乳酸转运体调控谷氨酸转运体在癫痫发病机制中的作用研究

Glutamate is the primary excitatory neurotransmitter in the central nervous system (CNS), mechanisms that promote glutamatergic neurotransmission could play important roles in the development of epilepsy. Increased levels of extracellular glutamate in fact have been observed in human epileptic serum. In vivo microdialysis studies also show elevated extracellular concentration of glutamate in the epileptogenic cortex as compared to the nonepileptogenic cortex in patients with temporal lobe epilepsy (TLE). These reports suggest that dysfunctional extracellular glutamate cycling and reuptake may play a important role in the genesis and maintenance of focal epileptic activity.Glutamate transporters, also referred to as excitatory amino acid transporters (EAAT), represent the sole mechanism of active reuptake of glutamate into the astrocytes and neurons and are essential to dampen neuronal excitation following glutamate release at synapses..Monocarboxylate transporters (MCT) facilitates the transport of monocarboxylate fuels (lactate and pyruvate) and acidic drugs, such as valproic acid, across cell membranes. A resent research shows that MCT1 is deficient on microvessels in the epileptogenic hippocampal formation in patients with medicationrefractory epilepsy. To further define the role of MCT in the pathophysiology of medicationrefractory epilepsy, we used immunohistochemistry、western blot and whole cell clamp analysis to localize and quantify the transporters in the hippocampal formation in three novel and highly relevant rat models of edicationrefractory epilepsy and in nonepileptic control animals. We were also to determine the expression and distribution of MCT in tissue samples from the refractory cortex of patients who had been surgically treated for refractory epilepsy. We compared these tissues with histologically normal samples from controls. In our present study, we found MCT4 was lost on hippocampus and piriform cortices in the lithium-pilocarpine model. In addition , we were able to coimmunoprecipitate MCT4 and EAAT1 in primary rat astrocytes. We also found RNAi-mediated inhibited of MCT4 can decrease the expression of EAAT1 in primary rat astrocytes. It is possible that EAAT1 is a substrate for MCT4, implying MCT4 may directly modulate EAAT1 in primary rat astrocytes. Therefore, we hypothesize that the loss of MCT in brain is mechanistically involved in the pathophysiology of intractable epilepsy, and propose that re-expression of MCT may represent a novel therapeutic approach for this disease.This study is supposed to confirm the above hypothesis. The results from this study will shed light on new strategic treatment for epilepsy.

癫痫的反复发作是长期困扰人类的顽疾，研究癫痫发作的调控机制具有重要现实意义。在癫痫致病机制研究中，谷氨酸清除障碍被公认为是最重要的致病机制之一。脑内高浓度的谷氨酸不仅可使神经系统兴奋性增高，且持续作用于受体造成神经细胞的损伤，目前关于癫痫病人脑内异常增高的谷氨酸水平的机制尚不清楚，因此探讨谷氨酸增高机制发展新的抗癫痫治疗策略具有重要意义。我们在回顾了谷氨酸转运体与癫痫、乳酸转运体与癫痫和谷氨酸转运体与乳酸转运体等方面研究进展基础上，结合预实验中观察到的现象，提出了乳酸转运体在脑内不仅有维持乳酸和丙酮酸正常转运保证胶质细胞-神经元能量交换功能，还可以通过调控谷氨酸转运体促进谷氨酸顺利清除的设想。本项目拟利用癫痫大鼠模型和临床癫痫组织样本，通过分子生物学、神经电生理记录、行为学等方法，揭示乳酸转运体调控谷氨酸转运体的病理生理学机制，也最终为乳酸转运体成为一个合理的抗癫痫药物靶点奠定理论基础。

癫痫病灶（发作起源区）组织糖代谢减弱而糖酵解增强，乳酸堆积。本项目研究发现乳酸转运体（MCT）的表达和功能的异常不仅影响了膜表面谷氨酸转运体（EAAT）的功能，而且通过乳酸堆积继发细胞外pH的变化间接影响酸敏感的离子通道（ASIC）的功能。我们的主要发现包括三个方面：（1）癫痫病灶组织中MCT4因其启动子区过度甲基化导致表达减少，MCT4的减少引起与其相互作用的EAAT1表达的下调；而MCT4和EAAT1的表达异常扰乱了神经元-星形胶质细胞之间能量代谢偶联途径，增加了乳酸堆积，诱导癫痫的发生。（2）间断缺氧激活细胞和大鼠的HIF-1α，激活的HIF-1α上调MCT，增加神经元-星型胶质细胞之间的乳酸转运，减少细胞间隙的乳酸含量，延长匹鲁卡品诱导的癫痫发作潜伏期和降低发作强度等级。（3）ASIC通道的开放可以被乳酸堆积引起细胞外pH的改变而影响。癫痫组织代谢异常（如糖代谢异常）通过转录因子TFCP2上调ASIC2a的表达水平，而ASIC2a表达量的改变不仅影响ASICs通道复合体的组成，而且直接影响神经元的兴奋性，增加了大鼠的癫痫易感性。综合本项目的研究发现，我们建立了“低代谢诱导癫痫发生”的假说：低代谢组织中代谢功能改变及其产物的异常可能通过影响基因转录增加神经元的兴奋性，继而诱导癫痫发生。具体来说，我们认为癫痫发生前期低代谢（如糖代谢减少）可能通过影响MCT启动子区甲基化、转录因子HIF-1α和TFCP2的表达，一方面干扰MCT对神经元-星形胶质细胞之间乳酸的转运和EAAT对细胞间隙堆积的乳酸的清除，另一方面上调ASIC2a促使神经元兴奋性的增加和对胞外pH变化响应的增强，通过（至少）这两种途径增加了癫痫发生的易感性。基于该假说，我们推测恢复低代谢脑区的正常代谢功能可能是阻止癫痫进一步发展的潜在手段，这为抗癫痫药物的开发提供新靶点和新思路。