Linkage of proteasomal impairment and autophagy activation by C9orf72 poly-GA aggregate formation

Abstract

Background Poly-GA, a dipeptide repeat protein unconventionally translated from GGGGCC repeat expansions in C9orf72, is abundant in C9orf72-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although the poly-GA aggregates have been identified in C9orf72-ALS/FTD neurons, the effects on UPS and autophagy, and their exact molecular mechanisms have not been fully elucidated. Methods We generated poly-GA mice,and treated poly-GA mice for 90 days by intraperitoneal injection rapamycin (4 mg/kg) every 2 days until behavioral analysis. GFAP (an astrogliosis marker), Iba1 (a microgliosis marker), GFP, p62, p-p62 (S349), p-p62 (S403) and LC3 proteins expression were quantified in brain tissues by immunohistochemistry, immunofluorescence, immunoblotting. These results were validated in vitro by protein mass spectrometry, co-immunoprecipitation and immunofluorescence. Results Herein, our in vivo experiments show that ploy-GA mice expressing 150 repeats but not 30 repeats GA exhibit significant aggregates in cells, behavioral deficits, and activate autophagy in the brain, in vitro results demonstrate that aggregated poly-GA induces proteasomal stress through directly binding proteasome subunit PSMD2 to recruit proteasome and impair its function, subsequently activates phosphorylation and ubiquitination of p62 to recruit autophagosome, ultimately leading to compensatory autophagy activation. While rapamycin treatment significantly improves the degenerative symptoms of mice expressing 150 repeats poly-GA, relieves neuronal injury and reduces neuroinflammation and aggregates in the brain. Conclusion In summary, we clarify for the first time the relationship of poly-GA between proteasome and autophagy: poly-GA aggregates recruit autophagosomes only when it forms complex with the proteasome and causes proteasomal stress. Our study provides support for further promoting the comprehension about the pathogenesis of C9orf72 and may bring a hint for the therapy.