Grp78和未折叠蛋白反应在脑梗死后丘脑自噬激活和继发变性中的作用研究

The mechanisms of secondary thalamic neurodegeneration induced by focal cerebral infarction remain unclear. Our previous studies have shown that the neuronal autophagy is activated in the ipsilateral thalamus of rats at seven days after cerebral infarction and is associated wtih the thalamic Aβ deposition and neuronal death, suggesting autophagy maybe a crucial mechamism underlying secondary thalamic neurodegenertion. How is neuronal autophagy activated in the ipsilateral thalamus following cerebral infarction? Recent studies indicate that the unfoled protein response (UPR) regulator Grp78 is required for the induction of neuronal autophagy after axonal degeneration. Our preliminary observations show that Grp78 level is elevated in the ipsilateral thalamus after cerebral infarction but the role of Grp78 in the induction of autophagy and cell death remains uncertain. In the present study, we aim to study the role of Grp78 in the neuronal autophagic activation and death in the ipsilateral thalamus after cerebral infarction. We first investigate the levels of Grp78 and its major effectors IRE1/Xbp1, PERK/eIF2α and ATF-6 in the ipsilateral thalamus after experimental cerebral cortical infarciton in rats. We then promote the overexpression of Grp78 in the ipsilateral thalamus using lentivirus vectors and investigate the levels of autophagic induction and neuronal death. The study will further elucidate the molecular mechanisms of secondary thalamic degeneration after focal cerebral infarction and may suggest a novel target for the development of a new therapy for cerebral infarction.

局灶性脑梗死引起非缺血区丘脑发生继发性变性的机制尚未阐明。我们之前的研究发现，大鼠大脑皮层梗死后7天同侧丘脑开始出现神经元自噬激活，参与丘脑的Aβ沉积和神经元死亡，表明自噬可能是脑梗死后丘脑远隔损害发生的重要机制。那么，脑梗死后丘脑神经元自噬是如何激活的呢？新近研究发现未折叠蛋白反应（UPR）调节蛋白Grp78参与轴索变性后细胞自噬的调控。我们的初步观察也发现大鼠脑梗死后同侧丘脑Grp78表达减少，但其在丘脑细胞自噬和死亡中的作用尚不明确。本课题先观察实验性大脑皮层梗死后同侧丘脑Grp78水平和其下游的IRE1/Xbp1通路、PERK/eIF2α通路、ATF-6通路的变化，说明脑梗死后丘脑UPR通路的改变；然后增强Grp78表达干预UPR通路，观察自噬水平和神经元数量的变化，来阐明Grp78在丘脑神经元自噬激活和死亡中的作用。研究这些问题，有助进一步明确脑梗死后丘脑继发性损害的发生机制。

局灶性脑梗死引起非缺血区域丘脑发生继发性变性，阻碍神经功能的恢复。我们之前的研究发现，大鼠大脑皮层梗死后7天同侧丘脑出现神经元自噬激活，参与丘脑的Aβ沉积和神经元死亡，表明自噬可能是脑梗死后丘脑远隔损害发生的重要因素。但脑梗死后丘脑神经元自噬激活的机制尚不清楚。本课题旨在探讨未折叠蛋白反应（Unfolded protein response, UPR）调节蛋白Grp78在丘脑神经元自噬激活和死亡中的作用。本课题采用易卒中型肾血管性高血压模型，建立右侧大脑中动脉远端闭塞模型。在丘脑注射过表达Grp78慢病毒，神经功能评分后使用尼氏染色、免疫组化、免疫印迹进行观察。结果发现：脑梗死后3天和7天，同侧丘脑Grp78主要表达于神经元和星形胶质细胞，蛋白水平较假手术组明显减少；相应地，MCAO后3天和7天，右侧丘脑eIF2α-P/eIF2α-T比例较假手术组表达明显增加，ATF4蛋白水平明显升高。ATF-4主要表达于神经元，与Grp78有共表达，但ATF-4下游的主要靶蛋白CHOP则未见明显变化；此外，ATF-6激活片段/全片段的比例，激活Xbp1 /总Xbp1的比例均未见明显增加；Grp78干预后，丘脑Grp78表达增多，ATF-4减少，自噬激活减少，神经元死亡减少，但神经功能评分未见明显改善。研究表明，脑梗死后丘脑Grp78和未折叠蛋白反应通路改变，参与丘脑神经元自噬和继发损害。