Snk-SPAR通路介导微波辐射后树突棘可塑性异常的机制研究

The mechanisms for learning and memory deficits induced by microwave radiation remain undetermined. Studies suggested that plasticity of dendritic spines is intimately related to learning and memory ability and many central nervous system diseases in which Snk-SPAR pathway critically adjusted the synaptic reorganization. In the previous study, it was found that microwave radiation could decrease the density of dendritic spines and the length of dendrites per neuron and influence the morphology of dendritic spines in hippocampus. Accordingly, we assumed that Snk-SPAR pathway may be involved in the process of the abnormalities of dendritic spines morphology and function induced by microwave exposure. In this study, taking Snk-SPAR pathway as a target, we aimed to explore the mechanisms of microwave exposure-induced dendritic spines plasticity abnormality. Firstly, we planned to establish the animal and hippocampal neuron models, and evaluate the.effects of microwave on the expression and phosphorylation of Snk and SPAR, ubiquitination of SPAR. Secondly, in the hippocampal neuron model, we planned to assess the effects of the radiation on upstream molecules of Snk-SPAR pathway (CaN、CIB、Cdk5、RasGRF1、SynGAP and PDZGEF1) and ubiquitin-proteasome pathway (SCFβ-TRCP). Then RNA interference, gene transfection methods and inhibitors of Snk, CaN, Cdk5 molecules and Ubiquitin-proteasome would be applied to interfere in the Snk-SPAR pathway to further study the role of Snk-SPAR pathway in the process. To conclude, the purpose of this study was to clarify the role of Snk-SPAR pathway in the learning and memory function deficits induced by the microwave radiation and provide new candidate targets for prevention and treatment of brain injury by microwave radiation.

微波辐射致学习记忆障碍的机制尚不明确。树突棘可塑性与学习记忆功能密切相关，其中Snk-SPAR 通路在活化依赖性突触重构中起关键作用。研究表明，微波辐射可致海马神经元树突棘密度降低，形状改变，树突片段长度减少，因此推测Snk-SPAR 通路在微波辐射所致的树突棘形态和功能异常中发挥重要作用。本项目拟以Snk-SPAR 通路为切入点，探讨微波辐射致树突棘可塑性异常的机制。建立微波辐射致树突棘可塑性异常的动物和细胞模型，研究微波辐射对该通路中Snk、SPAR的表达及其磷酸化、SPAR泛素化降解的影响；细胞水平进一步研究微波辐射对Snk-SPAR通路上游分子CaN、CIB及Cdk5等的影响；给予关键分子拮抗剂、RNA干扰和基因转染等干预，研究微波辐射通过Snk-SPAR 通路影响树突棘形态和功能的机制。为阐明微波辐射致学习记忆障碍的机制奠定基础，并为微波辐射脑损伤的防治提供新的候选靶点。

中枢神经系统是微波辐射的敏感靶部位之一。海马是学习记忆的重要部位，突触可塑性是学习记忆的神经基础。SNK-SPAR途径在神经元活化诱导的突触重构中发挥关键作用。本研究采用Wistar大鼠和原代海马神经元，建立微波辐射后树突棘可塑性异常的动物和细胞模型。在动物水平，通过对SNK-SPAR途径蛋白的表达、磷酸化以及相互作用的检测，研究微波辐射对SNK-SPAR途径的影响；在细胞水平，检测微波辐射后该途径及其上游分子的改变，针对改变的环节，给予抑制剂，明确该途径在微波辐射致大鼠海马神经元可塑性异常中的作用；最后，通过树突棘结构和功能的验证，阐明SNK-SPAR途径介导微波辐射后树突棘可塑性异常的机制。结果表明，在动物水平：大鼠空间学习记忆能力降低；海马神经元树突棘微刺内吞，突触后致密物厚度和长度减少，齿状回颗粒细胞树突棘密度减少，CA区锥体细胞成熟树突棘比例降低；SNK上调促使SPAR的降解和PSD-95丢失，使树突棘成熟受阻，突触连接弱化，大鼠学习记忆能力下降；在细胞水平：大鼠原代海马神经元树突棘密度和成熟树突棘所占百分比减少；兴奋性氨基酸递质释放减少；SNK-SPAR途径稳态破坏：CaN低表达引起SNK转录因子p-CREB表达升高和核转位，SNK mRNA显著上调，SNK蛋白表达升高，SNK和SPAR磷酸化、泛素化水平升高，Cdk5在此过程中发挥促进作用；SNK下游Ras/Rap家族蛋白表达失衡；SNK是微波辐射致树突棘可塑性异常的有效作用靶点；SPAR磷酸化位点改变可能是影响其与PSD-95作用的具体方式；MAP2与SPAR相互作用为微波辐射致树突棘可塑性异常的机制研究提供新思路。综上，本研究阐明微波辐射后海马神经元树突棘可塑性的改变规律，系统分析了微波辐射后树突棘形态功能的改变与学习记忆障碍的相关性；发现海马颗粒细胞树突棘密度降低和锥体细胞成熟树突棘比例降低是微波辐射致学习记忆损伤的重要结构基础；发现SNK激活和由此造成的SPAR降解及PSD-95丢失是微波辐射后树突棘可塑性改变的重要机制；其中SNK是微波辐射致树突棘可塑性异常的有效作用靶点，这为微波辐射所致脑损伤的防护和治疗提供了新思路。