前扣带回突触前小窝蛋白1通过调节谷氨酸释放介导神经病理性疼痛的机制研究

Neuropathic pain is a common disease and generally defined as a chronic pain with a high morbidity in clinical diagnosis. Progressing studies showed that the anterior cingulate cortex（ACC） is a critical region for modulating of chronic pain. High excitability of neurons is the cytological basis in regulation of chronic neuropathic pain in ACC. We had reported that the NMDA receptor 2B subunit trafficking via caveolin-1 (Cav-1), a scaffolding protein in postsynaptic of ACC neurons, and the variation of correlated intracellular signaling pathway played an important role in high excitability of ACC neurons during neuropathic pain and the regulation of pain behavior(J Neurosci, 2015). Further more, we also found that Cav-1 expressed highly in presynaptic membrane of glutamatergic neurons and regulated the release of glutamate. According to a series of preliminary experimental results and previous studies, we propose that the regulation of glutamatergic neurotransmitter release by the interaction of Cav-1 and NMDAR2B in presynaptic of ACC neurons mediate high excitability of ACC neurons in neuropathic pain mice. This project focuses on glutamate release in presynaptic, to clarify the effect and mechanism for regulation of glutamate release by Cav-1 from the overall, cellular and molecular level in neuropathic pain mice. The achivement of this project will offer a new molecular target for development of drugs.

慢性神经病理性疼痛是临床常见和多发疾病，现有资料显示ACC区是调控神经病理性疼痛的关键脑区，ACC神经元高兴奋性是其发挥调控作用的细胞学基础。我们近期研究表明，ACC神经元突触后膜的支架蛋白小窝蛋白1（Cav-1）介导的NMDA受体2B亚基转位（Trafficking）及其相关胞内信号通路在神经病理性疼痛ACC神经元高兴奋性和调节痛行为中发挥重要作用（J Neurosci, 2015）。深入研究还发现，Cav-1也大量表达在ACC谷氨酸能神经元突触前膜，且调控谷氨酸释放。根据系列预实验结果及以往的研究我们提出：ACC神经元突触前Cav-1与NMDAR2B相互作用调节谷氨酸神经递质释放介导神经病理性疼痛ACC神经元高兴奋性。本项目以突触前谷氨酸释放为切入点，从整体、细胞和分子水平阐明Cav-1对谷氨酸释放的调节在神经病理性疼痛中的作用及机制，该研究为药物开发提供新的分子靶点。

慢性神经病理性疼痛是临床常见和多发疾病，现有资料显示ACC区是调控神经病理性疼痛的关键脑区，ACC神经元高兴奋性是其发挥调控作用的细胞学基础。我们近期研究表明，ACC神经元突触后膜的支架蛋白小窝蛋白1（Cav-1）介导的NMDA受体2B亚基转位（Trafficking）及其相关胞内信号通路在神经病理性疼痛ACC神经元高兴奋性和调节痛行为中发挥重要作用（J Neurosci, 2015）。基于以往研究，本项目首先采用免疫双标技术，用激光共聚焦显微镜观察 Cav-1 与突触前膜标记物（Synapsin 1 ）和谷氨酸能神经元标记物（VGLUT 1 ，vesicular glutamate transporter 1)共定位显示Cav-1存在于谷氨酸能神经元突触前膜，且CCI 小鼠前扣带回谷氨酸能神经元突触前膜 Cav-1 较 Sham 组表达显著增高。并用生化试剂盒检测显示CCI小鼠前扣带回谷氨酸释放明显高于Sham组，分别在CCI小鼠或Sham组小鼠ACC区立体定位注射Cav-1 siRNA或Lenti-Cav-1，会分别导致ACC区脑片谷氨酸释放明显降低或增高。为进一步研究Cav-1对谷氨酸释放的影响，本研究采用了化学遗传学方法激活或抑制小鼠 ACC 区谷氨酸能神经元可升高或降低谷氨酸释放并缩短或延长小鼠热缩爪潜伏期以及升高或降低ACC 区谷氨酸能神经元 Cav-1 的表达。为了发现与Cav-1竞争性结合的小肽，本研究还发现在原代培养的ACC 区神经元用慢病毒为载体过表达Cav-1 至第三天，可明显增加培养基中谷氨酸的含量，在培养基中加入NR2B 特异抑制剂Ro25-69819（100nM）30min，可抑制Cav-1 过表达引起的谷氨酸释放增多；Cav-1 和 RIM1 蛋白能够产生免疫共沉淀。该研究可为研发理想的镇痛药物提供新的有益靶标。