p75NTR基因rs2072446多态性所致错义突变在阿尔茨海默病发生中的作用及机制研究

P75 neurotrophin receptor (p75NTR) mediates Aβ-induced neurotoxicity in the pathogenesis of Alzheimer’s disease (AD). Recently, we found that p75NTR increase the production of Aβ; meanwhile, the soluble extracellular domain (ECD) generated with the digestion of enzyme can inhibit the aggregation of Aβ and block Aβ-induced neurotoxicity. We also revealed that rs2072446 located in the fourth exon within p75NTR coding gene was related to the increased risk of AD. Moreover, the level of ECD was significantly reduced in the plasma of mutant allele carrier. The location of this mutant amino acid is in close proximity to the cleavage site leading to ECD shedding as well as the binding site of Aβ. Function analysis also indicated that this mutation is damage to the structure of p75NTR. Therefore, we speculate that the mutation caused by rs2072446 may influence the shedding of ECD or the binding of Aβ to p75NTR, thus promoting the progression of AD. In this project, biosensor technique will be applied to detect the affinity of Aβ for mutant or wild type p75NTR. We intended to investigate the effect of this mutation on the shedding of ECD and Aβ-induced neurotoxicity by transfection of mutant p75NTR gene to cultured hippocampus neurons from p75NTR-/- mice. And the mutant p75NTR gene will be further transfected to the brain of p75NTR-/-APPSwe mice in vivo to study the role of rs2072446 in the pathogenesis of AD. Besides, we will observe the relationship between rs2072446 and ECD level, AD biomarkers and cognition function through follow-up visits of mutant allele carriers and control group. This project is of great importance to reveal the pathogenesis mechanism of AD.

阿尔茨海默病（AD）中神经营养因子受体p75NTR介导Aβ的神经毒性。我们发现，AD脑内p75NTR促进Aβ产生；p75NTR释放的胞外段（ECD）具有抑制Aβ沉积和拮抗Aβ毒性的保护作用；p75NTR基因rs2072446多态性增加AD风险，且突变携带者血液ECD降低。该突变氨基酸靠近ECD释放酶切位点和Aβ结合区，功能分析提示其对蛋白结构影响显著，故推测该突变可能通过影响ECD释放或p75NTR与Aβ的结合能力而促进AD发生。本项目拟采用生物传感器检测rs2072446对p75NTR结合Aβ能力的影响；采用突变型p75NTR基因转染原代p75NTR敲除小鼠海马神经元，探讨其对ECD释放的影响；进一步转染p75NTR敲除AD小鼠，探讨该突变对Aβ代谢和神经毒性作用的影响；最后通过人群研究，探讨该突变与ECD、AD标志物和认知功能变化的相关性。本项目对于揭示AD发生机制具有重要意义。

β淀粉样蛋白（Aβ）是阿尔茨海默病（AD）的核心致病物质。神经营养因子受体p75NTR作为Aβ的关键受体，在促进Aβ产生和沉积、介导Aβ神经毒性等AD发病环节中发挥了重要作用。p75NTR经剪切后释放的胞外段（p75ECD）具有抑制Aβ产生、阻止Aβ聚集和拮抗Aβ神经毒性的作用，是一种内源性AD保护性分子，因而p75ECD释放障碍可促进AD的发生发展。本项目研究发现：p75NTR编码基因第四号外显子上的rs2072446多态性位点可显著增加中国汉族人群的AD患病风险；将构建的野生型AAV、不剪切型AAV和突变型AAV转染HEK293T细胞后发现，rs2072446所致错义突变（S205L）可增加p75NTR介导的Aβ神经毒性，并抑制p75ECD释放；同时，rs2072446多态性位点与AD患者脑萎缩程度、脑内葡萄糖代谢率、Aβ沉积量及脑脊液生物标志物均存在显著相关性；p75NTR与Aβ特异结合的结构域为胞外段四个半胱氨酸富集区（CRD）中的CRD2和CRD4。本项目通过分析p75NTR与Aβ结合的结构域，以及rs2072446所致错义突变（S205L）对p75ECD 释放、p75NTR介导Aβ神经毒性和AD病程进展的影响，进一步阐明了p75NTR在AD发生中的作用及机制，为AD治疗策略提供了新思路。