Soluble TREM2 regulation of microglial function in Alzheimer disease

项目摘要 阿尔茨海默病（AD）是美国第六大死亡原因， 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， 到2050年将达到1.1万亿美元。AD的病因仍然难以捉摸，可能是多因素的。最大的风险 已知的因素是年龄、遗传和遗传。全基因组关联研究表明， TREM 2的变体与发展成AD的风险的2至4倍相关。TREM 2是一种先天免疫 在小胶质细胞中表达的受体，已显示与磷脂、凋亡细胞、脂蛋白 和淀粉样蛋白-β（Aβ）的寡聚体形式。这种蛋白质已被证明是小胶质细胞增殖所必需的， 存活和吞噬Aβ。TREM 2是不具有胞内信号传导结构域的跨膜蛋白; 信号转导需要与DAP 12结合。在小胶质细胞活化和成簇后，TREM 2被切割， 所述膜产生可溶性TREM 2（sTREM 2）。由于膜结合的TREM 2和sTREM 2都是 在AD的不同阶段共存，很难区分每种形式的程度 独立地对疾病病理学有贡献。最近显示鞘内给药后， sTREM 2与小胶质细胞结合，传递细胞内信号，促进存活，改善认知功能， 并减轻Aβ病理学;据推测sTREM 2通过一种推定的但未知的受体起作用。 在这项提案中，我们将利用CRISPR/Cas9全基因组筛选来鉴定结合sTREM 2的受体 并促进信号转导。确定的候选基因将在人类小胶质细胞中进行验证。 细胞和基因敲除小鼠模型将被生成，以进一步探索每一个在发展中的重要性。 AD及其病理生理学。这些模型将作为工具，使我们能够确定sTREM 2 独立于膜结合的TREM 2调节小胶质细胞。这一发现将成为 旨在调节sTREM 2受体的新疗法。sTREM 2也被证明与Aβ结合 斑块我们将利用这一观察结果并开发sTREM 2-可结晶级分（sTREM 2-Fc）融合蛋白。 结合Aβ斑块并促进小胶质细胞活化和吞噬作用的蛋白质类。sTREM 2-Fc融合体 将使用来自5XFAD小鼠的脑切片来验证蛋白质，所述小鼠积累AD的典型A β斑块。我们 还将进行鞘内注射，这将使我们能够评估sTREM 2-Fc在消除 斑块和活化的小胶质细胞。总之，这些都是新的方法，可能有助于开发治疗方法 其增强清除和预防淀粉样蛋白-β斑块形成以治疗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.