内质网蛋白SIL1对NMDA受体运输的调节机制及其对突触可塑性的影响

Endoplasmic Reticulum (ER) is an important organelle that responsible for the synthesis, folding and assembly of transmembrane proteins and secretory proteins. Recent years, it is found that the proper function of ER is of required for the plasticity of central nervous system. The non-canonical trafficking is a newly discovered trafficking pathway for dendritic localized proteins, in which the nascent proteins were synthesized in dendrite but not soma, and thus the nascent protein could quickly be transported into synapse. Several important dendritic localized neuronal receptors, including the NMDA receptor, have been proved to be transported non-canonically. However, it is not clear whether or not the non-canonical trafficking is required for the neuronal plasticity, for example long-term potentiation (LTP) and long-term depression (LTD). In other words, the physiological function of neuronal non-canonical pathway is unknown yet. Recent works on the genetics of disease has found a single gene SIL1, the mutation of which resulted in Marinesco-Sj?gren Syndrome (MSS). The MSS is an autosomal recessively inherited neurodegenerative disorder characterized by cerebellar ataxia, cataracts and mental retardation. SIL is the nucleotide exchange factor of Bip, which is a multi-functional ER chaperone. It is interesting that a single mutation of an ER localized protein causes disorders of central nervous system. It is possible that the mutation of SIL1 disrupted the local synthesis in ER and thus leading to the mental retardation. In this project, we set out a serious of experiments to explore this hypothesis and thus to make clear the physiological function of local synthesis in neuron. Since we have found in our earlier study that the GluN2A subunit of NMDA receptor was the substrate of Bip and thus whose synthesis and assembly with GluN1 may be regulated by SIL1. It is well-known that the NMDA receptor plays key roles in multiple high level functions of the central nervous system, including LTP and LTD, both of which are considered as the molecular basis of learning and memory. Thus in this project we will focus on studying the deficits in the synthesis, trafficking and function of NMDA receptor, especially the GluN2A-containing NMDA receptor resulted from SIL1 mutation. Through this project, we hope to uncover the molecular mechanism underling the mental retardation of MSS, and to proved evidence for the physiological importance of dendritic ER-involved local synthesis of neuron.

依赖于内质网的树突局部合成可以为突触快速提供新生蛋白质，这一过程被称为非经典运输途径。然而，这一途径是否参与保障诸如长时程增强、长时程减弱等可塑性变化的顺利完成，也即其功能意义，目前并不清楚。内质网分子伴侣蛋白Bip的辅助因子SIL1的突变将会导致Marinesco-Sjogren综合症，该疾病经典症状之一为智力发育障碍。而我们的前期研究显示，NMDA受体的GluN2A亚基受可塑性刺激,在Bip介导下经由非经典途径运输表达。基于以上两点，我们认为SIL1的突变可能因为干扰了GluN2A的非经典运输而引起智力发育障碍。在本项目中，我们将重点研究SIL1的功能缺陷对于Bip的底物蛋白--NMDA受体GluN2A亚基的表达、运输及功能的影响，尤其是对神经元可塑性刺激调节含GluN2A的NMDA受体的突触表达的过程及NMDA受体参与的学习记忆功能的影响，从而揭示非经典运输在中枢神经系统的功能意义。

背景：NMDA受体是中枢神经系统重要的兴奋性受体，在受体合成装配完成后，需要准确定位到树突，突触外或突触上，并且还要响应可塑性刺激，动态调整其表达。这一过程受到细胞内、外复杂机制的精确调控，并且在中枢可塑性和学习记忆过程中发挥重要作用。我们前期研究发现，NMDA受体存在非经典运输，即它的GluN2A亚基可以在树突内置网中合成，并且在LTP刺激下迅速上膜表达。但是其分子机制未阐明，其生理意义也不明确。方向：在本项目中，我们研究了内质网分子伴侣蛋白Bip和它的辅助因子SIL1在NMDA受体运输表达过程中发挥的作用，以及这一调控机制对个体中枢可塑性的重要意义。主要内容、重要结果和关键数据：我们首先发现，Bip参与了NMDA受体GluN2A亚基的非经典运输。我们设计了干扰多肽阻断了Bip和GluN2A的相互作用之后，GluN2A无法响应可塑性刺激，其动态表达无法实现，并且小鼠的恐惧记忆受损。我们继而研究了是否SIL1作为Bip的辅助因子，参与了该过程。然而我们发现，SIL1并不参与成熟神经元中NMDA受体的非经典运输，但是参与了GluN2A的发育表达。沉默SIL1之后，发育过程中神经元成熟无法完成，表现为NMDA受体亚型组成异常，并且小鼠空间学习记忆能力受损。最后，我们进一步分析了发生这一现象的分子机制，发现沉默SIL1后Reelin信号通路受损，通过注射磷酸化多肽激活Reelin信号通路可以部分挽回小鼠的学习记忆损伤。科学意义：首先，本项目研究证实了非经典运输对中枢可塑性的重要意义。其次，本项目首次发现SIL1介导NMDA受体的成熟，并且阐明了其分子机制，拓展了SIL１蛋白的功能，并且为SIL１基因突变导致智力发育障碍的机制提供了新的研究视角。