星形胶质细胞A2AR-A1R异聚化介导的谷氨酸转运失衡在缺血性脑损伤中的作用及其分子机制

The excessive extracellular accumulation of glutamate leads to excitotoxicity and ultimately results in cell death during cerebral. The inhibition of glutamate-induced excitotoxicity has been a pototial therapeutic target for the treatment of stroke for many years, however, all the clinical trials based on it have failed. Our previous research has found that the inhibition of adenosine A2AR decreased the express of GLT-1, the extracellular glutamate level was lowered and cerebral infarct volume was reduced. We have also proved that such neuroprotection was associated with activation of A1R. However, the underlying mechanism of the interaction between these two receptors modulating the GLT-1 in cerebral ischemia remains to be elucidated. According to the latest research and the early study of our group,we assume that the excessive release of adenosine facilitates the A2AR-A1R heteromerization in astrocyte during acute cerebral ischemia, which would inhibit the activation of A1R and decrease the expression of GLT-1 of astrocyte, resulting in worsening of cerebral ischemic injury. The first aim of the present work was to clarify whether the high concentration ADO could facilitate the A2AR and A1R heteromerized, which would impact on A1R activity in co-transfected HEK-293 cell by FRET and Co-IP. Then, we will also use astrocyte and neuron co-culture and gene knock out technique to explore the degree of A2AR-A1R heteromers formation, A1R activity, p38MAPK-PPARγ signal pathway 、GLT-1 expression, extracellular glutamate level, and impact of neuron apoptosis and cerebral infarct volume after block the A2AR in vitro and vivo . The research will further elucidate the impact of A2AR-A1 heteromerization on the modulation of glutamate transport and underlying mechanism of ischemic brain injury, which ultimately provide new thoughts and new treatment target of neuroprotection in ischemic brain injury or stroke.

脑缺血时谷氨酸兴奋性毒性是神经元死亡的关键环节。通过调控谷氨酸转运来干预兴奋性毒性因不影响谷氨酸正常传递而备受关注。我们在青年基金项目中发现阻断腺苷A2AR可通过上调缺血区谷氨酸转运体GLT-1的表达来减轻缺血性脑损害，其保护作用与A1R活化有关。但两种受体相互作用对GLT-1的调控机制不清。结合文献提出如下假设：脑缺血时大量释放的腺苷促进星形胶质细胞上A2AR-A1R异聚化，两种受体对GLT-1的调控失衡，导致细胞外谷氨酸蓄积并启动神经元损伤。本课题拟在A2AR及A1R共转染细胞中观察不同浓度腺苷对A2AR-A1R异聚化的影响；制作脑缺血在体、离体模型，研究A2AR、A1R的异聚化状态、p38MAPK-PPARγ信号通路的变化以及对GLT-1功能和神经元凋亡的影响。阐明星形胶质细胞A2AR-A1R异聚化调控谷氨酸转运失衡在缺血性脑损伤中的作用机制，为缺血性脑保护治疗提供新的作用靶点。

1.按计划执行情况：本项目研究工作按照计划顺利执行。我们成功构建了A2aAR基因敲除鼠，并研究了星形胶质细胞胞膜中A1AR、A2aAR的相互作用。检测缺血条件对星形胶质细胞上A2AR-A1R异源二聚体、A2AR、A1R的表达分布的影响，对p38MAPK、PPARγ表达及对GLT-1功能的影响。建立小鼠MCAo模型，检测A2AR-A1R异源二聚体形成、p38MAPK、PPARγ、GLT-1表达；完成各组小鼠脑梗死体积、死亡率的检测。.2.研究目标完成情况。. 目标1：阐明急性脑缺血条件下，腺苷A1AR、A2AR相互作用导致GLT-1表达与功能下降，进而导致细胞外谷氨酸稳态破坏并启动神经元兴奋性毒性损害的分子调控机制；.完成情况：我们证实了A1AR和A2aAR在OGD以及腺苷刺激下存在相互作用，即形成A1AR-A2aAR异二聚体。进一步研究表明A1AR-A2aAR异二聚体可抑制GLT-1（也叫EAAT2）的表达，进而导致细胞内外谷氨酸稳态破坏。.目标2：证实我们所推测的调控GLT-1表达的p38MAPK-PPARγ信号通路。.完成情况：我们明确了OGD环境下星形胶质细胞胞膜表面形成的A1AR-A2aAR异二聚体会激活p38、抑制PPARγ，进而激活GLT1上游的Yin Yang 1(YY1)蛋白，影响GLT-1的表达。应用PPARγ激动剂、YY1 siRNA以及过表达GLT-1 均可调控OGD环境下星形胶质细胞中GLT-1的表达水平，进而影响细胞内谷氨酸水平。