NEK1 haploinsufficiency impairs ciliogenesis in human iPSC-derived motoneurons and brain organoids

Abstract

Primary cilia are microtubule-based organelles acting as specialized signalling antennae that respond to specific stimuli to maintain cellular integrity and homeostasis. Recent studies indicate defective primary cilia in post-mortem human brains and animal models of neurodegenerative conditions, including Amyotrophic Lateral Sclerosis (ALS). Heterozygous loss-of-function mutations (LOF) in NEK1 gene are present in about 1% of familial and sporadic ALS cases. The protein kinase NEK1 regulates various cellular processes, including ciliogenesis, but a clear link between NEK1 LOF mutation in ALS and primary cilia is unknown. In this study we generated a human iPSC line carrying a NEK1 LOF mutation by gene editing, leading to NEK1 protein haploinsufficiency. In differentiated iPSC-motoneurons (MNs) we observed that primary cilia were significantly shorter in NEK1-LOF iPSC-MNs compared to wild-type (WT) iPSC-MNs and that also the percentage of ciliated iPSC-MNs was significantly decreased in NEK1-LOF cells. We also investigated ciliogenesis in NEK1-LOF iPSC-brain organoids confirming that primary cilia were thinner with no apparent alteration in the ultrastructure by transmission electron microscopy. Our data suggest that NEK1 protein plays a role in regulating ciliogenesis in both 2D and 3D human iPSC-derived neuronal models and that NEK1 LOF mutations associated to ALS, leading to NEK1 haploinsufficiency and likely to reduced kinase activity, impair primary cilium formation. The involvement of ciliogenesis dysfunction in ALS deserves further investigation providing novel therapeutic targets and strategies to be addressed for this incurable disease.