ZnT3-ACh-AMPK-mTOR信号通路对脊髓损伤后自噬的调节及机制的研究

AMPK-mTOR signaling pathway plays an important role in autophagy after spinal cord injury (SCI). Zinc transporter protein 3 (ZnT3) can regulate the release of ACh of nerve presynaptic membrane, which promotes the recovery of neurological function. Previous studies have found that the ZnT3 and local ACh expression levels were significantly increased, the expression levels of ZnT3 and autophagy were positive correlativity, on the contrary, the expression of ZnT3 and mTOR protein was negative correlativity after SCI. Therefore, we have supposed that ZnT3 may induced autophagy by ACh-AMPK-mTOR signaling pathway. In our study, we will establish SCI model by utilizing ZnT3 knockout mouse, detect the recovery of their locomotor and nerve function. Besides, we will observe morphological changes of the cells and synapse after culturing spinal cord primary neuronal cells. Furthermore, we will observe the ultrastructure, proliferation and energy metabolism in the spinal motor neuron cells after ZnT3 gene silencing or overexpression, and detect the activity of autophagy after injury. Finally, our study will illuminate the specific molecular mechanisms on regulation of autophagy with ZnT3-ACh-AMPK-mTOR signaling pathway after SCI. The study will provide new therapeutic targets and scheme for SCI, and offer a strong basis of theoretical and experimental foundation for fundamental research of SCI.

AMPK-mTOR信号通路在脊髓损伤(SCI)后自噬中发挥重要作用，而锌转运蛋白3（ZnT3）可调节神经突触前膜释放ACh促进神经功能恢复。前期研究发现：SCI后ZnT3表达及局部ACh含量均明显增高，ZnT3与自噬水平呈正相关关系，而与mTOR表达呈负相关关系。因此我们提出：ZnT3可能通过ACh-AMPK-mTOR诱导SCI后自噬发生。本项目将利用ZnT3基因敲除小鼠构建SCI模型，检测此类小鼠SCI后运动神经功能的恢复情况；并培养其原代神经元细胞，观察研究损伤处理后的细胞及突触形态变化；探讨基因沉默/过表达对脊髓运动神经元细胞株损伤后超微结构、增殖及能量代谢的影响，检测自噬相关蛋白表达的变化；阐明SCI后调控自噬的ZnT3-ACh-AMPK-mTOR信号通路的具体分子机制。本项目的顺利进行将为SCI提供新的治疗靶点及方案，为SCI的基础研究提供有力的理论学依据和实验学基础。

脊髓损伤(spinal cord injury)是指由于外界直接或间接因素导致脊髓损伤，在损害的相应节段出现各种运动、感觉和括约肌功能障碍，肌张力异常及病理反射等的相应改变。脊髓损伤的程度和临床表现取决于原发性损伤的部位和性质。本研究团队分别研究锌处理后对于锌转运体的调控作用，以及对于脊髓损伤功能恢复、治疗影响及其作用机制进行了深入研究。本课题利用脊髓打击损伤模型，随机分为假手术组，溶剂组，给锌组，研究锌干预对脊髓损伤动物行为学影响，利用Western blot技术检测锌干预对锌转运体3以及自噬的表达影响，以及AMPK-mTOR信号转导通路的变化，初步探寻作用机制。利用尼氏染色和免疫荧光观察脊髓前角运动神经元形态；电镜检测锌干预对脊髓损伤神经元的自噬小体的变化；利用蛋白凝胶电泳、免疫荧光以及PCR等技术检测AMPK-mTOR信号转导通路的变化。结果显示：给与锌离子治疗后，能够诱导Znt3的表达。再此基础上，自噬特异性蛋白表达增强，锌治疗组小鼠脊髓组织自噬小体数量增多，自噬流增强。除此之外，我们的结果还提示，锌离子还可以通过自噬发挥对泛素化修饰的影响，以及通过发挥抗氧化应激的作用抑制炎性小体NLRP3的表达。因此提示，适量锌可能参与脊髓损伤修复。结论：锌处理能显著降低小鼠脊髓的继发性损伤，促进运动功能的恢复，其机制可能与促进自噬的高表达，增强泛素化水平，抑制焦亡小体活性有关。其作用机制可能成为脊髓损伤治疗的新途径。