DDX17 is involved in DNA damage repair and modifies FUS toxicity in an RGG-domain dependent manner.

Mutations in the RNA binding protein, Fused in Sarcoma (FUS), lead to amyotrophic lateral sclerosis (ALS), the most frequent form of motor neuron disease. Cytoplasmic aggregation and defective DNA repair machinery are etiologically linked to mutant FUS-associated ALS. Although FUS is involved in numerous aspects of RNA processing, little is understood about the pathophysiological mechanisms of mutant FUS. Here, we employed RNA-sequencing technology in Drosophila brains expressing FUS to identify significantly altered genes and pathways involved in FUS-mediated neurodegeneration. We observed the expression levels of DEAD-Box Helicase 17 (DDX17) to be significantly downregulated in response to mutant FUS in Drosophila and human cell lines. Mutant FUS recruits nuclear DDX17 into cytoplasmic stress granules and physically interacts with DDX17 through the RGG1 domain of FUS. Ectopic expression of DDX17 reduces cytoplasmic mislocalization and sequestration of mutant FUS into cytoplasmic stress granules. We identified DDX17 as a novel regulator of the DNA damage response pathway whose upregulation repairs defective DNA damage repair machinery caused by mutant neuronal FUS ALS. In addition, we show DDX17 is a novel modifier of FUS-mediated neurodegeneration in vivo. Our findings indicate DDX17 is downregulated in response to mutant FUS, and restoration of DDX17 levels suppresses FUS-mediated neuropathogenesis and toxicity in vivo.

Muscleblind acts as a modifier of FUS toxicity by modulating stress granule dynamics and SMN localization.

Mutations in fused in sarcoma (FUS) lead to amyotrophic lateral sclerosis (ALS) with varying ages of onset, progression and severity. This suggests that unknown genetic factors contribute to disease pathogenesis. Here we show the identification of muscleblind as a novel modifier of FUS-mediated neurodegeneration in vivo. Muscleblind regulates cytoplasmic mislocalization of mutant FUS and subsequent accumulation in stress granules, dendritic morphology and toxicity in mammalian neuronal and human iPSC-derived neurons. Interestingly, genetic modulation of endogenous muscleblind was sufficient to restore survival motor neuron (SMN) protein localization in neurons expressing pathogenic mutations in FUS, suggesting a potential mode of suppression of FUS toxicity. Upregulation of SMN suppressed FUS toxicity in Drosophila and primary cortical neurons, indicating a link between FUS and SMN. Our data provide in vivo evidence that muscleblind is a dominant modifier of FUS-mediated neurodegeneration by regulating FUS-mediated ALS pathogenesis.

Mining Disaggregase Sequence Space to Safely Counter TDP-43, FUS, and α-Synuclein Proteotoxicity.

Hsp104 is an AAA+ protein disaggregase, which can be potentiated via diverse mutations in its autoregulatory middle domain (MD) to mitigate toxic misfolding of TDP-43, FUS, and α-synuclein implicated in fatal neurodegenerative disorders. Problematically, potentiated MD variants can exhibit off-target toxicity. Here, we mine disaggregase sequence space to safely enhance Hsp104 activity via single mutations in nucleotide-binding domain 1 (NBD1) or NBD2. Like MD variants, NBD variants counter TDP-43, FUS, and α-synuclein toxicity and exhibit elevated ATPase and disaggregase activity. Unlike MD variants, non-toxic NBD1 and NBD2 variants emerge that rescue TDP-43, FUS, and α-synuclein toxicity. Potentiating substitutions alter NBD1 residues that contact ATP, ATP-binding residues, or the MD. Mutating the NBD2 protomer interface can also safely ameliorate Hsp104. Thus, we disambiguate allosteric regulation of Hsp104 by several tunable structural contacts, which can be engineered to spawn enhanced therapeutic disaggregases with minimal off-target toxicity.

Overriding FUS autoregulation in mice triggers gain-of-toxic dysfunctions in RNA metabolism and autophagy-lysosome axis.

Mutations in coding and non-coding regions of FUS cause amyotrophic lateral sclerosis (ALS). The latter mutations may exert toxicity by increasing FUS accumulation. We show here that broad expression within the nervous system of wild-type or either of two ALS-linked mutants of human FUS in mice produces progressive motor phenotypes accompanied by characteristic ALS-like pathology. FUS levels are autoregulated by a mechanism in which human FUS downregulates endogenous FUS at mRNA and protein levels. Increasing wild-type human FUS expression achieved by saturating this autoregulatory mechanism produces a rapidly progressive phenotype and dose-dependent lethality. Transcriptome analysis reveals mis-regulation of genes that are largely not observed upon FUS reduction. Likely mechanisms for FUS neurotoxicity include autophagy inhibition and defective RNA metabolism. Thus, our results reveal that overriding FUS autoregulation will trigger gain-of-function toxicity via altered autophagy-lysosome pathway and RNA metabolism function, highlighting a role for protein and RNA dyshomeostasis in FUS-mediated toxicity.

