Proteomics analysis of FUS mutant human motoneurons reveals altered regulation of cytoskeleton and other ALS-linked proteins via 3′UTR binding

Abstract

increasing evidence suggests that in Amyotrophic Lateral Sclerosis (ALS) mutated RnA binding proteins acquire aberrant functions, leading to altered RNA metabolism with significant impact on encoded protein levels. Here, by taking advantage of a human induced pluripotent stem cell-based model, we aimed to gain insights on the impact of ALS mutant fUS on the motoneuron proteome. Label-free proteomics analysis by mass-spectrometry revealed upregulation of proteins involved in catabolic processes and oxidation-reduction, and downregulation of cytoskeletal proteins and factors directing neuron projection. Mechanistically, proteome alteration does not correlate with transcriptome changes. Rather, we observed a strong correlation with selective binding of mutant fUS to target mRNAs in their 3′UtR. novel validated targets, selectively bound by mutant fUS, include genes previously involved in familial or sporadic ALS, such as VCP, and regulators of membrane trafficking and cytoskeleton remodeling, such as ASAP1. These findings unveil a novel mechanism by which mutant fUS might intersect other pathogenic pathways in ALS patients' motoneurons. The motoneuron disease Amyotrophic Lateral Sclerosis (ALS) has been linked to mutations in several RNA binding proteins (RBPs) and altered RNA metabolism 1,2. ALS mutations affecting nuclear localization of the RBP FUS are regarded as a primary event, eventually leading to motoneuron death by unknown mechanisms 3,4. Accordingly, FUS mutations that cause higher levels of mislocalization in the cytoplasm (such as the P525L) are associated to more aggressive and juvenile ALS pathology 5. However, little is known about the effects of abnormal FUS accumulation in the cytoplasm on its RNA targets. A change in the translatome has been previously observed in neurons derived from mouse embryonic stem cells ectopically overexpressing human FUS-R495X protein, which is another mutant FUS mislocalized to the cytoplasm 6. Alteration of protein translation was also studied upon oxidative stress and ectopic expression of wild-type (WT) or R495X mutant FUS in neuroblastoma cells 7. However, overexpression of WT FUS at non-physiological levels is known to produce toxic effects per se 8. Moreover, since ALS specifically affects motoneurons 9 , disease-relevant targets might be missed in in vitro models based on non-motoneuronal cells. The effect of mutant FUS on the proteome of human motoneurons remains unexplored.

FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

“…S10D; Ribo- Fus −/− : fold change < 0, P < 0.05, 21 genes). We then compared the RiboTag to published mass spectrometry on iPSC-derived FUS P525L MNs used throughout this work ( 35 ). We found seven human orthologs, with FMRP binding sites at the transcript level, to be down-regulated ( z score > 1 in both datasets) in both Ribo- Fus ∆ 14/ ∆ 14 and FUS P525L/P525L MN mass spectrometry (respective mouse genes: Dnm1 , Usp9x , Trim2 , Pld3 , Foxk2 , Arfgef1 , and Tmod2 ; table S3).…”

“…Human iPSCs used in this study are the isogenic FUS WT/WT and FUS P525L/P525L lines that were derived and maintained as described ( 58 ) and differentiated into spinal MNs as described ( 35 , 59 ). Briefly, iPSCs stably transduced with a piggyBac vector carrying inducible Ngn2, Isl1, and Lhx3 (NIL) transgenes dissociated to single cells with Accutase (Thermo Fisher Scientific) and plated in Nutristem XF/FF medium (Biological Industries) supplemented with 10 μM ROCK inhibitor (Enzo Life Sciences) on Matrigel (BD Biosciences) at a density of 100,000 cells/cm 2 .…”

Proximity-based labeling reveals DNA damage-induced N-terminal phosphorylation of fused in sarcoma (FUS) leads to distinct changes in the FUS protein interactome

“…The FUS P525L mutation was first identified in 2012 and causes a severe form of juvenile ALS (Conte et al , 2012; Zhou et al , 2020). FUS P525L robustly increases cytoplasmic localization of FUS and alters the transcriptome, proteome, and the spliceosome in multiple model systems (De Santis et al ., 2017; Garone et al , 2020; Humphrey et al , 2020). Therefore, the APEX2-FUS P525L mutant 1) served as a positive control for FUS cytoplasmic localization, 2) provided insight into the pathogenic nature of ALS-linked mutations, and 3) was a useful comparison to determine if FUS PM resembles a pathogenic phenotype (Monahan et al ., 2017; Rhoads et al ., 2018).…”

“…Proteomic analysis is a powerful tool that has revealed how pathogenic ALS-linked mutations (e.g. FUS P525L and R495X) may shift the proteome toward pathology (Baron et al , 2019; Garone et al ., 2020; Kamelgarn et al ., 2016). While these past studies have informed the role that a genetic mutation can have on protein-protein interactions, pathogenic FUS mutations only account for 4% of ALS cases and a handful of FTD cases (Broustal et al ., 2010; Renton et al , 2014; Rohrer et al , 2009; Snowden et al ., 2011).…”

ALS-FUS mutation affects the activities of HuD/ELAVL4 and FMRP leading to axon phenotypes in motoneurons

“…Axonal degeneration is a key feature in the ALS pathophysiology and occurs prior to the motor phenotype in patients [21][22][23]. The levels of cytoskeletal proteins and factors directing neuron projection are changed in FUS mutant hiPSC-derived MNs [8], and aberrant branching and axonal outgrowth have been recently identified across ALS mutations and model systems, underlying their importance in early disease pathogenesis [24][25][26][27][28]. Spinal MNs isolated from adult SOD1 mutant mice at pre-symptomatic stages displayed significant increase in axon outgrowth, in terms of length and branching complexity, and acute expression of mutant SOD1 in WT MNs was sufficient to increase axonal regeneration [29].…”