NEK1 loss-of-function mutation impairs ciliogenesis in iPSC-motoneurons

Mutations in NEK1 gene account for nearly 3% of familial and sporadic ALS cases. The survival time of heterozygous loss-of-function (LOF) mutation carriers is significantly shorter than that of missense variant carriers. NEK1 codes for a protein kinase involved in several biological pathways, including DNA damage repair, mitochondrial functionality and ciliogenesis. However, the pathomechanisms whereby NEK1 LOF leads to neurodegeneration in ALS have not been clarified so far. Aim of our study was to investigate the biological effects of NEK1 LOF mutations using motoneurons derived from human induced pluripotent stem cells (iPSC-MN). Primary fibroblasts from a healthy control were reprogrammed into iPSC and a NEK1 LOF mutation (p.Arg261Profs19) was introduced through CRISPR/Cas9 gene editing, leading to NEK1 protein haploinsufficiency. The DNA damage response was evaluated after exposing iPSC-MN to the radiomimetic agent Neocarzinostatin, that causes DNA double-stranded breaks. NEK1 LOF iPSC-MN showed a similar capacity to repair DNA breaks as control cells, but they exhibited abnormalities in mitochondrial structure and biogenesis. Our findings also revealed that a decreased number of NEK1 LOF iPSC-MN formed cilia, which were also significantly shorter, compared to control iPSC-MN. We also generated 3D brain organoids to evaluate whether impairment of cilia formation and mitochondria organization is also confirmed or influenced by non-cell autonomous mechanisms. Altogether our results support that NEK1 serves as a hub signalling kinase for mitochondrial functionality and cell fate determination via cilia organelles. The involvement of ciliogenesis dysfunction in ALS might also provide novel targets and therapeutic strategies to be further tested in iPSC-derived 2D and 3D disease models.