EPIGENETIC MARKS AND PATHOLOGICAL FEATURES ASSOCIATED TO MUTANT C9ORF72 GENE IN AMYOTROPHIC LATERAL SCLEROSIS: AN IN VITRO STUDY IN PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS AND MOTOR NEURONS

The expansion of the hexanucleotide repeat sequence GGGGCC (>30 repeats) in the first intron of C9ORF72 gene is the main genetic cause of two neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The 5’ promoter of C9ORF72 gene has been found hypermethylated in 30% of C9ORF72 positive (C9+) ALS/FTLD patients and never in unexpanded patients and healthy controls. Promoter methylation seems to have a neuroprotective role from RNA toxicity and RAN translated dipeptide repeats (DPRs). The aim of this study has been to characterize C9+ ALS patient-derived induced pluripotent stem cells (iPSC) and differentiated motor neurons (iPSC-MN) correlating epigenetic marks of gene promoter and of the GC-rich repeat expansion (HRE) to C9ORF72-related pathological features (gene expression, RNA foci and DPRs). We initially studied 3 different C9+ iPSC lines which were cultured for up to 40 passages in vitro and, at each timepoint (10th, 20th, 30th and 40th passage), DNA was extracted for genetic and epigenetic characterization, RNA was obtained for Q-PCR analyses and slides were fixed for C9ORF72 RNA foci count. C9+ iPSCs were also differentiated into motor neurons for three times and iPSC-MNs were harvested for the same molecular characterization as for iPSCs. We observed a down-regulation of the two HRE-harboring mRNA isoforms (V1 and V3) both in iPSCs and iPSC-MNs when the promoter was methylated, while RNA foci number showed no correlation with methylation state. Moreover, we found that the epigenetic pattern of promoter methylation could change after C9+ iPSC reprogramming and through differentiation into iPSC-MNs. When we extended our analysis to a cohort of 8 different C9+ iPSC lines, we observed that both epigenetic marks and HRE length may influence RNA foci formation. Our study reports for the first time in C9+ iPSC and iPSC-MNs that promoter methylation can be considered a possible therapeutic target and corroborates that patient-derived cells represent a suitable model for further studies on C9ORF72-related neurodegeneration.