阿尔茨海默病易感基因TREM2的精细作图及其致病机制研究

Recently, two large-scale studies have revealed that R47H, a rare variant within exon 2 of triggering receptor expressed on myeloid cells 2 (TREM2), increases susceptibility to late-onset Alzheimer's disease (LOAD) with an odds ratio similar to that of the apolipoprotein E ε4 allele in Caucasian population, suggesing that TREM2 represents an important susceptibility gene for LOAD. To date, the assoication of TREM2 with LOAD risk in Han Chinese remains unclear, and few studies have focused on the role of TREM2 in AD pathogenesis. In our preliminary studies, we provided the first evidence that TREM2 was associated with LOAD risk in Han Chinese, whereas the frequency and distribution of TREM2 variants were quite different with those in Caucasians, possibly due to the differences in genetic background. Meanwhile, we demonstrated that the expression of TREM2 might be affected by Aβ levels, and TREM2 was able to modulate the activation and function of microglia, the major type of macrophage in brain that was implicated in AD pathogenesis. Based on these findings, we intend to finely map TREM2 to identify risk variants for LOAD in a large Han Chinese cohort. In addition, to fully elucidate the role of TREM2 in AD pathogenesis, we investigate the impact of Aβ oligomer on the expresson and downstream signaling of TREM2 in vitro and in vivo first. Afterwards, we intend to manipulate TREM2 expression using lentivirus vectors to reveal its modulation on activation and function of microglia as well as AD-related neuropathology and congitive impairment. The completion of this project will provide theoretic support for early warning and diagnosis of AD in Han Chinese, and will offer novel target for therapies and durg development of this disease via uncovering its underlying pathogenesis.

近期，两项大规模高加索人群二代测序研究发现TREM2为AD新的易感基因，其稀有变异R47H可增加患病风险3倍以上，这与APOEε4接近。我们前期率先证实TREM2与汉族人群AD易感性存在一定关联，但未检测到R47H变异，提示TREM2的SNP存在种族差异。同时，我们发现TREM2可参与调控小胶质细胞激活状态和功能，且其表达可能受Aβ影响。在此基础上，本项目拟对汉族AD患者和健康对照的TREM2重点区域进行第二代高通量测序，筛选出AD易感SNP，以全面揭示汉族人群TREM2与AD易感性的关系。此外，我们拟运用Aβ刺激离体和在体小胶质细胞，观察其对TREM2表达水平及下游信号转导的作用，同时运用慢病毒为载体的基因过表达/干扰技术，在细胞层面和活体层面对TREM2进行靶向干预，研究其对小胶质细胞激活状态及功能的调控和对AD病理特征及认知功能的影响，以全面阐明TREM2参与AD病理进程的分子机制。

本项目在前期建立的汉族人阿尔茨海默病遗传资源库基础上，应用第二代高通量测序技术对TREM2重要功能区进行精细作图，首次发现TREM2基因H157Y突变可增加AD发病风险11倍，该发现已被ALZFORUM MUTATIONS数据库收录，并被发表在Nat Genet和Alzheimers Demen中的两项AD大规模的全外显子测序研究证实，该突变亦可显著增加高加索人AD患病风险4.7和2.16倍。我们进一步对TREM2在AD发病中的作用及机制进行了深入研究，我们应用APPswe/PS1dE9和P301S等AD转基因小鼠，首次证实上调TREM2可以增强小胶质细胞对Aβ的吞噬能力，抑制小胶质细胞炎症因子的产生和分泌，下调周边神经元内tau激酶活性，从而改善AD的病理变化和认知功能。此外，我们还发现TREM家族的另一关键成员TREM1的rs6910730G风险变异，可以显著降低外周血单核细胞对Aβ的吞噬能力。同时，我们应用体内、外实验和APP/PSEN1转基因小鼠模型研究发现：靶向沉默TREM1可衰减小胶质细胞对Aβ 的吞噬作用，显著增加AD小鼠脑内Aβ病理；而靶向上调TREM1及应用TREM1激动性抗体均可易化小胶质细胞对Aβ的吞噬作用，改善AD小鼠脑内Aβ病理及空间学习记忆能力。本项目的研究成果揭示了TREM1和TREM2在AD发生中的作用机制，为AD的防治提供了新靶点。本研究成果在Acta Neuropathol、Neuropsychopharmacology等杂志共发表SCI收录论文5篇，目前已被Cell、Lancet Neurology等高影响力国际期刊引用200余次。本项目研究成果获批发明专利1项，荣获中华医学会医学科技奖三等奖1项，中华医学会青年科技奖1项，山东省科技进步二等奖2项，青岛市自然科学奖一等奖1项，青岛市科技进步二等奖1项。本项目研究成果还受邀在美国神经病学年会、日本神经病学会议等国内外会议上发言并获得10项国际学术奖励。在本项目的支持下，项目负责人赴美国加州大学旧金山分校神经内科完成博士后工作，并于2016年入选山东省“泰山学者”和青岛市“创业创新领军人才”。同时本项目共培养博士3名，硕士4名。研究成果已在多家大型三甲医院中推广应用，为后续深入探讨AD发生机制和防治措施奠定了坚实基础，取得了显著的社会效益。