秀丽线虫中MADD-4通过突触后分化调控蛋白调节GABABR的突触后定位的机制研究

The metabotropic type B γ-aminobutyric receptor (GABABR) is one member of G protein-coupled recepter family C. Deficiency in GABABR is linked to several neuropsychiatric diseases such as epilepsy and schizophrenia. Thus GABABR is an important therapeutic target against these diseases.To date, however, the molecular mechanisms of GABABR localization at postsynaptic site is largely unknown although GABABR clusters and localizes at both synaptic and extra-synaptic sites in mouse hippocumpus regions. In this project, we are planning to investigate the novel molecular mechanisms of GABABR postsynaptic localization by using Caenorhabditis elegans neuromuscular junction (NMJ) as a model system. The applicant previously generated a GFP-GBB-1 knock-in strain using MosTIC genome editing technique and found that GFP-GBB-1 formed clusters and localized post-syntaptically at both cholinergic and GABAergic NMJs. In madd-4 null mutant lacking an extracellular matrix protein MADD-4 or unc-40 null mutant lacking the netrin receptor DCC (Deleted in Colorectal Cancer), however, GABABR mislocalized at extra-synaptic regions of muscle cells suggesting that MADD-4 and UNC-40 coordinately regulate GABABR postsynaptic localization. By using genetics, immunofluorescence staining, confocal imaging in living animals, quantitative analysis of fluorescence and electrophysiological recordering techniques, we are planning to investigate and elucidate the molecular and genetic mechanisms of GABABR post-synaptic localization and funtion at NMJs. Under this project support, we might not only uncover a complete novel molecular mechanism for G-protein coupled GABAB receptor postsynaptic localization and its new function in muscle cells, reveal the new function of extracellular matrix protein Punctin/MADD-4 and UNC-40/DCC, but also provide some insights into understanding the pathology of some neuropsychiatric diseases related to abnormal localization and function of GABABR.

代谢型γ-氨基丁酸B型受体(GABABR)与多种神经和精神疾病密切相关。已有研究表明GABABR在大脑海马区聚集成簇并定位在突触和突触外位点，然而其定位的分子机制不明。申请人前期发现在进化上保守的秀丽隐杆线虫GABABR聚集成簇并定位在胆碱能和GABA能神经肌肉接头突触后位点。但在细胞外基质蛋白MADD-4或UNC-40编码基因缺失的遗传背景下，GABABR的突触后定位却明显异常，提示MADD-4与UNC-40共同调控GABABR突触后位点的正确定位，申请人由此提出MADD-4通过突触后分化调控蛋白调节GABABR的突触后定位假说，并将采用遗传学，免疫荧光标记与共聚焦成像等技术研究GABABR突触后定位全新的分子机制。通过本项目的研究，将揭示MADD-4与UNC-40等蛋白的新功能，从而为揭示GABABR的突触后位点定位和功能异常所导致的相关神经与精神疾病的机理奠定一定的理论基础。

本项目采用遗传学，免疫荧光标记，活体共聚焦成像与荧光定量分析技术深入研究MADD-4调控GABABR突触后定位全新的分子机制，研究和分析了运动神经元支配对突触后GABABR的突触定位的影响研究，在缺失胆碱能运动神经元支配的unc-3突变体中，GABABR仅定位在GABA能突触后位点，而在缺失GABA能运动神经元支配的unc-30的突变体中，GABABR仅定位在胆碱能突触后位点，这些结果提示在线虫神经肌肉机头突触前的运动神经元可能除表达分泌MADD-4细胞外基质蛋白对突触后GABABR的正确定位进行调控以外，还表达分泌另外一种蛋白，并和MADD-4共同调控突触后GABABR的突触定位。. 另外，通过缺血脑切片进行的核酸适配体筛选方法，我们成功筛选到了能够靶向缺血脑切片的核酸适配体LCW17，并且鉴定出其靶标蛋白为Vigilin蛋白。 核酸适配体LCW17可以作为一个检测初期脑缺血疾病，靶向治疗和疾病发生机制研究的有效分子工具。研究和鉴定了ENO1蛋白参与了脑缺血进程，并利用体外系统，发现ENO1及其酶解产物PEP在缺氧缺糖的病理发生过程中，对神经元的树突及树突棘有保护作用，从而为进一步深入理解脑卒中导致神经损伤的病理机制提供了新机制。.研究和阐释了GABA能突触通过肌肉表达分泌胰岛素样因子INS-31调控肠道固有免疫反应机制，从而揭示了突触—肌肉胰岛素—肠道免疫这一全新的信号轴；研究和发现了转录因子BATRF3/ZIP-10通过抑制保守的PMK-1/p38丝裂原激活蛋白信号通路削弱宿主对细菌感染的抵抗力；细胞外基质蛋白MADD-4与免疫球蛋白结构域蛋白OIG-1协同抑制突触重塑的遗传机制。