谷氨酸受体和转运体在NSSR1参与缺血性神经损伤保护中的作用及机制研究

谷氨酸兴奋毒是脑缺血导致神经元损伤的一大主要机制。存在于神经元细胞膜上的谷氨酸受体和胶质细胞膜上的谷氨酸转运体，是影响兴奋毒性的两大关键因素。两类蛋白的表达都受前体mRNA剪接调控，产生功能不同的亚型，但其调控机制尚不清楚。我们的前期研究提示，剪接因子NSSR1 可能通过调控谷氨酸转运体GLAST或GLT1，或AMPA受体的GluR-2亚基的基因转录或亚型剪接来降低脑缺血引起的兴奋毒性神经元损伤。本项目将综合利用分子生物学、组织化学和电生理检测等方法，利用NSSR1条件性敲除小鼠的脑缺血模型，以及离体培养的小鼠海马星型胶质细胞或神经元氧糖剥夺（或谷氨酸刺激）模型，通过比较NSSR1基因敲除前、后的海马星型胶质细胞上GLAST和GLT1的亚型表达和转运功能、以及神经元上GluR-2的亚型表达及AMPA受体的电生理特性和钙离子通透性的差异，来探讨NSSR1参与缺血脑内神经保护的功能和机制。

Glutamate induced excitotoxicity is an important mechanism which leads to ischemic neuronal injury. The glutamate receptors located on the neuronal membrane and the glutamate transporters located on the astrocytic membrane are the two major factors that affect the excitotoxicity. The expression of both proteins is subject to pre-mRNA splicing regulation, producing multiple isoforms with different functions. However, the regulation mechanism is unknown. The results of our previous studies suggest that pre-mRNA splicing factor NSSR1 might reduce the excitotoxicity neuronal injury induced by cerebral ischemia via regulating the alternative splicing of GluR-2 subunit of AMPA receptor, glutamate transporters GLAST or GLT1. In the proposed project, molecular biological, histochemical and electrophysiological analyses will be comprehensively used to investigate the protective function and mechanism of NSSR1 in ischemic brain. The transient global cerebral ischemic model of NSSR1 conditional knockout mice, and the oxygen-glucose deprivation model (OGD) or glutamate stimulated model of cultured hippocampal neurons or astrocytes will be used. The expression level of the GLAST and GLT1 isoforms, the glutamate-transporting ability of the GLAST and GLT1 in astrocytes, the expression level of the GluR-2 isoforms, the electrophysiological characteristics and the ability of calcium response of the AMPA receptor in neurons will be characterized to determine the differences between the NSSR1 conditional knockout mice and the control mice. The results will provide molecular basis for understanding the mechanism underlying the protective function of NSSR1 against ischemic neuronal injury.