星形胶质细胞Ndrg2调节“谷氨酸-谷氨酰胺循环”在缺血性神经损伤中的作用

How to effectively prevent and alleviate ischemic brain injury is one of the major focuses of current neuroscience. We reported for the first time the ndrg2 gene in 1999 and provided the evidence that in the nervous system ndrg2 is predominantly expressed in astrocytes. The expression level of ndrg2 was significantly up-regulated after ischemic brain injury, indicating its potential involvement during the pathogenesis. Recently we found that NDRG2 was able to regulate the activity of astrocytes by increasing extracellular fluid concentrations of glutamate and lowering glutamine levels. Accumulated data have shown that glutamate-glutamine cycle led by astrocyte could clear accumulated glutamate in cell gap during ischemic brain injury and thereby protects neurons from excitotoxicity injury. On the above-mentioned basis, we aim to further study the role of NDRG2 in glutamate-induced excitatory neurotoxicity and to verify the potential mechanism that inhibition of NDRG2 expression could improve the glutamate-induced excitotoxicity injury, by using the function, morphology and molecular biology techniques including the astrocyte specific ndrg2 knockout mice, the co-culture model of primary nerve cells, lentiviral transfection and interference, liquid chromatography, microdialysis. Our systematic analysis should shed light on a new target for the development of more effective prevention and treatment strategy on the ischemic brain injury.

如何有效预防和减轻缺血性脑损伤是目前神经科学研究的重点。本课题组于1999年在国际上率先发现ndrg2基因，证实其在脑组织特异性表达于星形胶质细胞；在缺血性脑损伤后其表达显著上调，提示其参与缺血性脑损伤。我们最新研究发现NDRG2能够调节星形胶质细胞活性，升高细胞外液谷氨酸浓度,降低谷氨酰胺水平。而星形胶质细胞主导的"谷氨酸-谷氨酰胺循环"在缺血性脑损伤时可清除细胞间隙蓄积的谷氨酸，使神经元免遭兴奋性毒性损伤。本课题拟在前期研究基础上，在体构建星形胶质细胞特异性ndrg2基因敲除小鼠，离体利用原代神经细胞共培养模型，采用慢病毒转染、高效液相色谱、在体微透析等功能、形态与分子生物学技术，证实NDRG2调控"谷氨酸-谷氨酰胺循环"的分子机制，探讨NDRG2在谷氨酸诱导的兴奋性神经毒性中的作用机制，抑制NDRG2的表达改善谷氨酸诱导的神经损伤，为开发更为有效的缺血性脑损伤防治策略提供新的靶点。

如何有效预防和减轻缺血性脑损伤是目前神经科学研究的重点。本课题组于1999年在国际上率先发现ndrg2基因，证实其在脑组织特异性表达于星形胶质细胞；在缺血性脑损伤后其表达显著上调，提示其参与缺血性脑损伤。成功构建了星形胶质细胞特异性ndrg2基因敲除小鼠，发现缺血性损伤后ndrg2-/-小鼠脑梗死容积明显大于野生型小鼠，行为学评分明显差于野生型小鼠，而利用慢病毒恢复NDRG2表达则能显著改善神经损伤。在体微透析研究发现ndrg2-/-小鼠缺血损伤前后突触间隙谷氨酸含量明显高于野生型小鼠，而利用慢病毒恢复NDRG2表达，则能降低谷氨酸含量。离体原代共培养星形胶质细胞和神经元，发现ndrg2-/-星形胶质细胞谷氨酸清除率明显降低，且谷氨酸神经损伤效应加剧。表达于星形胶质细胞的谷氨酸转运体EAAT1和EAAT2在谷氨酸清除中发挥重要作用，其功能驱动依赖于离子泵Na+/K+-ATPase维持的跨膜Na+梯度。本课题证实ndrg2-/-星形胶质细胞谷氨酸转运体EAAT1、EAAT2及Na+/K+-ATPase β1表达显著降低，恢复NDRG2表达则能恢复谷氨酸转运体和Na+/K+-ATPase β1的表达。课题组最终通过免疫共沉淀和生物信息学技术，进一步证实NDRG2与Na+/K+-ATPase β1 存在相互作用，并筛选出NDRG2与Na+/K+-ATPase β1的作用位点位于NDRG2的141-160氨基酸序列，进而合成Tat-NDRG2(aa141-160)，证实其能够显著增加ndrg2-/-星形胶质细胞Na+/K+-ATPase β1的表达与稳定性，增强星形胶质细胞谷氨酸清除能力。同时，Tat-NDRG2(aa141-160)能够显著增加ndrg2-/-小鼠谷氨酸转运体和Na+/K+-ATPase β1的表达。本课题利用转基因动物和慢病毒证实了特异性表达于星形胶质细胞的ndrg2能够调控谷氨酸转运体EAAT1与EAAT2的表达，同时与Na+/K+-ATPase β1相互作用维持跨膜Na+梯度驱动谷氨酸转运体摄取突触间隙谷氨酸，减轻缺血性损伤中兴奋性毒性损伤，从而发挥神经保护作用，并最终筛选出NDRG2与Na+/K+-ATPase β1的作用位点，合成Tat-NDRG2(aa141-160)辅证了上述假说，为开发更为有效的缺血性脑损伤防治策略提供新的靶点。