Nogo-A inhibits necdin-accelerated neurite outgrowth by retaining necdin in the cytoplasm

Nogo-A has been identified in the central nervous system as an inhibitor for axonal regeneration. Previous works have mainly focused on Nogo-A in oligodendrocytes and the roles of neuronal intracellular Nogo-A remain elusive. To gain deep insight into the physiological functions of Nogo-A, a yeast two-hybrid screening was performed with Nogo-66 as bait. We identified a new interaction between Nogo-66 and necdin. Mutagenesis analysis revealed that the central region of necdin was indispensable for the interaction of necdin with Nogo-66. The interaction was further confirmed by co-immunoprecipitation in neural tissues and cultured cortical neurons. Morphological evidence showed that Nogo-A and necdin highly colocalized in rat cortical and dorsal root ganglia neurons. Ectopic expression of Nogo-A in HEK293 cells led to retention of necdin from the nucleus to the cytoplasm. Furthermore, overexpression of Nogo-A in PC12 cells and cultured cortical neurons inhibited necdin-accelerated neurite outgrowth. Meanwhile, necdin was found to be significantly sequestered in the cytoplasm of PC12 cells stably overexpressing Nogo-A. Together, these data suggest that Nogo-A is a novel necdin binding protein and inhibits necdin-accelerated neuronal neurite outgrowth by sequestering necdin in the cytoplasm.