TROY Interacts with Rho Guanine Nucleotide Dissociation Inhibitor α (RhoGDIα) to Mediate Nogo-induced Inhibition of Neurite Outgrowth

Background: TROY, together with the Nogo receptor, mediates Nogo-66 induced neurite outgrowth inhibition. Results: TROY binds to RhoGDI to activate RhoA and inhibit neurite outgrowth after Nogo-66 stimulation in cerebellar granule neurons. Conclusion: TROY/RhoGDI interaction transduces the inhibitory effects of Nogo-66 on neurite extension by activating RhoA. Significance: TROY/RhoGDI interaction plays a key role in RhoA activation and neurite outgrowth inhibition by Nogo-66. TROY can functionally substitute p75 to comprise the Nogo receptor complex, which transduces the inhibitory signal of myelin-associated inhibitory factors on axon regeneration following CNS injury. The inhibition of neurite extension relies on TROY-dependent RhoA activation, but how TROY activates RhoA remains unclear. Here, we firstly identified Rho guanine nucleotide dissociation inhibitor (RhoGDI) as a binding partner of TROY using GST pull-down combined with two-dimensional gel electrophoresis and mass spectra analysis. The interaction was further confirmed by coimmunoprecipitation in vitro and in vivo. Deletion mutagenesis revealed that two regions of the TROY intracellular domain (amino acids 234-256 and 321-350) were essential for the interaction with RhoGDI. Secondly, TROY and RhoGDI were coexpressed in postnatal dorsal root ganglion neurons, cortex neurons, and cerebellar granule neurons (CGNs). Thirdly, TROY/RhoGDI association was potentiated by Nogo-66 and was independent of p75/RhoGDI interaction. Fourthly, TROY/RhoGDI interaction was still able to activate RhoA when p75 was deficient. Furthermore, RhoA activation was decreased dramatically when TROY was knocked down in p75-deficient CGNs cells. Finally, RhoGDI overexpression abolished RhoA activation and following neurite outgrowth inhibition by Nogo-66 in both wild-type and p75-deficient CGNs. These results showed that the association of RhoGDI with TROY contributed to TROY-dependent RhoA activation and neurite outgrowth inhibition after Nogo-66 stimulation.