常染色体显性遗传家族性阿尔茨海默病候选基因XP2的功能研究

XP2 was a novel candidate gene of autosomal dominant inherited familial Alzheimer’s disease (AD) through exome sequencing analysis in our previous work. XP2 encoding protein belongs to the metalloprotease family. Its exact function remains unknown, however, the XP1 protein, which sharing as much as 60% homologous with XP2, has already been proved to participant in the degradation of Aβ42. Furthermore, we found that XP2 gene was highly expressed in hippocampus compared with other brain regions; the mutant residue was predicted to have functional impact by SIFT, Polyphen2 and MutationTaster. Moreover, overexpression of XP2-RNNC led to a higher Aβ42 level compared with that of wild-type XP2 in APPsw cell model. Taken together, these results suggest that XP2 has important physiological function and may play an important role in the pathogenesis of AD. In this research, we intend to clarify whether the mutants of XP2 impact its expression, stabilization, enzymatic activity, subcellular location, APP processing, Aβ degradation or cell apoptosis in cell models. In the meanwhile, brain stereotactic injection strategy will be performed to observe the behavior and histopathology alterations of APP23 transgenic mice after injecting lentivirus contained either XP2 RNAi, XP2 wild-type rescue or XP2 mutant rescue vectors into their hippocampus regions. All these might help us to understand the physiological function of XP2 and reveal its role in AD pathology, and provide new clues of pathogenesis of AD.

XP2基因是本课题组在前期工作中发现的家族性阿尔茨海默病（Alzheimer’s disease, AD）新的候选基因，该基因编码蛋白为金属蛋白酶，功能尚不清楚，与其具有60%同源性的另一基因XP1已在功能上证实与AD发病相关，且参与了Aβ的降解通路。前期工作中我们已证实该蛋白在小鼠海马区高表达；功能预测提示在家系内发现的错义突变具有致病性；在AD细胞模型中过表达突变型XP2时Aβ42水平较野生型明显增高，提示XP2蛋白可能参与了AD的致病过程。本课题拟在此基础上，在细胞水平研究XP2基因突变对蛋白稳定性、酶活性、亚细胞定位、APP剪切、Aβ降解及细胞凋亡等的影响；同时采用慢病毒脑定位注射策略，在体分析XP2基因RNAi、野生型及突变型XP2蛋白对Aβ生成和降解及对APP23转基因小鼠病理及行为学的影响，明确XP2蛋白在AD发病过程中的可能作用，为揭示AD致病机制提供新的线索。

阿尔茨海默病（Alzheimer’s Disease, AD）又称为老年痴呆症，是一种常见的、与年龄密切相关的神经退行性疾病，Aβ异常聚集和沉积是AD发病机制中的重要环节。前期研究中，我们发现一个排除已知致病基因及风险基因突变的晚发性AD（Late onset AD, LOAD）大家系，其中3名患者携带ECE2基因R186C序列变异，且该变异在家系内共分离。在本研究中，我们纳入741例AD患者及545例正常对照进行ECE2基因序列分析，在AD患者中共发现ECE2基因罕见变异17个，在正常对照中发现ECE2基因罕见变异5个，通过与ExAc亚洲人群数据库比对，未发现单个位点罕见变异与AD患病风险相关，通过Burden检测发现罕见变异在AD患者中的富集程度是正常对照的5.51倍（P = 1.59E-04）。在细胞水平研究中，我们发现ECE2蛋白具有降解Aβ的功能，而ECE2基因R186C和F751S突变可导致ECE2酶活性下降，并导致ECE2蛋白降解Aβ的能力受损，是有害性变异。我们进一步评估了R186C突变对AD小鼠模型的影响，在AD模型小鼠海马中过度表达野生型ECE-2降低了其脑内淀粉样蛋白负荷和斑块形成，改善了学习和记忆障碍；然而，过表达R186C突变型ECE-2蛋白没有这种营救效应。综上所述，结果表明ECE-2肽酶突变通过破坏Aβ降解而参与AD的发病，ECE2的过度表达减轻了AD的表型。本研究表明ECE2是AD发生发展的风险基因，ECE2的激活药物可能是AD治疗领域一个有前景的策略。