Defective primary cilia in NEK1 and C9ORF72 iPSC-derived motoneurons and brain organoids

Dysfunctions in primary cilia, non-motile cytoplasmatic organelles acting as a sensory-hub in post-mitotic cells, are recently emerging as a possible pathomechanism in amyotrophic lateral sclerosis (ALS). SOD1G93A mutant mice show decreased cilia number in the spinal cord, C9ORF72 knock-down causes longer cilia formation and the two minor ALS genes NEK1 and C21ORF2 cause human ciliopathies in a recessive condition. Aim of this work was to investigate the role of primary cilia in the pathogenesis of ALS, in particular related to mutations in NEK1 and C9ORF72 genes, using motoneurons differentiated from human induced-pluripotent stem cells (iPSC-MNs) and iPSC-brain organoids. From two mutant ALS patients we generated an iPSC line carrying the C9ORF72 hexanucleotide repeat expansion (HRE) and a line carrying both a non-sense mutation in NEK1 and a concurrent C9ORF72 HRE. By CRISPR/Cas9 genome editing we introduced a heterozygous NEK1 loss-of-function (LOF) mutation in a wild-type (WT) healthy control line. . 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 the cilium length in the NEK1 LOF, C9ORF72 single mutant and C9ORF72/NEK1 double mutant iPSC-MNs compared to the WT controls, with no differences among the three distinct mutant lines. Moreover, NEK1 LOF iPSC-brain organoids at day 60 showed thinner cilia compared to the WT control analyzed by transmission electron microscopy. The mechanistic link between NEK1 haploinsufficiency, C9ORF72 HRE and alterations in primary cilia formation remains to be further elucidated in order to define the role of these organelles in ALS pathogenesis.