Soluble TREM2 regulation of microglial function in Alzheimer disease

项目总结 阿尔茨海默病(AD)是美国第六大致死原因，超过580万人 美国人患有这种疾病。今年，AD和其他痴呆症的估计成本为3050亿美元，以及 到2050年可能会上升到1.1万亿美元。阿尔茨海默病的病因仍然难以捉摸，可能是多因素造成的。最大的风险 已知的影响因素有年龄、遗传和遗传。全基因组关联研究表明， TREM2的变异与AD发病风险的两到四倍有关。TREM2是一种先天免疫 在小胶质细胞中表达的受体，已被证明与磷脂、凋亡细胞、脂蛋白、 和低聚形式的淀粉样蛋白-β(Aβ)。这种蛋白质已被证明是小胶质细胞增殖所必需的， Aβ的存活和吞噬作用。TREM2是一种跨膜蛋白，不含细胞内信号域； 信号转导需要与DAP12结合。在小胶质细胞激活和聚集时，TREM2被从 产生可溶性TREM2(STREM2)的膜。由于膜结合TREM2和sTREM2 在AD的不同阶段共存，很难区分每种形式的程度 独立为疾病病理学做出贡献。最近有研究表明，鞘内给药后， STREM2与小胶质细胞结合，传递细胞内信号，促进生存，改善认知功能， 和减轻β的病理；据推测，sTREM2通过一个假定的但未知的受体发挥作用。 在这项提案中，我们将利用CRISPR/Cas9全基因组筛选来鉴定与sTREM2结合的受体 并促进信号转导。已确定的候选基因将在人类小胶质细胞中得到验证 将生成细胞和基因敲除小鼠模型，以进一步探讨每种模型在发育中的重要性 阿尔茨海默病及其病理生理学。这些模型将作为工具，使我们能够确定sTREM2如何 独立于膜结合的TREM2调节小胶质细胞。这样的发现将成为 旨在调节sTREM2受体的新疗法。STREM2也被证明与β结合 斑块。我们将利用这一观察结果，开发sTREM2组分可结晶(sTREM2-FC)融合 结合Aβ斑块并促进小胶质细胞激活和吞噬的蛋白质。STREM2-Fc融合 蛋白质将使用5XFAD小鼠的大脑切片进行验证，这些小鼠积累了AD典型的A斑块。我们 还将进行鞘内注射，这将使我们能够评估sTREM2-FC在消除 斑块和激活的小胶质细胞。总而言之，这些都是可能有助于开发治疗方法的新方法 增强清除和防止淀粉样蛋白-β斑块形成用于治疗AD。

PROJECT SUMMARY Alzheimer's Disease (AD) is the 6th leading cause of death in the United States and more than 5.8 million Americans suffer from this disease. This year the estimated cost of AD and other dementias is $305 billion, and could rise to $1.1 trillion by 2050. The cause of AD remains elusive and is likely multifactorial. The greatest risk factors are known to be age, genetics, and inheritance. Genome-wide association studies have shown that variants of TREM2 are associated with two to four times the risk of developing AD. TREM2 is an innate immune receptor expressed in microglia that has been shown to interact with phospholipids, apoptotic cells, lipoproteins, and oligomeric forms of amyloid-β (Aβ). This protein has been shown to be required for microglial proliferation, survival, and phagocytosis of Aβ. TREM2 is a transmembrane protein with no intracellular signaling domains; signal transduction requires binding to DAP12. Upon microglial activation and clustering, TREM2 is cleaved from the membrane generating soluble TREM2 (sTREM2). Since both membrane-bound TREM2 and sTREM2 coexist during different stages of AD, it has been difficult to differentiate the extent of which each form independently contributes to disease pathology. It was recently shown that after intrathecal administration, sTREM2 binds to microglia, transmitting intracellular signals, promoting survival, improving cognitive function, and mitigating Aβ pathology; it has been postulated that sTREM2 acts through a putative yet unknown receptor. In this proposal we will utilize a CRISPR/Cas9 whole genome screen to identify the receptor that binds sTREM2 on microglia and promotes signal transduction. Identified candidate genes will be validated in human microglial cells and knockout mouse models will be generated to further explore the importance of each in the development of AD and its pathophysiology. These models will serve as tools that will permit us to identify how sTREM2 modulates microglia independently from membrane-bound TREM2. Such discovery will serve as the basis for novel therapeutics aimed at modulation of the sTREM2 receptor. sTREM2 has also been shown to bind Aβ plaques. We will exploit this observation and develop sTREM2-fraction crystallizable(sTREM2-Fc) fusion proteins that bind Aβ plaques and promote activation of microglia and phagocytosis. The sTREM2-Fc fusion proteins will be validated using slices of brains from 5XFAD mice that accumulate A plaques typical of AD. We will also perform intrathecal injections, which will allow us to evaluate the efficacy of sTREM2-Fc in eliminating plaques and activating microglia. Taken together, these are novel approaches that may help to develop therapies that enhance clearance and prevention of amyloid-β plaque formation for the treatment of AD.