Neurofilaments are synthesized in the nerve cell body and transported bidirectionally along axons by slow axonal transport. Direct observations of neurofilament transport indicate that the slow rate is due to rapid movements interrupted by prolonged pauses. Previous studies suggest that the neurofilament motors include dynein/dynactin and kinesin-1A (KIF5A). Recently, mutations in kinesin-1A have been linked to one of the dominantly inherited forms of hereditary spastic paraplegias (SPG10), suggesting that neurofilament transport may be disrupted in SPG10. To explore this hypothesis, I investigated the effect of an SPG10 point mutant, N256S-kinesin-1A, on neurofilament transport. Previous studies have shown that this mutant has impaired velocity and processivity in vitro. I transfected cultured mouse cortical neurons with GFP-tagged neurofilament protein M, with or without mutant or wild type kinesin-1A, and then analyzed neurofilament transport in axons using live-cell time-lapse imaging after 8-12 days in culture. The N256S mutant decreased anterograde neurofilament transport flux (from 136 to 35 µm/axon/hr) by decreasing anterograde transport frequency (from 3.9 to 1.0 moving filaments/hr) without any statistically significant effect on anterograde transport velocity. Consistent with previous observations from the Brown lab on neurofilament transport in kinesin-1A knockout neurons, retrograde neurofilament transport flux was also decreased (from 111 to 85 µm/axon/hr). This was due to decreased retrograde transport frequency (from 3.1 to 1.9 moving filaments/hr) in spite of a statistically significant increase in retrograde transport velocity (from 0.32 to 0.41 µm/s). I conclude that the N256S-kinesin-1A mutant disrupts bidirectional movement of neurofilament in cultured mouse cortical neurons.
I also studied the interaction between neurofilaments and motors. While there is evidence for an interaction between neurofilaments and dynein/dynactin, there is no such evidence for kinesin-1A. Therefore, I examined the interaction of kinesin-1A and dynein/dynactin (p150 subunit) with neurofilaments using immunoprecipitation with magnetic beads. About 0.1% of the total kinesin-1A and about 0.5% of the total p150 co-immunoprecipitated with neurofilaments from mouse brain homogenate. In addition, about 0.1% of the neurofilament protein co-immunoprecipitated with kinesin-1A, and about 1.0% with p150. To test the specificity of the interaction, I designed a sequential immunoprecipitation approach, in which I first precipitated all the neurofilaments, and then I performed a second immunoprecipitation with the same antibody using the immuno-depleted supernatant from the first immunoprecipitation. I found neurofilament antibody failed to precipitate kinesin-1A and p150 from the neurofilament-depleted supernatant in the second immunoprecipitation, even though both kinesin-1A and p150 were present in abundance. The reciprocal sequential immunoprecipitation experiment, using kinesin-1A or p150 antibody, yielded similar results. I also found that kinesin-1A and p150 also co-purified and co-immunoprecipitated with neurofilaments from mouse and rat spinal cords. Finally, I found that kinesin-1A interacted with each neurofilament subunit in HEK cells. Together, these data suggest a specific interaction between neurofilaments and kinesin-1A and confirm the previously reported interaction between neurofilaments and dynein/dynactin.
In conclusion, I present several lines of evidence suggesting that kinesin-1A is a neurofilament motor. Mutations in kinesin-1A, such as SPG10 mutations, may disrupt neurofilament transport in patients, which may contribute to the etiology of this disease.