Wild-type FUS corrects ALS-like disease induced by cytoplasmic mutant FUS through autoregulation

Abstract

Mutations in FUS, an RNA-binding protein involved in multiple steps of RNA metabolism, are associated with the most severe forms of amyotrophic lateral sclerosis (ALS). Accumulation of cytoplasmic FUS is likely to be a major culprit in the toxicity of FUS mutations. Thus, preventing cytoplasmic mislocalization of the FUS protein may represent a valuable therapeutic strategy. FUS binds to its own pre-mRNA creating an autoregulatory loop efficiently buffering FUS excess through multiple proposed mechanisms including retention of introns 6 and/or 7. Here, we introduced a wild-type FUS gene allele, retaining all intronic sequences, in mice whose heterozygous or homozygous expression of a cytoplasmically retained FUS protein (FusΔNLS) was previously shown to provoke ALS-like disease or postnatal lethality, respectively. Wild-type FUS completely rescued the early lethality caused by the two FusΔNLS alleles, and improved age-dependent motor deficit and reduced lifespan associated with the heterozygous expression of FusΔNLS. Mechanistically, wild-type FUS decreased the load of cytoplasmic FUS, increased exon 7 skipping and retention of introns 6 and 7 in the endogenous mouse Fus mRNA, leading to decreased expression of the mutant mRNA. Thus, the wild-type FUS allele activates the homeostatic autoregulatory loop, maintaining constant FUS levels and decreasing the mutant protein in the cytoplasm. These results provide proof of concept that an autoregulatory competent wild-type FUS expression could protect against this devastating, currently intractable, neurodegenerative disease.

A scalable screening platform for phenotypic subtyping of ALS patient-derived fibroblasts

“…4a-b). We divided sporadic cell lines into high scoring (sporadic+) and low scoring (sporadic-) groups and conducted gene set enrichment analysis 38 for both groups along with FUS-ALS cell lines (Fig. 4b-c).…”

“…4). However, gene set enrichment analysis 38 highlighted pathways that were consistent between fibroblasts and spinal cords (19 pathways enriched in all of FUS-ALS, sporadic+, spinal cord; 28 pathways enriched in one of FUS-ALS, sporadic+, spinal cord; Methods). For example, pathways common to fibroblasts and spinal cord included nonsense-mediated decay and RNA metabolism, which have been previously implicated in ALS 39 (Fig.…”

“…In contrast, the reversion of FUS-ALS towards WT depended on reduced cytosolic FUS levels, consistent with observed patient cases with higher cytosolic levels 31 . Decreased nuclear and increased cytosolic FUS levels in FUS-ALS have led to hypotheses of loss- and/or toxic gain-of-function (respectively) for FUS 29,38,39 . While it is not the goal of our current study to answer mechanistic questions around loss-vs. gain-of-function, the distinctions that the models captured will provide the flexibility to evaluate disease-causing phenotypes.…”

“…Gene set enrichment analyses (GSEAs) were run using the R package fgsea to assess the enrichment of Reactome pathways 38 . Genes were ranked by -log10 unadjusted p-value from our differential expression analysis.…”