BRIEF RESEARCH REPORT article
Front. Genet.
Sec. Stem Cell Research
Functional validation of the novel KIF5A p.R17Q VUS reveals defective axonal transport in iPSC-motoneurons from a SPG10 patient
Serena Santangelo 1
Valeria Casiraghi 2
Claudia Fallini 3
Sabrina Invernizzi 2
Silvia Peverelli 1
Martina Bertocchi 2
Monica Feole 4
Marta Cozzi 2
Stefania Magri 5
Angelo Poletti 2
Patrizia Bossolasco 1
Franco Taroni 5
Vincenzo Silani 2,1
Antonia Ratti 2,1
1. Istituto Auxologico Italiano Istituto di Ricovero e Cura a Carattere Scientifico, Milan, Italy
2. University of Milan, Milan, Italy
3. University of Rhode Island, Kingston, United States
4. Cedars-Sinai Medical Center, Los Angeles, United States
5. Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy
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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.
Summary
Keywords
IPSC, KIF5A, Motor Neurons, SPG10, VUS
Received
23 December 2025
Accepted
18 February 2026
Copyright
© 2026 Santangelo, Casiraghi, Fallini, Invernizzi, Peverelli, Bertocchi, Feole, Cozzi, Magri, Poletti, Bossolasco, Taroni, Silani and Ratti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Antonia Ratti
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