The amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD)-linked RNA-binding protein called fused in sarcoma (FUS) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian Target of Rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation as FUS-deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants, R521G and P525L, associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling, and that ALS-linked FUS-mutants can cause a toxic gain-of-function in the cytoplasm by repressing the translation of mRNA at polyribosomes.

Genetic mutations in nitrogen permease regulator-like 2 (NPRL2) are associated with a wide spectrum of familial focal epilepsies, autism, and sudden unexpected death of epileptics (SUDEP), but the mechanisms by which NPRL2 contributes to these effects are not well known. NPRL2 is a requisite subunit of the Gap Activity TOward Rags 1 (GATOR1) complex, which functions as a negative regulator of mammalian Target Of Rapamycin Complex 1 (mTORC1) kinase when intracellular amino acids are low.Here we show that loss of NPRL2 expression in mouse excitatory glutamatergic neurons causes seizures prior to death, consistent with SUDEP in humans with epilepsy. Additionally, the absence of NPRL2 expression increases mTORC1-dependent signal transduction and significantly alters amino acid homeostasis in the brain. Loss of NPRL2 reduces dendritic branching and increases the strength of electrically stimulated action potentials in neurons. The increased action potential strength is consistent with elevated expression of epilepsy-linked, voltage-gated sodium channels in the NPRL2-deficient brain. Targeted deletion of NPRL2 in primary neurons increases the expression of sodium channel Scn1A, whereas treatment with the pharmacological mTORC1 inhibitor called rapamycin prevents Scn1A upregulation. These studies demonstrate a novel role of NPRL2 and mTORC1 signaling in the regulation of sodium channels, which can contribute to seizures and early lethality.Significance Statement: NPRL2 is a requisite subunit of the epilepsy-linked GATOR1 complex that functions as a negative regulator of mTORC1 kinase when intracellular amino acids are limited. Here we report the generation and characterization of a new neurological model of GATOR1-dependent mTORopathy, caused by the loss of NPRL2 function in glutamatergic neurons. Loss of NPRL2 increases mTORC1 signal transduction, significantly alters amino acid homeostasis in the brain, and causes SUDEP.In addition, loss of NPRL2 increases the strength of electrically stimulated action potentials and the expression of epilepsy-linked sodium channels. These data reveal an unanticipated link between 3 intracellular amino acid signaling by NPRL2 and a novel mTORC1-dependent regulation of sodiumchannel expression in epilepsy.