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

Cytoskeletal and axonal transport deficits are among the 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 ALS, missense mutations in the N-terminal motor domain are associated with hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth (CMT2) diseases. We identified the novel mutation c.50G>A (p.R17Q) in the ATP-binding motor domain of KIF5A gene in a patient diagnosed with HSP and generated induced-pluripotent stem cells (iPSCs). Aim of our work was to study the effect of this KIF5A mutation functionally in iPSC-motoneurons (iPSC-MNs) to unravel the different pathomechanisms in HSP and ALS. By CRISPR/Cas9 gene editing of the patient-derived mutant KIF5A iPSCs, we generated the isogenic wild-type iPSC line, as well as an iPSC line with a loss-of-function mutation (p.N20Kfs*4) in heterozygous state (KIF5A+/-). The mutant KIF5A iPSCs efficiently differentiated into motoneurons, as shown by the expression of typical motoneuronal markers. However, they displayed axonal swellings, sign of axonal degeneration, and increased levels of neurofilament by Western blot analysis compared to the isogenic wild-type iPSC-MNs. Western blot analysis revealed a similar amount of KIF5A protein in mutant p.R17Q iPSC-MNs compared to the isogenic control, suggesting that the mutation does not cause the protein to aggregate or misfold, while KIF5A+/- iPSC-MNs showed KIF5A protein haploinsufficiency, as expected. Morphological analysis of mitochondria performed using the MitotrackerTM dye revealed increased aspect ratio values along neurites in KIF5A+/- iPSC-MNs and an increased mitochondrial density near the soma of p.R17Q iPSC-MNs compared to the isogenic control. Finally, preliminary live-imaging analysis for mitochondria showed a decrease in mean mitochondrial velocity and mitochondrial distance in both KIF5A+/- and p.R17Q iPSC-MNs compared to the isogenic control. In conclusion, we here report the use of iPSC-MNs derived from an HSP patient carrying a novel mutation in KIF5A N-terminal domain as a valuable disease model to further elucidate the different pathomechanisms associated with KIF5A-related disorders.