FAM134B调控的内质网自噬在癫痫神经损伤中的作用及机制研究

Seizure-induced endoplasmic reticulum damage makes epileptic neurons more sensitive to injury. Therefore, timely removal of damaged endoplasmic reticulum is important for neuronal survival in epilepsy. Appropriate endoplasmic reticulum autophagy (ER-phagy) can selectively remove damaged endoplasmic reticulum to maintain the steady-state intracellular enviroment. However, the function and mechanism of ER-phagy in seizure-induced neuronal injury is still unknown. Recent studies have revealed that FAM134B is closely with ER-phagy. Our previous study also found that the levels of FAM134B expression and ER-phagy were both significantly increased in hippocampus of epileptic rats. So we speculate FAM134B may affect the development of epilepy by regulation of ER-phagy. Using in vitro primary cultured hippocampal neuron epileptic discharge and in vivo PILO (pilocarpine, PILO)-induced rat model of temporal lobe epilepsy, We will detect the effect of FAM134B on ER-phagy, endoplasmic reticulum injury and apoptosis at the molecular, cellular, tissue and animal levels by molecular biology and transmission electron microscope by constructing lentiviral vectors to regulate the expression of FAM134B. In addition, we will further prove the signaling cascade of FAM134B by regulation of autophagy (induction or inhibition of autophagy) and Calpain (inhibition of Calpain). Taken together, this study will clarify the molecular mechnism of epileptic neuronal injury through new viewpoint and provide new targets for treatment of epilepsy.

癫痫发作后内质网受损导致神经元对痫性损伤更敏感，及时清除受损内质网对神经元存活至关重要。适当的内质网自噬可选择性清除受损内质网以维持细胞内环境稳态，但内质网自噬在癫痫神经损伤中的作用及机制目前并不清楚。最近研究发现FAM134B蛋白与内质网自噬关系密切，我们前期研究也发现癫痫大鼠海马FAM134B和自噬水平均明显升高，因此推测FAM134B可能通过对内质网自噬的调控进而影响癫痫的发生发展。为验证该假说，本研究拟利用体外培养海马神经元癫痫放电及PILO诱导的大鼠颞叶癫痫模型，构建慢病毒载体干预FAM134B表达，采用分子生物学、透射电镜等技术，从分子、细胞、组织及动物等水平观察对内质网自噬、内质网损伤和细胞凋亡等影响；再给予调控自噬（自噬诱导剂或抑制剂）和钙蛋白酶（抑制剂）干预，进一步证明FAM134B发挥作用的信号级联。本课题将从新的视角阐明癫痫神经损伤的分子机制，为癫痫治疗提供新的靶点。

适当的内质网自噬可选择性清除受损内质网以维持细胞内环境稳态，最近研究发现FAM134B与内质网自噬关系密切。本研究利用体外培养海马神经元癫痫放电及PILO诱导的大鼠颞叶癫痫模型，通过构建慢病毒载体干预FAM134B表达，采用分子生物学、透射电镜等技术观察对动物行为学、内质网自噬、内质网损伤和细胞凋亡等影响，并利用选择性自噬抑制剂（3-methyladenine, 3-MA）干预进一步确定其作用级联。课题进展顺利，相关研究成果目前已发表 SCI 或中文核心期刊论文 10 篇（其中第一作者2篇）。研究结果如下：.结果.1. 在无镁诱导海马神经元癫痫放电模型中，构建慢病毒载体干预FAM134B表达，并通过调控自噬（3-MA）干预后发现：.1）FAM134B过表达可通过增强内质网自噬，减少内质网Ca2+释放和细胞内ROS生成，缓解癫痫诱导的内质网应激，从而对癫痫海马神经元发挥保护作用，而抑制FAM134B表达则发挥相反作用。表明FAM134B可缓解癫痫诱导的内质网应激和海马神经元凋亡。.2）采用自噬抑制剂3-MA预处理使内质网应激相关蛋白GRP78和CHOP的表达增加，神经元活力降低，显著抵消了FAM134B过表达对癫痫海马神经元内质网应激和凋亡的保护作用。表明FAM134B通过促进内质网自噬，从而缓解内质网应激和海马神经元凋亡。.3）FAM134B过表达可通过抑制Cyt C释放和caspase-3活化发挥抗凋亡作用。表明FAM134B通过线粒体凋亡途径对癫痫海马神经元发挥保护作用。.2. 在PILO诱导急性癫痫大鼠模型中，构建慢病毒载体干预FAM134B表达后发现：.1）4组大鼠发作潜伏期及致痫成功率无统计学差异。透射电镜结果显示FAM134B过表达组内质网结构损伤减轻，抑制FAM134B表达内质网结构损伤加重，可见内质网断裂。.2）FAM134B过表达组Beclin1表达增加。表明FAM134B过表达可增强内质网自噬。.综上所述，本课题深入研究了FAM134B调控的内质网自噬在癫痫神经损伤中的作用及相关机制，发现FAM134B可减轻癫痫诱导的内质网应激和海马神经元凋亡，而内质网自噬在其过程中发挥重要作用，为癫痫治疗提供了潜在的新靶点。