The chaperone HSPB8 removes insoluble forms of the C9ORF72 RAN translated dipeptides responsible for ALS/FTD

Introduction: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases characterized similar pathogenic mechanisms. These in- volves the contribution of TAR DNA binding protein (TARDBP/TDP-43) and the fused in sarcoma gene (FUS). Recently, the causative gene C9ORF72 has identi ed in 50% of ALS/ FTD familial cases. Diseases are linked to an expansion of the repeated G4C2 hexanu- cleotide sequence in C9ORF72 gene. A loss-of-function mechanism in C9ORF72 ALS/ FTD is suggested by the reduction of all three C9ORF72 mRNA variants in patient tissue samples. In parallel the gain-of-toxic-function may be well described by repeated RNA me- diated toxicity that sequesters RNA and protein in RNA foci and impairs nucleocytoplasmic traf cking. Although the G4C2 expansion is localized in the 5’ untranslated region of the C9ORF72 transcript, it drives an unconventional ATG independent translation, known as “RAN translation”. Thus leads to the synthesis of ve different di-peptide repeat proteins (DPRs) (named polyGA, polyGP, polyGR, polyPR, polyPA), which are not “classical” mis- folded proteins. DPRs, like misfolded proteins, are processed by protein quality control (PQC) system to prevent their aggregation and toxicity by enhancing their degradation via proteasome and/or autophagy. 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 classi- cal misfolded aggregation-prone proteins via autophagy. Methods: We have developed a stable transfected and inducible SH-SY5Y cells with low DPRs protein levels and a transiently transfected immortalized motoneurons (NSC34 cells) with higher DPRs protein levels for each DPR. We compared the biochemical behaviour of the ve DPRs by vitality assays, immuno uorescence (IF), western blot (WB) and lter retardation assay (FRA). Results: Five days after induction we observed an increase of 4-8% of dead cells in each SH-SY5Y lines. We deeply evaluated the biochemical behaviour of the ve DPRs in NSC34 cell. In these 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 signi cantly de- creased the accumulation of most DPRs insoluble species while HSPB8 silencing increase the level of PBS insoluble DPRs. Moreover, we observe that proteasome degrades polyGP insoluble forms when autophagy is inhibited by an altered retrograde transport. Conclusions: Collectively these data show that activation of PQC prevents DPRs accumu- lation. Particularly, HSPB8 induction might represent a valid approach to counteract DPRs accumulation, to reduce DPR-mediated toxicity and to improve motoneuron viability by PQC activation.