ndrg2基因调控星形胶质细胞可塑性在脑缺血中的作用

Stroke has become “the first killer" in our country. Current studies of neuroprotection focus on the neuron survival following stroke, which, though extensive, have not made any breakthrough. Recent years, astrocyte has become the new therapeutic target for stroke treatment. We have previously discovered that ndrg2 gene was specifically expressed in the astrocytes of brain tissue, and this expression was significantly up-regulated following ischemic brain injury. Recently, our research group has successfully established astrocyte-specific ndrg2 knock out mice. We found that the cell body of ndrg2-/- astrocyte became swelled and the cell processes significantly shortened. From a functional standpoint, the glutamate and aspartate concentration in synaptic cleft were significantly increased and ndrg2-/- mice exhibited aggravated ischemic brain injury. These findings prompt our belief that ndrg2 plays an important role in ischemic injury and may affect astrocytes excitability and synapse transmission by regulating structural plasticity in astrocytes. Nevertheless, the underlying mechanism remains unclear. Therefore, by using electrophysiology, two-photon in vivo imaging and other technologies, this program is designed to investigate the effect of ndrg2 on astrocyte structure plasticity, analyze the molecular mechanism and explore the influence of these astrocyte structure plasticity changes on astrocyte function and synapse transmission in ndrg2-/- mice, Overall, our systematic analysis will help to clarify whether ndrg2 plays an important role in ischemic brain injury by regulating the astrocyte plasticity, which will definitely provides a novel therapeutic target for stroke treatment.

脑卒中是目前我国“第一杀手”，既往研究多聚焦于神经元存活，未取得突破进展，星形胶质细胞已成为卒中治疗新靶点。我们前期研究发现ndrg2基因在脑组织特异表达于星形胶质细胞，脑缺血后其表达显著上调。近期申请者构建了星形胶质细胞特异性ndrg2-/-小鼠，发现星形胶质细胞胞体变圆，突起显著缩短，突触间隙谷氨酸和天冬氨酸浓度增高，而脑缺血后，ndrg2-/-小鼠损伤较重，提示ndrg2可通过调控星形胶质细胞结构可塑性影响其兴奋性及突触传递，在脑缺血中发挥重要作用，但具体分子机制有待深入研究。本课题拟利用ndrg2-/-小鼠，采用电生理、活体成像等技术，研究ndrg2对星形胶质细胞结构可塑性的作用特点并分析其分子机制，深入探讨这种结构可塑性对星形胶质细胞兴奋性及突触传递的影响，最终阐明ndrg2通过调控星形胶质细胞可塑性在脑缺血中发挥作用的性质和机理，为开发更为有效的脑卒中治疗策略提供新靶点。

星形胶质细胞在突触可塑性和神经递质调控中发挥重要作用，已成为卒中治疗的新靶点。前期研究发现ndrg2在脑组织特异性表达于星形胶质细胞，脑缺血后其表达显著上调。本课题成功构建了ndrg2基因敲除小鼠（ndrg2-/-），发现缺血性脑损伤后ndrg2-/-小鼠脑梗死容积明显大于野生型小鼠（ndrg2+/+），且ndrg2-/-小鼠海马星形胶质细胞末端突起明显减少变短。原代培养ndrg2-/-小鼠星形胶质细胞，并利用慢病毒转染恢复Ndrg2-/-星形胶质细胞中NDRG2的表达（Ndrg2-/-+LV-Ndrg2），发现与Ndrg2+/+星形胶质细胞典型的星形状形态相比，Ndrg2-/-星形胶质细胞胞体扁平宽大、突起缩短变粗且几乎无分支，而慢病毒转染恢复NDRG2蛋白表达后，星形胶质细胞恢复其典型的星形状形态。蛋白质谱分析结果显示：Ndrg2-/-星形胶质细胞中Rho-GTP酶多个上游调控分子（GEFs和GAPs等）和下游效应分子蛋白表达水平发生显著变化，提示NDRG2可能通过Rho-GTP酶信号通路介导星形胶质细胞形态改变；深入研究发现ndrg2缺失显著增加α-Actinin4蛋白表达而降低GFAP蛋白表达，并且ndrg2缺失显著增加RhoA活性而降低Rac1活性，而慢病毒转染可显著逆转上述骨架蛋白α-Actinin4和GFAP蛋白表达变化以及上述Rho-GTP酶表达变化。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，且ndrg2-/-小鼠谷氨酸转运体EAAT1、EAAT2表达显著下降，离体原代星形胶质细胞和神经元共培养亦证实ndrg2-/-星形胶质细胞谷氨酸清除率明显降低，ndrg2-/-星形胶质细胞增加谷氨酸损伤效应。小鼠脑片电生理结果显示ndrg2-/-小鼠神经元自发性兴奋性突触后电流（sEPSC）频率显著升高，幅度略微升高。矿场实验亦发现ndrg2-/-小鼠的活动范围和活动速率较ndrg2+/+小鼠均明显增高，提示其神经兴奋性增高。综上所述，本课题成功构建了星形胶质细胞特异性ndrg2基因敲除小鼠（ndrg2-/-），通过在体和离体研究发现ndrg2调节Rho-GTP酶信号通路调控星形胶质细胞结构可塑性，证实ndrg2通过调控谷氨酸转运体蛋白表达，改变突触间隙谷氨酸浓度和突触放电，参与脑缺血损伤中神经元兴奋性毒性损伤。