Heterogeneous splicing patterns resulting from KIF5A variants associated with amyotrophic lateral sclerosis.

Single nucleotide variants (SNVs) in the gene encoding Kinesin Family Member 5A (KIF5A), a neuronal motor protein involved in anterograde transport along microtubules, have been associated with amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive and fatal neurodegenerative disease that primarily affects motor neurons. Numerous ALS-associated KIF5A SNVs are clustered near splice site junctions of the penultimate exon 27 and are predicted to alter the carboxy-terminal (C-term) cargo-binding domain of KIF5A. Mis-splicing of exon 27, resulting in exon exclusion, is proposed to be the mechanism by which these SNVs cause ALS. Whether all SNVs proximal to exon 27 result in exon exclusion is unclear. To address this question, we designed an in vitro minigene splicing assay in HEK293 cells which revealed heterogeneous site-specific effects on splicing: only 5' splice site (5'ss) SNVs resulted in exon skipping. We also quantified splicing in select CRISPR-edited human stem cells differentiated to motor neurons and in neuronal tissues from a 5'ss SNV knock-in mouse, which showed the same result. Moreover, survival of representative 3' splice site, 5'ss, and truncated C-term variant KIF5A (v-KIF5A) motor neurons was severely reduced compared to wildtype motor neurons, and overt morphological changes were apparent. While total KIF5A mRNA levels were comparable across cell lines, total KIF5A protein levels were decreased for v-KIF5A lines, suggesting an impairment of protein synthesis or stability. Thus, despite the heterogeneous effect on RNA splicing, KIF5A SNVs similarly reduce the availability of the KIF5A protein, leading to axonal transport defects and motor neuron pathology.