Neuron-to-Neuron Transfer of FUS in Drosophila Primary Neuronal Culture Is Enhanced by ALS-Associated Mutations.

The DNA- and RNA-binding protein fused in sarcoma (FUS) has been pathologically and genetically linked to amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Cytoplasmic FUS-positive inclusions were identified in the brain and spinal cord of a subset of patients suffering with ALS/FTLD. An increasing number of reports suggest that FUS protein can behave in a prion-like manner. However, no neuropathological studies or experimental data were available regarding cell-to-cell spread of these pathological protein assemblies. In the present report, we investigated the ability of wild-type and mutant forms of FUS to transfer between neuronal cells. We combined the use of Drosophila models for FUS proteinopathies with that of the primary neuronal cultures to address neuron-to-neuron transfer of FUS proteins. Using conditional co-culture models and an optimized flow cytometry-based methodology, we demonstrated that ALS-mutant forms of FUS proteins can transfer between well-differentiated mature Drosophila neurons. These new observations support that a propagating mechanism could be applicable to FUS, leading to the sequential dissemination of pathological proteins over years.

Protein arginine methyltransferase 1 and 8 interact with FUS to modify its sub-cellular distribution and toxicity in vitro and in vivo.

Amyotrophic lateral sclerosis (ALS) is a late onset and progressive motor neuron disease. Mutations in the gene coding for fused in sarcoma/translocated in liposarcoma (FUS) are responsible for some cases of both familial and sporadic forms of ALS. The mechanism through which mutations of FUS result in motor neuron degeneration and loss is not known. FUS belongs to the family of TET proteins, which are regulated at the post-translational level by arginine methylation. Here, we investigated the impact of arginine methylation in the pathogenesis of FUS-related ALS. We found that wild type FUS (FUS-WT) specifically interacts with protein arginine methyltransferases 1 and 8 (PRMT1 and PRMT8) and undergoes asymmetric dimethylation in cultured cells. ALS-causing FUS mutants retained the ability to interact with both PRMT1 and PRMT8 and undergo asymmetric dimethylation similar to FUS-WT. Importantly, PRMT1 and PRMT8 localized to mutant FUS-positive inclusion bodies. Pharmacologic inhibition of PRMT1 and PRMT8 activity reduced both the nuclear and cytoplasmic accumulation of FUS-WT and ALS-associated FUS mutants in motor neuron-derived cells and in cells obtained from an ALS patient carrying the R518G mutation. Genetic ablation of the fly homologue of human PRMT1 (DART1) exacerbated the neurodegeneration induced by overexpression of FUS-WT and R521H FUS mutant in a Drosophila model of FUS-related ALS. These results support a role for arginine methylation in the pathogenesis of FUS-related ALS.

Arginine methylation by PRMT1 regulates nuclear-cytoplasmic localization and toxicity of FUS/TLS harbouring ALS-linked mutations.

Mutations in FUS/TLS (fused in sarcoma/translated in liposarcoma) cause an inheritable form of amyotrophic lateral sclerosis (ALS6). In contrast to FUS(WT), which is concentrated in the nucleus, these mutants are abnormally distributed in the cytoplasm where they form inclusions and associate with stress granules. The data reported herein demonstrate the importance of protein arginine methylation in nuclear-cytoplasmic shuttling of FUS and abnormalities of ALS-causing mutants. Depletion of protein arginine methyltransferase 1 (PRMT1; the enzyme that methylates FUS) in mouse embryonic fibroblasts by gene knockout, or in human HEK293 cells by siRNA knockdown, diminished the ability of ALS-linked FUS mutants to localize to the cytoplasm and form inclusions. To examine properties of FUS mutants in the context of neurons vulnerable to the disease, FUS(WT) and ALS-linked FUS mutants were expressed in motor neurons of dissociated murine spinal cord cultures. In motor neurons, shRNA-mediated PRMT1 knockdown concomitant with the expression of FUS actually accentuated the shift in distribution of ALS-linked FUS mutants from the nucleus to the cytoplasm. However, when PRMT1 was inhibited prior to expression of ALS-linked FUS mutants, by pretreatment with a global methyltransferase inhibitor, ALS-linked FUS mutants were sequestered in the nucleus and cytoplasmic inclusions were reduced, as in the cell lines. Mitochondria were significantly shorter in neurons with cytoplasmic ALS-linked FUS mutants, a factor that could contribute to toxicity. We propose that arginine methylation by PRMT1 participates in the nuclear-cytoplasmic shuttling of FUS, particularly of ALS6-associated mutants, and thus contributes to the toxic gain of function conferred by these disease-causing mutations.

Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS.

TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.