Toll样受体4对海马神经元发育的影响及机制

Epilepsy is the third most common chronic brain disorder. Epileptic seizures affect 1-2% of the global population and remain the subject of concentrated neuroscientific investigation. To date, about 30% of epilepsy cases are resistant to current therapies. Rapidly growing body of evidence that supports the involvement of inflammation and immune responses in epileptogenesis. Toll-like receptors (TLRs) are pattern recognition receptors that play essential roles in innate immunity. TLR4 is the first TLR identified and activated specially by bacterial lipopolysaccharide (LPS). TLR4 plays an important role in developing epileptic seizures. Hippocampus is particularly vulnerable to damages caused by ischemia, hypoxia and trauma, and the consequent neuroinflammation, which can lead to epilepsy. The abnormal synaptic transmission, dendritic development and synaptogenesis as well as the synaptic plasticity of hippocampal neurons lead to imbalance of excitatory and inhibitory neuronal circuitry, which is considered as one of the underlying mechanisms of epilepsy. TLR4 is expressed in neurons and astrocytes, where they possibly mediate different signaling pathways after activation by LPS. Increasing evidence indicates that TLR4 affects neuronal survival, neurite growth, and hippocampus-dependent spatial reference memory in an inflammation-dependent manner, suggesting that TLR4 may participate in epileptic seizure through affecting neuronal development and function as well as synaptic plasticity. But the nature of detailed effects and mechanisms remain to be elucidated. In this study, we will explore the effects of TLR4 on hippocmapl neuronal development, synaptic plasticity, hippocampus-dependent learning and memory, and its roles in seizure susceptibility in mice, as well as the related underlying signaling pathway of TLR4, to demonstrate the important molecular and cellular mechanisms of TLR4-mediated immune response in epileptic seizures.

癫痫是中枢神经系统（CNS）常见疾病之一。CNS炎症和免疫反应是癫痫发生和持续发作的一个重要因素。Toll样受体(TLR)是模式识别受体，在先天免疫应答中起关键作用。TLR4是最早发现的TLR亚型，可被细菌脂多糖特异激活。TLR4在神经元和胶质细胞均有表达，但可能具有不同信号途径。TLR4参与癫痫发病，但具体作用及机制尚待阐明。海马是癫痫易感区，神经元突触发育、传递及可塑性异常会导致兴奋与抑制神经环路失衡，这也是癫痫发病机制之一。激活TLR4可能影响神经元发育、存活及海马相关学习记忆功能，提示TLR4可通过影响神经元发育和突触可塑性而介入癫痫发病。本项目将采用形态学、分子细胞生物学、电生理和行为学及基因敲除小鼠研究TLR4对海马神经元发育、突触可塑性及癫痫发作的影响及其机制，旨在揭示TLR4介导的免疫反应参与癫痫发病的神经生物学机制，也是将来进一步研究新型癫痫动物模型及癫痫疗法的重要前提。

早期发育阶段癫痫发作的高发性表明发作与癫痫的发生可能与大脑发育密切相关。发育过程中很多危险因素，如免疫刺激，可以影响大脑的发育，提示免疫激活可能是导致大脑发育异常继而引发癫痫的一个关键因素。星形胶质细胞参与调节神经元及其环路的发育过程，其异常发育和功能可诱发包括癫痫在内的许多神经系统发育障碍疾病。此外，模式识别受体Toll样受体4（TLR4）在星形胶质细胞中表达，而TLR4激活与癫痫的发生密切相关。但是在发育过程中星形胶质细胞TLR4激活是否通过调控神经元与突触的发育而造成癫痫发作的易感性这一过程及其机制尚不清楚。在此项研究中，我们发现TLR4配体脂多糖（lipopolysaccharide，LPS）通过激活星形胶质细胞免疫反应，促进发育关键期（出生后第2周）幼年小鼠海马神经元兴奋性突触的发生，从而增加小鼠的癫痫发作易感性。星形胶质细胞TLR4激活通过髓样分化因子88（myeloid differentiation primary response protein 88，MyD88）-依赖性途径上调星形胶质细胞ERK1/2以及phospho-ERK1/2的水平。组成性上调星形胶质细胞ERK1/2的表达可以在不激活TLR4的情况下促进海马神经元上兴奋性突触的发生。特异性敲除星形胶质细胞MyD88或者抑制星形胶质细胞ERK1/2则可以挽救LPS引起的海马神经元兴奋性突触发育异常，并使小鼠的癫痫发作易感性恢复正常。综上所述，我们的研究发现1）发育关键期激活星形胶质细胞可以增加小鼠的癫痫发作易感性；2）星形胶质细胞TLR4-MyD88-ERK信号通路和海马神经元兴奋性突触发生相关；3）激活星形胶质细胞的ERK足以促进海马神经元兴奋性突触发生；4）抑制星形胶质细胞TLR4通路可以使免疫刺激造成的癫痫发作易感现象恢复。本研究探明了在出生后发育过程中由星形胶质细胞上TLR4介导的、在外周免疫刺激诱导癫痫发作易感性中发挥重要作用的信号通路。这一信号通路也可能与中枢神经系统直接感染引起的儿童发作与癫痫的发生密切相关。大多数与儿童期发作和癫痫的发生相关的病原体是由MyD88依赖性的TLRs识别，因此，MyD88依赖的ERK1/2激活可以作为诸多免疫激活相关疾病的共同病理机制。