A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy

Abstract

Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.

Induction Of Chronic Stress Reveals An Interplay Of Stress Granules And TDP-43 Pathological Aggregates In Human ALS Fibroblasts And iPSC-Neurons

“…Likewise, we detected a significantly increased number of LC3-TIAR-positive vacuoles after chronic ARS exposure in both control (1,6 vs 0,5 in untreated controls) and mutant fibroblasts (0,9 vs 0,5 in untreated mC9ORF72) (Additional file 1: Figure S6). The increase of double-labeled vacuoles was significantly lower in mC9ORF72 fibroblasts than in healthy control ones, supporting evidence that C9ORF72 haploinsufficiency occurring in mutant C9ORF72 ALS patients impairs autophagy [13,37].…”

“…Remarkably, it has been demonstrated that C9ORF72 colocalizes with Ubiquilin-2 and LC3-positive vesicles and the ratio of the autophagosome markers LC3II/LC3I is altered in C9ORF72 knocked-down cells [51]. Additionally, C9ORF72 protein was recently described to form a complex with the autophagy receptor p62, which associates with symmetrically methylated arginine proteins enriched in SG, controlling SG removal by autophagy [13]. All these data, together with present findings, suggest that an impairment of the p62-dependent clearance of SG components may be amplified in ALS patients with C9ORF72 repeat expansions, due to the haploinsufficiency of C9ORF72 protein observed in such patients [52].…”

“…From a biophysical point of view, assembly of SG has been shown to occur via a liquid-liquid phase separation (LLPS) process through which soluble cytoplasmic protein/RNA complexes condensate into dynamic liquid droplets [8,9] by weak and reversible interactions of the LCDs present in several RBPs, including TDP-43 [10]. SG usually disassemble after stress removal through a chaperone-mediated mechanism, while a small fraction of aberrant SG, which accumulates defective ribosomal products (DRiPs), fails to completely disassemble and is degraded by autophagy [11][12][13]. Some evidence also suggest participation of the proteasome system in SG removal [9,36], so that an interaction between chaperones, autophagy and proteasome system plays an important role in SG dynamic [14].…”

Reduced autophagy upon C9ORF72 loss synergizes with dipeptide repeat protein toxicity in G4C2 repeat expansion disorders

“…Furthermore, accumulation of DPR proteins is prevented by their constant degradation through autophagy. As the C9ORF72 protein regulates autophagy (Amick et al, 2016;Sellier et al, 2016;Sullivan et al, 2016;Ugolino et al, 2016;Webster et al, 2016;Xiao et al, 2016;Yang et al, 2016;Jung et al, 2017;Chitiprolu et al, 2018;Ho et al, 2019), we tested whether C9ORF72 modulates DPR expression. Importantly, reduced expression of C9ORF72 increases the accumulation and toxicity of DPR proteins expressed under their natural sequences.…”

Arginine valency inC9ORF72dipolypeptides mediates promiscuous proteome binding that stalls ribosomes, disable actin cytoskeleton assembly and impairs arginine methylation of endogenous proteins

“…Arginine methylation has been reported to be abnormal in patients with C9ORF72 mutations, including the presence of arginine-dimethylated enriched inclusions (42,43). Arginine residues are commonly methylated to regulate biological activity of many cellular processes and are important in particular in histones which are enriched GO terms in our datasets.…”