Autophagy mediated HSPB8 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. Recently, the discovery of the causative gene C9ORF72 has linked the 50% of ALS/FTD familial cases to an expansion of the repeated GGGGCC sequence in C9ORF72 gene. C9ORF72 haploinsufficiency may represent a loss-of-function mechanism. In parallel, a gain-of-function mechanism is suggested by interaction of repeated RNA sequence that sequesters RNA and protein in RNA foci and impairs nucleocytoplasmic trafficking. Moreover, GGGGCC expansion drives an unconventional ATG independent translation, known as RAN translation, that leads to the synthesis of five biochemical different di-peptide repeat proteins (DPRs) that despite their lack of function, can be considered as unconventional 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. To enhance DPRs protein level, we deeply evaluated biochemical behaviour of the five DPRs in immortalized motoneurons transfected with DPRs. By immunofluorescence, western blot and filter retardation assay we found that in NSC34 cells, although 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 8 (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 despite they are unconventional misfolded proteins. Furthermore, HSPB8 silencing increases the level of PBS insoluble DPRs. Thus, the induction of HSPB8 might represent a valid approach to prevent DPR-mediated accumulation and toxicity in order to improve motoneuron viability.