Functional validation of the novel KIF5A p.R17Q VUS reveals defective axonal transport in iPSC-motoneurons from a SPG10 patient.
Publication Year:
2026
PubMed ID:
41836058
Funding Grants:
Public Summary:
Inside our brain cells, a tiny "motor protein" called KIF5A acts like a delivery truck, traveling down long nerve pathways to transport vital cargo like energy-producing mitochondria and waste-clearing lysosomes. In this study, scientists investigated a patient with a rare genetic mutation in this protein—labeled a "variant of unknown significance" because doctors weren't sure if it actually caused their neurodegenerative disease, Spastic Paraplegia 10 (SPG10). To test it, researchers grew the patient's nerve cells in a lab using stem cells, and used CRISPR gene-editing to create an identical, healthy version of the cells for comparison. They discovered that the mutation severely damaged the cell's delivery system: the motor proteins got stuck at the far ends of the nerves, causing traffic jams and structural swelling, which drastically slowed down the travel distance and speed of vital cell cargo. By proving exactly how this mutation breaks down axonal transport, the study officially confirms this genetic variant is a dangerous cause of disease, while showing that stem-cell models are a powerful tool for diagnosing mystery genetic mutations in patients.
Scientific Abstract:
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.