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，我们通过显示TREM 2 R47 H与LOAD显著相关，提供了在LOAD中涉及TREM 2的强有力证据。在我们超过11，000例受试者的联合病例对照系列中，R47 H的比值比为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TREM 2错义变体D87 N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R47 H相关的风险从AD扩展到FTD和PD，这表明TREM 2可能是一种一般的神经退行性疾病风险基因。TREM 2是小胶质细胞中的跨膜信号传导受体，已知其与其衔接蛋白TYROBP（也称为DAP 12）一起发挥作用以实现凋亡神经元的非炎性吞噬作用。疾病相关的TREM 2变体可能通过改变这种跨膜受体的正常功能而发挥作用。然而，已知可溶形式的TREM 2（sTREM 2）在CSF中是可检测的，并且在患有多发性硬化和CNS炎症的患者中增加。R47 H和D87 N变体位于TREM 2的细胞外结构域中，因此将存在于sTREM 2中。在这里，我们探索了疾病相关的R47 H和D87 N变体可能对sTREM 2而不是全长TREM 2（FL-TREM 2）发挥部分或全部疾病修饰作用的假设。sTREM 2可以通过两种不相互排斥的机制产生：FL-TREM 2的蛋白水解切割和外显子4的选择性剪接，外显子4包含跨膜结构域。我们的初步数据和最近发表的研究表明，在培养的细胞中，sTREM 2是通过蛋白水解裂解产生的。R47 H和D87 N可以通过改变这种蛋白水解切割从而改变可溶性和FL-TREM 2的相对量来起作用。如果sTREM 2作为触发FL-TREM 2信号传导的配体的诱饵受体起作用，则R47 H和D87 N可以通过改变sTREM 2与这些信号传导配体的相互作用而另外起作用。为了在体外评估这些机制，我们将通过夹心ELISA在表达人WT、R47 H和D87 N TREM 2的细胞系和原代小鼠小胶质细胞培养物中定量sTREM 2和全长TREM 2。为了评估sTREM 2的功能作用，我们将产生WT、R47 H和D87 N TREM 2的可溶形式，并在分析与凋亡神经元的直接结合和吞噬作用的功能测定中测试它们的作用。在补充的体内研究中，我们将使用来自马约诊所衰老研究的样本来评估（i）sTREM 2与R47 H变体之间的关联和（ii）sTREM 2与转化为MCI或AD之间的关联。在脑样品中，我们将评估R47 H与sTREM 2、FL-TREM 2、sTREM 2/FL-TREM 2比率、Braak分期、A的生物化学测量以及淀粉样蛋白和tau病理学的免疫组织病理学测量的关联。我们还将分析与非AD脑相比AD中的sTREM 2、FL-TREM 2和sTREM 2/FL-TREM 2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.