✍🏼 Megan G Pino , Kelly A Rich , Nicholas J Hall , Meredith L Jones , Ashley Fox , Karin Musier-Forsyth , Stephen J Kolb

🏠 Department of Neurology, The Ohio State University Wexner Medical Center, Columbus, Ohio, USA

📑 Human Molecular Genetics (2023)

Abstract
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 the motor neurons. Numerous ALS-associated KIF5A SNVs are clustered near the 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 human embryonic kidney 293 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 clustered, regularly interspaced, short palindromic repeats-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, the survival of representative 3′ splice site, 5’ss, and truncated C-term variant KIF5A (v-KIF5A) motor neurons was severely reduced compared with wild-type motor neurons, and overt morphological changes were apparent. While the total KIF5A mRNA levels were comparable across the cell lines, the total KIF5A protein levels were decreased for v-KIF5A lines, suggesting an impairment of protein synthesis or stability. Thus, despite the heterogeneous effect on ribonucleic acid splicing, KIF5A SNVs similarly reduce the availability of the KIF5A protein, leading to axonal transport defects and motor neuron pathology.

The authors used the WOLF microfluidic cell sorter to isolate single cells for downstream molecular analysis. Following dissociation of cultures into single-cell suspensions and preparation for fluorescence-based detection, the WOLF sorter was employed to perform single-cell sorting of fluorescently labeled cells into individual wells of 96-well plates, enabling isolation of targeted cell populations while preserving viability and RNA quality. The authors highlight that sorting was conducted at low pressure (<2 psi) to maintain cell integrity and improve RNA preservation—important for sensitive gene expression and splicing assays in induced pluripotent stem cell (iPSC) models of ALS. This allowed subsequent generation of high-quality single-cell data for analyzing KIF5A variant effects on splicing heterogeneity across individual neurons and glia.