Role of soluble TREM2 and its R47H and D87N variants in neurodegenerative disease

描述（由申请人提供）：作为国际合作的一部分，我们提供了强有力的证据，表明TREM2 R47H与LOAD显著相关，表明TREM2与LOAD有关。在我们超过11,000名受试者的联合病例对照系列中，R47H的优势比为4.0 (P=6.6x10-9)，与众所周知的APOE¿4等位基因相似。在这个系列中，第二个TREM2错义变体D87N也显示出显著的相关性。值得注意的是，我们最近参与的一项研究表明，与R47H相关的风险从AD扩展到FTD和PD，这表明TREM2可能是一种普遍的神经退行性疾病风险基因。TREM2是小胶质细胞中的一种跨膜信号受体，已知与其接头蛋白TYROBP（也称为DAP12）一起作用，影响凋亡神经元的非炎性吞噬。与疾病相关的TREM2变异可能通过改变这种跨膜受体的正常功能而起作用。然而，已知TREM2的可溶性形式在脑脊液中可检测到，并且在多发性硬化症和中枢神经系统炎症患者中增加。R47H和D87N变异位于TREM2的胞外结构域，因此会出现在TREM2中。在这里，我们探索了一种假设，即疾病相关的R47H和D87N变异可能对TREM2而不是全长TREM2 （FL-TREM2）发挥部分或全部的疾病修饰作用。strem - 2可以通过两种不相互排斥的机制产生：FL-TREM2的蛋白水解裂解和包含跨膜结构域的外显子4的选择性剪接。我们的初步数据和最近发表的一项研究表明，在培养细胞中，sTREM2是通过蛋白水解裂解产生的。R47H和D87N可以通过改变这种蛋白水解裂解作用，从而改变可溶性和FL-TREM2的相对量。如果strem - 2作为一个诱饵受体，可以触发fl - trem - 2的信号，那么R47H和D87N可能通过改变strem - 2与这些信号配体的相互作用而起额外的作用。为了在体外评估这些机制，我们将在表达人WT、R47H和D87N TREM2的细胞系和原代小鼠小胶质细胞培养物中，用夹心ELISA法定量sTREM2和全长TREM2。为了评估TREM2的功能作用，我们将生成WT、R47H和D87N TREM2的可溶性形式，并在分析凋亡神经元直接结合和吞噬的功能分析中测试它们的作用。在补充的体内研究中，我们将使用来自梅奥诊所衰老研究的样本来评估(i) sTREM2与R47H变异之间的关系，（ii） sTREM2与转化为MCI或AD之间的关系。在脑样本中，我们将评估R47H与sTREM2、FL-TREM2、sTREM2/FL-TREM2比值、Braak分期、A¿生化指标以及淀粉样蛋白和tau病理的免疫组织病理学指标的关系。我们还将分析AD大脑中与非AD大脑相比的sTREM2、FL-TREM2和sTREM2/FL-TREM2比率。

DESCRIPTION (provided by applicant): As part of an international collaboration, we provided strong evidence implicating TREM2 in LOAD by showing that TREM2 R47H is significantly associated with LOAD. In our combined case-control series of over 11,000 subjects, R47H has an odds ratio of 4.0 (P=6.6x10-9) similar to that of the well-known APOE ¿4 allele. In this series, a second TREM2 missense variant, D87N, also shows significant association. Remarkably, a recent study in which we participated shows that the risk associated with R47H extends beyond AD to FTD and PD, indicating that TREM2 may be a general neurodegenerative disease risk gene. TREM2 is a transmembrane signaling receptor in microglia known to function with its adaptor protein TYROBP (also known as DAP12) to effect non-inflammatory phagocytosis of apoptotic neurons. The disease-associated TREM2 variants may well act by altering the normal functioning of this transmembrane receptor. It is known, however, that soluble forms of TREM2 (sTREM2) are detectable in CSF, and are increased in patients with multiple sclerosis and CNS inflammation. The R47H and D87N variants are located in the extracellular domain of TREM2 and will, therefore, be present in sTREM2. Here, we explore the hypothesis that the disease-associated R47H and D87N variants may exert some or all of their disease-modifying effects on sTREM2 rather than on full-length TREM2 (FL-TREM2). sTREM2 could be generated by two mechanisms that are not mutually exclusive: proteolytic cleavage of FL-TREM2 and alternative splicing of exon 4, which contains the membrane-spanning domain. Our preliminary data and a recently published study show that, in cultured cells, sTREM2 is produced by proteolytic cleavage. R47H and D87N could act by altering this proteolytic cleavage thereby altering the relative amount of soluble and FL-TREM2. If sTREM2 functions as a decoy receptor for ligands that trigger signaling by FL-TREM2, then R47H and D87N may act additionally by altering the interaction of sTREM2 with these signaling ligands. To evaluate these mechanisms in vitro, we will quantitate sTREM2 and full-length TREM2 by sandwich ELISA in cell lines and in primary mouse microglia cultures expressing human WT, R47H, and D87N TREM2. To evaluate functional effects of sTREM2, we will generate soluble forms of WT, R47H, and D87N TREM2, and test their effects in functional assays analyzing direct binding to and phagocytosis of apoptotic neurons. In complementary in vivo studies, we will employ samples from the Mayo Clinic Study of Aging to evaluate associations (i) between sTREM2 and the R47H variant and (ii) between sTREM2 and conversion to MCI or AD. In brain samples, we will evaluate the association of R47H with sTREM2, FL- TREM2, the sTREM2/FL-TREM2 ratio, Braak stage, biochemical measures of A¿, and immunohistopathological measures of amyloid and tau pathology. We will also analyze sTREM2, FL-TREM2 and the sTREM2/FL-TREM2 ratio in AD as compared to non-AD brains.