Autophagic removal of aggregating dipeptides produced in C9ORF72 related neurodegenerative diseases

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases characterized by partial clinical overlap. Similar pathogenic mechanisms are involved with the contribution of TAR DNA binding protein (TARDBP/TDP-43) and the fused in sarcoma gene (FUS). Recently, the discovery of the causative gene C9ORF72 has clarified the ALS and FTD as a spectrum disorder. The 50% of ALS/FTD familial cases are linked to an expansion of the repeated G4C2 hexanucleotide 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 (haploinsufficiency). In parallel the gain of toxic function may be well described by repeated RNA mediated toxicity that sequesters RNA and protein in RNA foci and impairs nucleocytoplasmic trafficking. Moreover, although the G4C2 expansion is localized in an untranslated region of the C9ORF72 transcript, it drives an unconventional ATG independent translation, known as RAN translation. That leads to the synthesis of five different di-peptide repeat proteins (DPRs), which are not "classical" misfolded proteins. DPRs as misfolded protein may be processed by protein quality control (PQC) system to prevent their aggregation and toxicity by enhancing their degradation via proteasome and/or autophagy. We have developed a stable transfected and inducible SH-SY5Y cell line for each DPR. We compare the biochemical behaviour of the five DPRs. We observe that low expression level of stable transfection is insufficient to reach a detectable protein level in western blot or in filter retardation assay. Nevertheless, 5 days after induction we observe an increase of 4-8% of dead cells in each line. In order to enhance the DPRs protein level, we deeply evaluated the biochemical behaviour of the five DPRs in immortalized motoneurons transiently transfected with DPRs. 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 analysed the role of the small heat shock protein HSPB8, a protective protein that reduces a large variety of classical misfolded aggregation-prone proteins. We observed that HSPB8 overexpression significantly decreased the accumulation of most DPRs insoluble species while HSPB8 silencing increase the level of PBS insoluble DPRs. Thus, the induction of HSPB8 might represent a valid approach to prevent DPR-mediated toxicity and improve motoneuron viability.