Effect of NEK1/C9ORF72 double mutation on DNA damage response in neural stem cells and motoneurons derived from Amyotrophic Lateral Sclerosis patient

Emerging evidence suggest that DNA damage and impairment of DNA damage response (DDR) are implicated in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). C9ORF72 repeat expansion, the main genetic cause of ALS, associates to DDR defects in motoneurons derived from human induced pluripotent stem cells (iPSC). Moreover, mutations in NEK1 gene involved in DDR and maintenance of chromosomal stability, have been found in ALS patients. 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). We aim to study the effect of the double mutation in C9ORF72 and NEK1 genes on DNA damage repair. We reprogrammed primary fibroblasts from the double mutant C9ORF72-NEK1 patient into iPSC, then we characterized and differentiated iPSC into neural stem cells (NSC). We induced DNA damage with the radiomimetic agent Neocarzinostatin in NSC from the double mutant C9ORF72-NEK1, 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. FISH analysis revealed a significant higher number of pathological C9ORF72 RNA foci in the double mutant C9ORF72-NEK1 compared to three different mutant C9ORF72 iPSC lines. 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 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. 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. We are now investigating the DDR in iPSC-motoneurons that represents a more differentiated and post-mitotic neuronal model. Our study aims to better understand the possible interplay between NEK1 and C9ORF72 genes and assess the relevance of DNA damage and DDR as novel and druggable pathomechanisms in ALS.