PSD-93调节卒中后突触后膜-胶质细胞间的兴奋性环路研究

Exitotoxicity is the initial insult contributing to the pathogenesis of ischemic stroke. Communication between postsynapse and glia plays a critical role in this progress. Postsynaptic density 93 (PSD-93) is located in the postsynaptic density region and belongs to the membrane associated guanylate kinase family. Our previous studies confirmed that PSD-93 knock out reversed the .neurological deficits and decreased the infarct size in experimental stroke model. Interestingly, our studies showed that PSD-93 could regulate the expression of GLT-1 and SR in glia, both of which directly affected the level of excitotoxic neurotransmitters. Based on the previous work, we are going to perform experiments with the purpose of illustrating two questions: 1) We want to confirm PSD-93 .knock out protects against ischemic injury through regulating postsynapse-glia excitotoxic loop. 2) We want to dig out the underlying mechanism through which PSD-93 regulates postsynapse-glia excitotoxic loop. To be specifically, we want to know the way through which PSD-93 affects the level and function of GLT-1 and SR. Our study extended the excitotoxic study from neuron to neuron-glia era, which will broad the current vision on excitotoxicity and ultimately provide therapeutic strategies for clinical treatment.

兴奋性毒性是缺血性脑损伤的最早病理机制，突触后膜-胶质细胞间的相互作用对兴奋性毒性调控起着关键作用。PSD-93位于突触后膜电子致密区，是膜相关鸟苷酸激酶家族成员之一。我们前期研究发现PSD-93基因敲除能明显改善缺血性卒中小鼠的神经功能，且PSD-93影响胶质细胞上调控兴奋性递质的关键分子GLT-1和SR的表达。本课题基于前期工作，采用野生和PSD-93基因敲除小鼠体内外缺血模型，利用病理学、分子生物学等技术进一步从以下方面进行研究：1）证实PSD-93调控突触后膜-胶质细胞的兴奋性毒性环路，并建立其与缺血性卒中的密切相关性；2）找到PSD-93调控突触后膜-胶质细胞兴奋性环路的机制，具体探讨PSD-93影响胶质细胞上GLT-1和SR表达和功能的分子机制，将兴奋性毒性的研究由神经元转向神经元-胶质细胞，为兴奋性毒性的机制研究开拓新思路，为缺血性卒中的临床治疗提供新策略。

研究背景：缺血性卒中是高发病率、高死亡率、高致残率以及高复发率的疾病，给社会和家庭带来了沉重的负担。兴奋性毒性是缺血性脑损伤最早期发生的病理反应。PSD-93在这毒性反应中具有重要作用。PSD-93位于突触后膜，属于鸟苷酸激酶家族。胶质细胞和神经元细胞共同参与调节突触间隙中兴奋性毒性的传递。胶质细胞表达谷氨酸转运子 1 (glutamate transporter 1, GLT-1)，它将突触间隙的谷氨酸转运到胶质细胞内，降低突触间隙中的谷氨酸浓度。而突触间隙中的另外一种兴奋性促进递质D-丝氨酸的产生依赖于胶质细胞所分泌的丝氨酸消旋酶 (serine racemase, SR)。本研究将采用PSD-93基因敲除小鼠研究PSD-93介导的兴奋性毒性在缺血性脑损伤中的病理作用及其机制。研究方法：制备体内外脑缺血模型（体外采用原代神经元培养氧糖剥夺模型，Oxygen glucose deprivation, OGD; 体内采用小鼠大脑中动脉结扎模型，Middle cerebral artery occlusion, MCAO)。通过TTC染色测定脑梗死体积，Nissl染色检测组织缺损，Fluro-Jade B染色检测细胞凋亡，行为学评分等评估小鼠的恢复情况。MTT, LDH，Hochest染色检测神经元死亡情况。免疫荧光，Western Blot, 免疫共沉淀，PCR等技术检测分子表达，探讨药物作用的药理机制。研究结果：1) PSD-93基因敲除在小鼠MCAO模型中减轻缺血性脑损伤 - 降低梗死面积，提高行为学表现。2) PSD-93基因敲除抑制OGD所致的神经元损伤；3) PSD-93基因敲除抑制海马脑片的缺血长时程增强；4）PSD-93基因敲除在体减轻缺血性脑损伤后神经元细胞的凋亡；5）PSD-93基因敲除抑制Fyn所介导的NR2B的Tyr1472的磷酸化水平；6）PSD-93基因敲除能增加GLT-1的表达和降低SR的表达；7）PSD-93基因敲除可能通过JNK以及AKT通路调节GLT-1和SR的表达。结论：PSD-93基因敲除减轻缺血性脑损伤，一方面通过直接作用抑制Fyn所介导的NR2B的酪氨酸的磷酸化，同时能增加GLT-1的表达和降低SR的表达，抑制兴奋性毒性的级联放大反应。