细胞外基质蛋白MADD-4通过增强GABA能突触传递抑制固有免疫能力的机制研究

Multiple aspects of interactions between the nervous and immune systems have been demonstrated in homeostasis and neuropsychiatric disorders ranging from Alzheimer Disease (AD) to Autism Spectrum Disorder (ASD).However, the machinery and the role of neural chemical GABAergic synaptic transmission in the regulation of innate immunity remain unclear. ..In order to investigate the role of GABAergic neurotransmission in the intestinal innate immunity of Caenorhabditis elegans by using human opportunistic pathogen Pseudomonas aeruginosa PA14 to infect the nematode Caenorhabditis elegans as a model system, we found that the life span of animals infected by PA14 was significantly extended in ionotropic GABAA/unc-49 mutant, but not altered in metabotropic GABABR/gbb-1 or gbb-2 mutants as compared to that of wildtype, suggesting that GABAR/UNC-49 suppresses the innate immunity of C. elegans. Our tissue specific rescue data have shown that muscle, not intestine expressing GABAAR/UNC-49 restored the defect of lifespan in PA14 lawn, indicating that GABAAR/UNC-49 act in a cell autonomous manner. To test if the content of GABAergic synaptic components alter when worms are infected by PA14, we performed laser confocal microscopy imaging and then quantitatively analysed the intensity of postsynaptic GABAAR/UNC-49-tagRFP and presynaptic VAMP/SNB-1-GFP at 24 hours after PA14 infection. We found that the content of both postsynaptic GABAAR/UNC-49 and presynaptic VAMP/SNB-1 at GABAergic synapse increased upon PA14 infection as compared to OP50 control, suggesting that the strength of GABAergic synapse is increased upon PA14 infection. Since the extracellular matrix MADD-4 (with the long isoform MADD-4L and the short isoform MADD-4B) are key organizers of cholinergic and GABAergic postsynaptic membrane, respectively, to test if MADD-4B is involved in the GABAergic transmission mediated innate immunity of C. elegans, we performed survive assay of worms in full PA14 lawn NGM plate and found that madd-4B(tr185) mutant lacking the short isoform only and madd-4(kr270) mutant lacking both the long and short isoforms were resistant to PA14 infection, indicating that the short isoform MADD-4B suppresses the innate immunity. Our data also indicated that MADD-4B and GABAergic transmission act in the same pathway to inhibit the innate immunity of C. elegans. ..On the basis of current literature knowledge and our data, we hypothesize that the content of GABAergic synaptic components including postsynaptic neurotransmitter receptor GABAAR/UNC-49 and presynaptic VAMP/SNB-1, and the neuronal extracellular matrix MADD-4B are upregulated by an unknown transcriptional program stimulated by PA14. In turn, MADD-4B recruits more GABAAR/UNC-49 to GABAergic synapse and increases the strength of GABAergic synaptic transmission. Our preliminary data showed that the genetic pathway of the model is dependent of FOXO/DAF-16, but independent of p38 MAPK pathways involved in the innate immunity of C. elegans. ..Therefore, in this project, we are going to investigate the genetic, molecular and cellular mechanisms of MADD-4B mediating enhanced GABAergic synaptic transmission suppressing the innate immunity of C. elegans by using genetic and molecular techniques, RNAseq, patch clamp electrophysiology recording, laser confocal microscopy imaging and quantitative analysis.

研究表明神经与免疫系统之间存在多方面的相互作用，并与阿尔茨海默病和孤独症等多种神经精神疾病密切相关。但GABA能突触传递对固有免疫的作用及其调控机制还不清楚。我们前期结果表明GABAAR或MADD-4缺失均增强秀丽隐杆线虫对人源条件感染性病原菌PA14感染的固有免疫能力，PA14感染后GABA能突触传递增强。在前期研究基础上，本项目将采用Q-PCR、转录组测序、膜片钳电生理记录和活体共聚焦成像等技术深入研究病原菌感染线虫后，通过上调MADD-4与GABA能突触前与突触后结构与功能蛋白的表达及其突触定位，增强GABA能突触的传递，进而通过激活下游DAF-16及其靶基因、但不依赖于PMK-1信号通路，抑制机体的固有免疫能力。本项目的实施不仅研究和阐明细胞外基质蛋白MADD-4和GABA能突触传递调控固有免疫能力的遗传、分子与细胞新机制，而且拓展、丰富和加深我们对固有免疫神经系统调控机制的认识。

在该国家自然基金青年项目的资助下，我们首先开展了GABA能突触通过调节肌肉表达的胰岛素样因子INS-31，抑制线虫肠道先天免疫反应的研究。我们知道，GABA能突触传递是生物体中主要的抑制性信号通路，其在调控中枢神经系统生理机能方面扮演重要角色，并且可通过调节免疫细胞功能进而参与到自身免疫。然而，其在先天免疫调控中的作用仍不清楚。在本研究中，我们发现GABA能神经肌肉接头（NMJs）缺陷能够增强秀丽隐杆线虫对病原菌的抵抗能力。同时，病原菌的感染能够上调GABA能信号通路相关基因的表达并增强GABA能突触传递。我们鉴定到肌肉中表达的胰岛素样信号肽INS-31，其在肌肉中表达并作用于神经肌肉接头下游，以细胞非自主性方式抑制肠道先天免疫。我们的研究在生物机体内揭示了突触—肌肉胰岛素—肠道免疫这一全新的信号轴。该研究结果已经发表在美国科学院院刊PNAS杂志上（PNAS, 2021）。. 其次，我们还研究了细胞外基质蛋白MADD-4调节突触重塑的机制研究。由于突触重塑在健康和神经疾病中起着重要作用，虽然先前的研究表明，几个转录程序控制线虫突触重塑，但DD突触重塑的分子机制尚不清楚。本项目中，采用遗传学、分子生物学、生物化学以及共聚焦荧光成像与定量分析等技术，研究发现MADD-4的丢失表现出DD突触重塑的早熟启动时间。MADD-4L动态表达，MADD-4B持续表达于L1期DD神经元。在缺乏Pitx型同源同域转录因子UNC-30的UNC-30突变体中，MADD-4B和L亚型在L1期DD神经元中的表达水平显著降低。我们的进一步数据表明，MADD-4B和L亚型在物理上与OIG-1相互作用，并在OIG-1遗传途径中作用，调节DD突触重塑。我们的结果表明细胞外基质在突触可塑性中起着新的作用。该研究工作已经发表在化学神经科学杂志（ACS Chemical Neuroscience, 2021）。

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