Impact of Charcot-Marie-Tooth Type 2B Disease-Associated Rab7 Mutations on Signaling and Axonal Trafficking of NGF/TrkA

Abstract

Charcot-Marie-Tooth type 2B (CMT-2B) is a neurodegenerative disease characterized by terminal axonal death. Genetic analysis from human CMT-2B patients revealed four missense point mutations (L129F, K157N, N161T, V162M) in their genes encoding a small GTPase Rab7, a marker for late endosomes in the degradation pathway. The exact mechanism of how Rab7 mutants cause CMT-2B remains poorly understood. Here, we analyzed the effect of Rab7 mutants on the signaling and axonal transport of a nerve growth factor (NGF) receptor - TrkA. Fluorescent protein-engineered Rab7 and TrkA were transfected in rat embryonic dorsal root ganglia neuronal cells. Axonal transport of Rab7- and TrkA-containing endosomes was followed by time-stamped live cell fluorescence microscopy. We found that TrkA moves roughly twice as fast as Rab7s, among which CMT-2B associated Rab7 mutants outpace wt-Rab7. Curiously, endosomes co-transfected with both Rab7 and TrkA move even slower than those with singly transfected Rab7. Western blot analysis from Rab7/TrkA-cotransfected PC12 cells showed that the level of phosphorylated TrkA is lower in Rab7 mutants that that in wt-Rab7. Our results suggested that Rab7 mutants can potentially contribute to CMT-2B by dis-regulating NGF-TrkA signaling via perturbing the balance between retrograde and anterograde axonal transport processes. These results imply that axonal transport can be a potential treatment target for CMT-2B neurodegeneration.