Effect of the C9ORF72-NEK1 dobuble mutation on DNA damage response in patient-derived iPSC-motoneurons

Background Emerging evidence suggest that DNA damage and impairment of DNA damage response (DDR) are implicated in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). DDR defects have been described in motoneurons derived from induced pluripotent stem cells (iPSC) of ALS patients carrying C9ORF72 repeat expansions or NEK1 gene mutations. By mutational analysis we have identified an Italian ALS patient carrying a concomitant repeat expansion in C9ORF72 gene and a loss-of-function mutation in NEK1 gene (p.Ser1036Ter). Objectives Aim of our study was to evaluate the effect of the double mutation in C9ORF72 and NEK1 genes on DDR in patient-derived cells. Methods Primary fibroblasts from the double mutant C9ORF72-NEK1 patient were reprogrammed into iPSC, which were then differentiated into neural stem cells (NSC) and motoneurons (iPSC-MN). FISH analysis was conducted to evaluate RNA foci. DNA damage was induced with the radiomimetic agent Neocarzinostatin that induces DNA double stranded breaks and DDR was evaluated by quantitative image analysis of γH2A.X histone- and BP53-positive nuclear foci, used as markers of DNA damage. Results The double mutant C9ORF72-NEK1 iPSC revealed a significant higher number of C9ORF72 RNA foci compared to three different mutant C9ORF72 iPSC lines already available in our lab. Since a previous study described no difference in DDR in iPSC from mutant C9ORF72 compared to healthy controls, we obtained NSC as a more neural-committed cell model where the DDR could be better investigated. After Neocarzinostatin treatment in NSC from the double mutant C9ORF72-NEK1, three different C9ORF72 and two healthy control lines, we found similar amounts of γH2A.X and BP53 nuclear foci, independently from the presence of C9ORF72 or C9ORF72-NEK1 double mutation. To evaluate DDR in a more differentiated and post-mitotic neuronal model, we first quantified γH2A.X and BP53 nuclear foci in patients’ iPSC-MN in basal condition, which showed low and comparable amounts, with no significant differences among the experimental groups. After inducing DNA damage, we observed a similar increase of γH2A.X and BP53 foci number in all the analyzed cell lines. DNA damage was rescued in a time-frame between 6 and 10 hours after Neocarzinostatin removal, returning to the basal values after 24 hours, with no significant differences among all the analyzed iPSC-MN groups. Discussion Our data indicate that C9ORF72 and C9ORF72-NEK1 iPSC-MN show a similar capacity to repair DNA damage with no apparent biological impact of the NEK loss-of-function mutation on DDR. However, we do not exclude an age-dependent effect of DDR in iPSC-MN at different days of MN differentiation. A better understanding of the possible interplay between NEK1 and C9ORF72 genes will help assess the relevance of DNA damage and DDR as novel and druggable pathomechanisms in ALS.