Oxidative stress impairs processive motility of the axonal transport motor KIF1A

The kinesin-3 family member, KIF1A is an essential motor protein that carries out intracellular transport in neurons. Previous work has established that: (1) intracellular transport can be impaired in neurodegenerative diseases such as Alzheimer's and Parkinson's; and (2) oxidative stress is elevated in neurodegenerative diseases and during aging. To date there has not been a systematic study of the effects of reactive oxygen species on kinesin motor proteins. We hypothesized that oxidative stress can damage kinesin, leading to decreased motility. To test our hypothesis, we treated KIF1A in vitro with varying concentrations of hydrogen peroxide (H₂O₂), a common reactive oxygen species, and characterized the impacts on KIF1A function. Pretreatment of KIF1A with H₂O₂ at concentrations of 1 mM and higher decreased motility in microtubule gliding assays. In single-molecule assays KIF1A was impacted in two ways: a fraction of motors moved with slowed velocity, while a fraction of motors moved only diffusively with no net directionality. Nonreducing SDS-PAGE of oxidized kinesin showed higher molecular weight bands, consistent with disulfide-bonded dimers and higher-order species. Treating oxidized motors with reducing agents reversed this cross-linking and partially restored motility. Replacing cysteine residues in the motor domain reduced the effects of moderate oxidation but did not prevent the severe degradation of motility at the highest H₂O₂ concentrations, indicating there is irreversible oxidative damage beyond only cysteine residues. Our results suggest that KIF1A can be impacted by oxidative stress and raise the possibility that oxidized KIF1A may be involved in the pathogenesis of neurodegenerative diseases.