Unraveling the role of NEK1 on primary cilia using mutant iPSC models

Dysfunctions in primary cilia, non-motile cytoplasmatic organelles acting as a sensory-hub in post-mitotic cells, are emerging as a new pathomechanism in amyotrophic lateral sclerosis (ALS). Decreased cilia number and length are found in C9ORF72 patient’s post-mortem motor cortex and in spinal motoneurons differentiated from mutant iPSCs (iPSC-MNs). The two genes NEK1 and C21ORF2 are involved in ciliogenesis and cause human ciliopathies with a recessive mode of inheritance, while they are associated to ALS risk when present in heterozygous state. While the impact of ALS-associated C21ORF2 heterozygous variants on primary cilia has recently been investigated, the role of NEK1 variants remains to be further elucidated. In this work, we are using human iPSC models differentiated into motoneurons to unravel the role of mutant NEK1 gene on primary cilia in ALS pathogenesis. We firstly generated a NEK1 loss-of-function (LOF) iPSC model by introducing a heterozygous frameshift mutation in a wild-type (WT) healthy control line by CRISPR/Cas9 genome editing. In differentiated iPSC-MNs, image analysis of primary cilia by immunofluorescence staining for the adenylate cyclase III marker showed a significant decrease both in the percentage of cilia-positive cells and in cilium length in the NEK1 LOF cells, compared to the isogenic control iPSC-MNs. We next investigated two iPSC lines carrying two distinct nonsense mutations found in ALS patients and mapping in the central coiled-coil domain and in the C-terminal acidic domain, respectively, and which were inserted in a WT iPSC line by CRISPR/Cas9 genome editing. Immunofluorescence staining for the Arl13b cilium marker showed a decrease in the percentage of cilia-positive iPSC-MNs in both mutant cell lines compared to the isogenic WT one. Our data suggest that ALS-associated NEK1 LOF mutations have an impact on primary cilia formation. Future perspectives include the study of cilia functionality and of the mechanistic link between NEK1 haploinsufficiency and cilia alterations, in order to better define the role of these organelles in ALS pathogenesis.