Misfolded proteins causing Amyotrophic Lateral Sclerosis (ALS) and Spinobulbar Muscular Atrophy (SBMA) are actively removed by autophagy facilitation

Amyotrophic Lateral Sclerosis (ALS) and Spinobulbar Muscular Atrophy (SBMA) are fatal neurodegenerative diseases affecting motorneurons. Recently also muscular tissue was found to be involved at the onset and in the progression of these diseases. The molecular mechanism at the basis of these diseases is the loss of the right conformation of mutated proteins that misfold becoming toxic to cells. One important ALS related protein is the TAR-DNA-binding Protein (TDP43) in its truncated disease-associated form TDP43-25. SBMA, instead, is caused by a polyglutammine elongation in the androgen receptor protein (ARpolyQ). These misfolded proteins tend to aggregate forming high molecular weight species, hallmark of the disease. The protein quality control (PQC) system is in charge to maintain the protein homeostasis, trying to refold misfolded proteins trough the chaperone system or by degrading them via the ubiquitin proteasome system (UPS) or via autophagy. In this study we tried to modulate the PQC system in muscle cells in order to reduce the accumulation of misfolded toxic proteins. Initially, we analyzed the expression of some important elements of the PQC system (like Bag3, Bag1, HspB8, p62 and LC3) trough western blot, immunofluorescence and real time analysis. We demonstrated that autophagy is more activated in muscle C2C12 cells than in motorneuronal NSC34 cells. Successively, we investigate the aggregation of toxic misfolded protein in muscle cells. Trough filter retardation assay (FRA) we observe that both ARpolyQ and TDP43-25 aggregate in muscle C2C12 cells. Treating cells with an autophagic inhibitor we also found that autophagy is greatly involved in ARpolyQ aggregation. In the case of TDP43-25 we noted by FRA that TDP43 is mainly degraded trough UPS system, while autophagy is poorly involved. Finally, we try to reduce the presence of aggregates trough the well-known autophagic activator trehalose; surprisingly, we observe that ARpolyQ aggregation was reverted in presence of autophagic facilitation; on the contrary trehalose fails in the removal of TDP43 aggregates. When we overexpressed the small heat shock protein B8, a molecular chaperone that prevents aggregate ARpolyQ formation in motorneuronal model of the disease, we observed a reduction in ARpolyQ aggregation also in muscle cells. In conclusion we demonstrated that modulating autophagy could be a promising strategy in treating SBMA. On the contrary TDP43 homeostasis is in charge of proteasome. So it will be of interest to test drugs that could enhance proteasomal activity. GRANTS: Regione Lombardia; AFM-TELETHON, FRANCE; FONDAZIONE TELETHON, ITALY; FONDAZIONE CARIPLO, ITALY; FONDAZIONE ARISLA, ITALY; Ministero della Sanit\ue0, ITALY