The small heat shock protein B8 (HSPB8) removes dipeptides produced in C9ORF72-related neurodegenerative diseases

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases characterized by similar pathogenic mechanisms. These involves the TAR DNA binding protein (TARDBP/TDP-43) and the fused in sarcoma gene (FUS). Recently, the causative gene C9ORF72 has been identified in 50% of ALS/FTD familial cases. Diseases are linked to an expansion of the repeated G4C2 hexanucleotide sequence in C9ORF72 gene. It drives an unconventional ATG independent translation, known as “RAN translation”. Thus, leads to the synthesis of five different di-peptide repeat proteins (DPRs) (named polyGA, polyGP, polyGR, polyPR, polyPA), which are not "classical" misfolded proteins. DPRs, like misfolded proteins, are processed by protein quality control (PQC) system to prevent their aggregation and toxicity. In this work, we analysed the role of heat shock protein B8 (HSPB8), a protective chaperone that reduces the accumulation of a large variety of classical misfolded aggregation-prone proteins via autophagy. We have produced an inducible stably transfected SH-SY5Y cells with low DPRs protein levels and a transiently transfected NSC34 cells with higher DPRs protein levels. We compared the biochemical behavior of DPRs by vitality assays, immunofluorescence, western blot and filter retardation assay. After induction we observed an increase of 4-8% of dead cells in each SH-SY5Y lines. In NSC34 cells, we found that although the DPRs are mainly processed via autophagy, this system is unable to fully clear their aggregated forms, which tend to form PBS insoluble aggregates in basal condition. We observed that HSPB8 overexpression significantly decreased the accumulation of most DPRs insoluble species while HSPB8 silencing increase the level of PBS insoluble DPRs. Collectively these data show that activation of PQC prevents DPRs accumulation. HSPB8 induction might represent a valid approach to counteract DPRs accumulation, to reduce DPR-mediated.