Effect of NEK1/C9ORF72 double mutation on DNA damage response in patient-derived neural stem cells

Emerging evidence suggest that DNA damage and impairment of DNA damage response (DDR) are implicated in the pathogenesis of ALS. C9ORF72 repeat expansion associates to DDR defects in motoneurons from human induced pluripotent stem cells (iPSC) and mutations in NEK1 gene, involved in both DDR and maintenance of chromosomal stability, have been identified in ALS patients. Moreover, increased DNA damage and impaired DDR have been recently described in iPSC-motoneurons from an ALS patient carrying a loss-of-function mutation in NEK1 gene. By mutational analysis we identified an Italian ALS patient carrying a concomitant repeat expansion in C9ORF72 gene and a loss-of-function mutation in NEK1 gene (p.Ser1036Ter). In order to study the effect of the double mutation on DNA damage repair, we obtained primary fibroblasts from the patient then reprogrammed into iPSC. When we characterized the generated double mutant iPSC line, FISH analysis revealed a significant higher number of C9ORF72 RNA foci compared to three different mutant C9ORF72 iPSC lines already available in our laboratory. Since a previous study described no difference in DDR in iPSC from mutant C9ORF72 and healthy controls, we differentiated iPSC into neural stem cells (NSC) to obtain a neural committed cell model in which the DDR could be better investigated. We induced DNA damage with the radiomimetic agent Neocarzinostatin, that causes DNA double-stranded breaks, and compared the DDR in NSC from the double mutant C9ORF72-NEK1, the three different C9ORF72 and two healthy control lines. We quantified γH2A.X histone- and BP53-positive nuclear foci as markers of DNA damage and further divided NSC in four arbitrary categories according to the γH2A.X foci number per cell: (I) 2-5 foci, (II) 5-20 foci, (III) 20-30 foci and (IV) >30 foci. Our results showed that all NSC displayed low and comparable levels of DNA damage in basal condition without significant differences among the experimental groups. After DNA damage induction, we observed a similar increase of both γH2A.X- and BP53-positive nuclear foci in all the analyzed cell lines. DNA damage was rescued in a time-frame between 4 and 8 hours after Neocarzinostatin removal, returning to the basal values at 24 hours, with no significant differences among all the analyzed NSC lines. In conclusion, our preliminary results indicate that, although the C9ORF72-NEK1 iPSC showed increased pathological RNA foci, the induced DNA damage could be efficiently repaired in the highly-mitotic NSC, independently from the presence of C9ORF72 or C9ORF72-NEK1 double mutation. This suggests to further investigate DDR in a more differentiated and post-mitotic neuronal model, such as iPSC-motoneurons, to better understand the possible interplay between NEK1 and C9ORF72 genes. Our study aims to assess the relevance of DNA damage and DDR as novel and druggable pathomechanisms in ALS.