BAG1 prevents misfolded proteins accumulation when autophagy flux is blocked in neurodegenerative disorders

Different disease associated proteins, as SOD1 and TDP-43 in familial and sporadic amyotrophic lateral sclerosis and frontotem- poral dementia, or androgen receptor (AR) in spinal and bulbar muscular atrophy, tend to misfold and accumulate into aggregates in neurons. Protein quality control system prevents their aggregation and toxicity by enhancing their degradation via proteasome and/or autophagy. An efficient dynein mediated transport of misfolded proteins to the site of degradation is required as key point to control their aggregation and degradation. HSPB8 is a protective protein that reduces disease associated proteins aggregation by autophagy facilitation. Here we evaluated the HSPB8 effects on the recently discovered RAN translated poly-di-peptides (DPRs) from C9ORF72 gene. Using filter trap and western blot we observed that HSPB8 over-expression facilitates DPRs clearance even when proteasome is blocked. when we blocked the dynein retrograde transport by EHNA we found an alteration of SQSTM1/p62 and LC3 expression and localization. However, dynein inhibition reduced SQSTM1/p62 and LC3 levels induced by trehalose and drastically reduced the number of autophagosome per cell. Moreover, EHNA reduced the PBS insoluble fraction of mutated misfolded proteins and DPRs also when autophagy is blocked. This effect was counteracted by proteasome inhibition. Notably, EHNA selectively increased BAG1 mRNA (responsible for misfolded protein degradation via protea- some) in NSC34 and motoneuron derived from iPS cells, while exogenous BAG1 overexpression reduced misfolded species aggre- gation and BAG1 down-regulation blocked the EHNA effect. Moreover, EHNA increased mRNA and protein levels of chaperone mediated autophagy receptor Lamp2A, suggesting that CMA can restore the degradation of misfolded proteins with KFERQ-like motif that are internalized into lysosome by Lamp2A. Collectively, these data suggest that when autophagy flux is blocked, misfolded proteins can be re-routed by BAG1 to alternative degradative pathways.