Abstract：
Investigating novel mechanisms of neurite outgrowth via cytoskeleton is critical for developing therapeutic strategies against neural disorders. Rab3A is a vesicle-related protein distributed throughout the nervous system, but the detailed mechanism related to cytoskeleton remains largely unknown. Our previous reports show that spastin serves microtubule to regulate neurite outgrowth. Here, we asked whether Rab3A could function via modulating spastin during neuronal development. The results revealed that Rab3A colocalized with spastin in cultured hippocampal neurons. Immunoprecipitation assays showed that Rab3A physically interacted with spastin in rat brain lysates. Rab3A overexpression significantly induced spastin degradation; this effect was reversed by leupeptin- or MG-132- administration, suggesting the lysosomal and ubiquitin-mediated degradation system. Immunofluorescence staining further confirmed that Rab3A and spastin immune-colocalized with the lysosome marker lysotracker. In COS7 cells, Rab3A overexpression significantly downregulated spastin expression and abolished the spastin-mediated microtubule severing. Furthermore, overexpression inhibited while genetic knockdown of Rab3A promoted neurite outgrowth. However, this inhibitory effect on neurite outgrowth in hippocampal neurons could be reversed via co-transfection of spastin, indicating that Rab3A functions via its interaction protein spastin. In general, our data identify an interaction between Rab3A and spastin, and this interaction affects the protein stability of spastin and eliminates its microtubule severing function, thereby modulating neurite outgrowth.
摘要：
研究神经突通过细胞骨架生长的新机制对于开发针对神经疾病的治疗策略至关重要。Rab3A是一种分布在整个神经系统的囊泡相关蛋白，但是与细胞骨架相关的详细机制仍然未知。我们以前的报告表明，spastin服务于微管以调节神经突生长。这里,我们询问Rab3A是否可以通过调节痉挛在神经元发育过程中发挥作用。结果表明，Rab3A与spastin在培养的海马神经元中共定位。免疫沉淀实验表明，Rab3A与大鼠脑裂解物中的spastin发生物理相互作用。Rab3A过表达显着诱导spastin降解；这种作用被leupeptin或MG-132-给药逆转，提示溶酶体和泛素介导的降解系统。免疫荧光染色进一步证实Rab3A和spastin与溶酶体标记物lysotracker免疫共定位。在COS7细胞，Rab3A过表达显着下调spastin表达并消除spastin介导的微管切断。此外，过表达抑制，而Rab3A的遗传敲除促进神经突生长。然而,这种对海马神经元神经突生长的抑制作用可以通过spastin共转染逆转，表明Rab3A通过其相互作用蛋白spastin起作用。总的来说,我们的数据确定了Rab3A和spastin之间的相互作用，这种相互作用会影响spastin的蛋白质稳定性并消除其微管切断功能，从而调节神经突生长。
