Modeling a novel N-terminal mutation of KIF5A gene in patient-derived iPSC-motoneurons

Cytoskeletal and axonal transport deficits are among the primary pathways affected in several neurodegenerative disorders. The Kinesin family member 5A (KIF5A) belongs to a superfamily of microtubule motor proteins involved in the anterograde transport of synaptic vesicles, RNA granules, mitochondria and neurofilaments along dendrites and axons. Interestingly, while loss-of-function KIF5A gene mutations in the C-terminal cargo-binding domain are associated with Amyotrophic Lateral Sclerosis (ALS), missense mutations in the N-terminal motor domain are associated with hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth (CMT2) diseases. We recently identified the novel mutation c.50G>A (p.R17Q) in the ATP-binding motor domain of KIF5A gene in a patient diagnosed with HSP. Aim of our work was to model this novel mutation in iPSC-derived motoneurons to unravel the different pathomechanisms associated to KIF5A gene mutations in HSP and ALS. We reprogrammed primary fibroblasts from the KIF5A-mutated HSP patient into iPSC by Sendai virus kit. The obtained iPSC were fully characterized for the expression of stemness markers and for their pluripotency to spontaneously differentiate into the three germ layers. Karyotype analysis revealed no gross rearrangements during iPSC reprogramming. CRISPR/Cas9 gene editing was used to generate the isogenic wild-type KIF5A iPSC line, as well as an iPSC line with a loss-of-function mutation (p.Asn20Lysfs*4) in heterozygous state (KIF5A+/-). Whole exome sequencing excluded off-target variants upon gene-editing. The mutant KIF5A iPSCs efficiently differentiated into motoneurons, similarly to the isogenic wild type cells, as shown by the expression of typical motoneuronal markers. Western blot analysis revealed a similar amount of KIF5A protein in mutant p.R17Q iPSC-motoneurons compared to the isogenic control, suggesting that the mutation does not cause the protein to aggregate or misfold. As expected, KIF5A+/- iPSC-motoneurons showed half amount of KIF5A protein. We observed the appearance of axonal swellings in mutant KIF5A iPSC-motoneurons and our current effort is aimed to assess if the KIF5A p.R17Q mutation has any impact on mitochondrial and RNA-binding proteins axonal transport rate by live-cell imaging. In conclusion, we here report of the generation of iPSC-motoneurons from an HSP patient carrying a novel mutation in KIF5A N-terminal domain that will be a valuable disease model to further elucidate the different pathomechanisms associated with KIF5A-related disorders. (Supported by grant RF-2018-12367768 to FT, VS and AR).