C21ORF2 mutations point towards primary cilia dysfunction in amyotrophic lateral sclerosis.

Progressive loss of motor neurons is the hallmark of the neurodegenerative disease amyotrophic lateral sclerosis (ALS), but the underlying disease mechanisms remain incompletely understood. In this study, we investigate the effects of C21ORF2 mutations, a gene recently linked to ALS, and find that primary cilia are dysfunctional. Human patient-derived mutant C21ORF2 motor neurons have a reduced ciliary frequency and length. We report that C21ORF2 is located at the basal body of the primary cilium, and mutations associated with ALS alter this localization. Furthermore, we show that a reduction of C21ORF2 levels in cell lines and motor neurons is sufficient to cause fewer primary cilia and reduced cilial length. This ciliary dysfunction leads to defective downstream sonic hedgehog signalling and reduces the expression of cellular retinoic acid binding protein 1 (CRABP1), a protein involved in motor neuron maintenance and survival. In a compartmentalized co-culture system of motor neurons and muscle cells, these ciliary defects were associated with a reduced ability of neuromuscular junction formation. Interestingly, these cilia defects are seemingly not restricted to C21ORF2 ALS, as we also observed perturbed primary cilia in cultured motor neurons and post-mortem motor cortex from patients with the most common genetic subtype of ALS caused by repeat expansions in the C9ORF72 gene. Finally, overexpression of C21ORF2 in mutant C21ORF2 motor neurons rescued the ciliary frequency and length, CRAPBP1 expression and neuromuscular junction formation, confirming the importance of primary cilia for motor neuron function. These results point towards primary cilia dysfunction contributing to motor neuron degeneration in ALS and open new avenues for further research and interventions for this as yet untreatable disease.