Effect of Spastic Paraplegia Mutations in KIF5A Kinesin on Transport Activity

Hereditary spastic paraplegia (HSP) is a neurodegenerative disease caused by motoneuron degeneration. It is linked to at least 30 loci, among them SPG10, which causes dominant forms and originates in point mutations in the neuronal Kinesin-1 gene (KIF5A). Here, we investigate the motility of KIF5A and four HSP mutants. All mutations are single amino-acid exchanges and located in kinesin's motor or neck domain. The mutation in the neck (A361V) did not change the gliding properties in vitro, the others either reduced microtubule affinity or gliding velocity or both. In laser-trapping assays, none of the mutants moved more than a few steps along microtubules. Motility assays with mixtures of homodimeric wild-type, homodimeric mutant and heterodimeric wild-type/mutant motors revealed that only one mutant (N256S) reduces the gliding velocity at ratios present in heterozygous patients, whereas the others (K253N, R280C) do not. Attached to quantum dots as artificial cargo, mixtures involving N256S mutants produced slower cargo populations lagging behind in transport, whereas mixtures with the other mutants led to populations of quantum dots that rarely bound to microtubules. These differences indicate that the dominant inheritance of SPG10 is caused by two different mechanisms that both reduce the gross cargo flux, leading to deficient supply of the synapse.