ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects

Abstract

Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf ) as a target of FUS-R521C-associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions. IntroductionAutosomal dominant mutations in RNA/DNA binding protein fused in sarcoma/translocated in liposarcoma (FUS/TLS) have been causally linked to familial ALS (FALS). The main pathological features in FALS with FUS mutations are FUS-positive protein aggregates in neuronal cytoplasm and dendrites (1, 2). While the majority of these aggregates are identified in the spinal motor neurons, FUS-positive aggregates have also been found in neurons in cerebral cortex and brainstem nuclei (3, 4), raising the possibility that FUS mutations may have a broader impact on the functions of other neuronal subtypes. Consistent with these observations, a subset of FALS-FUS patients also exhibits cognitive impairments during the developmental or degenerative processes.Although the exact mechanism of FUS mutations remains unclear, several previous studies have provided compelling evidence that FUS can regulate DNA damage response, transcription, and RNA processing. For instance, fibroblasts and lymphocytes from fus-null mice show increased sensitivity to ionizing irradiation and genomic instability, respectively (5, 6). Consistent with these results, our recent study shows that WT FUS proteins are rapidly recruited to DNA damage foci in neurons, where it interacts with histone deacetylase 1 (HDAC1), a critical component in

Activity-dependent FUS dysregulation disrupts synaptic homeostasis

“…5). These data are consistent with transgenic mice harboring the R521C mutation under control of the Syrian hamster prion promoter (43). However, Qiu et al (43) reported only transgenic mice for FUS mutant R521C, without comparable wild-type transgenic animals.…”

ALS mutations in TLS/FUS disrupt target gene expression

“…Leading theories propose that ALS mutations cause pathological changes in gene expression/RNA processing Qiu et al 2014). However, whether this reflects reduced function of the mutated proteins, a toxic gain of function, or both is not known (Mackenzie and Neumann 2012).…”

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…Among the differentially regulated splicing events observed in mutant TDP43 transgenic mice were those involving neurexins 1 and 3, and GluA2-transcripts that are either direct targets of TDP43 or are processed by gene products that are regulated by TDP43 (i.e., ADAR) [14,36]. In contrast to results from mutant TDP43 transgenic mice, overexpression of mutant FUS in an animal model of ALS resulted in the retention of >1000 introns, significantly more than with WT FUS overexpression (177 introns) [63]. In addition, mutant FUS dramatically altered the splicing and expression of an essential neuroprotective agent, brainderived neurotrophic factor (BDNF).…”

“…In addition, mutant FUS dramatically altered the splicing and expression of an essential neuroprotective agent, brainderived neurotrophic factor (BDNF). Exogenous replacement of BDNF rescued dendritic and synaptic abnormalities in mutant FUS-overexpressing primary neurons [63], suggesting that abnormalities in RNA processing can be overcome and neuronal function improved in some instances by manipulating downstream signaling pathways.…”

“…In addition, targeted reduction in TDP43 or FUS produced disparate effects in NSC34 mouse motor neuron-like cells [62]. In an animal model of ALS, the overexpression of FUS bearing an ALS-associated mutation (R521C) significantly affected the expression of >700 genes [63]. Many of the upregulated genes in this context involved extracellular matrix or immune processes, while downregulated transcripts included those responsible for neuron projections and ion channel functions.…”