Data Sheet 1_Functional validation of the novel KIF5A p.R17Q VUS reveals defective axonal transport in iPSC-motoneurons from a SPG10 patient.pdf

Cytoskeletal alterations and axonal transport deficits are key factors in many neurodegenerative disorders. The neuronal kinesin family member 5A (KIF5A) is a microtubule-based motor protein critical for anterograde transport of RNA granules, organelles, and neurofilaments along axons and dendrites. Heterozygous missense and nonsense mutations in the N-terminal motor and stalk domains are associated with hereditary spastic paraplegia 10 (SPG10) and Charcot-Marie-Tooth disease type 2 (CMT2), while frameshift mutations in KIF5A C-terminal cargo-binding domain are linked to amyotrophic lateral sclerosis (ALS). We recently reprogrammed an iPSC line from a SPG10 patient carrying the novel missense variant c.50G>A (p.R17Q) in the KIF5A motor domain, classified as variant of unknown significance (VUS) and predicted to affect ATP binding. Here we gene-edited this mutant iPSC line by CRISPR-Cas9 to obtain an isogenic wild-type (WT) KIF5A cell line. We next examined functionally the impact of the p.R17Q VUS on KIF5A protein sub-cellular distribution and on axonal transport of mitochondria and lysosomes in differentiated iPSC-motoneurons (MNs). The presence of neurofilament-positive axonal swellings and an increased distribution of KIF5A protein in distal neurites was observed in the mutant p.R17Q compared to the WT KIF5A iPSC-MNs, indicating a likely defective axonal transport. The anterograde velocity and distance travelled by mitochondria and lysosomes along neurites was indeed significantly reduced in the mutant KIF5A iPSC-MNs compared to the WT ones. These findings demonstrate that the p.R17Q VUS is pathogenic, thereby extending the spectrum of KIF5A mutations causing SPG10 and support the use of patient-derived iPSC-MNs to functionally validate KIF5A-associated VUS.