星形胶质细胞mTOR通路异常激活在内侧颞叶癫痫中致病机制的研究

Temporal lobe epilepsy (TLE) is often medically intractable. The mTOR pathway is activated in several models of epilepsy and the mTOR inhibitor rapamycin has antiepileptogenic properties but the mechanism is still unclear. Although the proconvulsanteffects of activated mTOR signaling on neurons have been proposed, our current study demonstrated that mTOR activation was most prominent in reactive astrocytes in sclerotic hippocampus from patients with drug resistant TLE. We speculated that the astrocytic hyperactivation of mTOR pathway induced by seizures could disrupt glutamate buffering capacity of astrocytes and contribute to epileptogenesis. The proposal focuses on the role of mTOR overactivation during temporal lobe epileptogenesis. At the molecular and cellular level, we aim to figure out how the mTOR pathway regulates the protein expression of a critical glutamate transporter Glt1 in astrocytes. Using mouse model of temporal lobe epilepsy and mTOR inducible conditional knock-out mice, we hope to explain whetherastrocytic inhibition of mTOR pathway has anticonvulsant effect.

内侧颞叶癫痫是一类药物难治性癫痫。动物实验提示，mTOR通路是潜在的治疗靶标，但机制不清。以往研究集中于mTOR通路在神经元中的致痫机制，近期本组发现并首次报道了mTOR通路在患者致癫灶中的激活主要发生在星形胶质细胞中，且起始于癫痫潜伏期，引起国际同行关注。我们推测神经系统损伤导致mTOR通路异常激活，使星形胶质细胞对神经微环境中谷氨酸的调节能力下降，进而促进了癫痫发生。本申请将重点关注mTOR通路的激活在颞叶癫痫发生中的分子致病机制；在细胞分子水平研究mTOR通路的上下游在星形胶质细胞中调节谷氨酸转运分子Glt1的具体作用机理；利用海人酸颞叶癫痫模型小鼠以及mTOR基因的诱导性条件敲除小鼠探究在癫痫发生过程中针对星形胶质细胞mTOR通路的靶向抑制是否具有抗癫痫效果。

胚系和体细胞突变导致的哺乳动物雷帕霉素靶蛋白（mTOR）通路上关键基因突变与癫痫发作相关疾有密切关联。mTOR基因突变可以导致mTOR通路异常激活，尽管现有研究都表明其对神经元的影响与癫痫发生密切相关，但突变对胶质细胞的作用以及与癫痫发病之间的关联性罕有报道。本组前期报道了mTOR通路在颞叶癫痫患者致癫灶的星形胶质细胞中有过度激活的现象，本研究中，我们证实了星形胶质细胞删除mTOR基因可以阻止癫痫发作频率的升高。通过使用他莫昔芬诱导的条件性敲除系统和海人酸诱导的癫痫模型，我们成功在慢性癫痫小鼠上实现了星形胶质细胞特异性的mTOR基因敲除，敲除比例约为44%。敲除组小鼠表现出了较低的癫痫发作频率，并伴有星形胶质细胞增生缓解的现象，但并不影响苔状纤维发芽。在培养的原代星形胶质细胞中，我们发现mTOR通路可以通过泛素化方式调节谷氨酸转运体GLT1的蛋白质稳定性，并影响星形胶质细胞对体外谷氨酸的清除能力。本研究证实了mTOR在颞叶癫痫致癫灶星形胶质细胞中的激活具有促进癫痫发病的效果。研究成果发表于Molecular Neurobiology期刊，同时后续研究证实了Hsp90抑制剂能够阻碍GLT1蛋白酶体降解途径，能够更有效提高GLT1表达，研究成果发表于Journal of Experimental Medicine期刊。