The role of TREM2 in Alzheimer's disease pathogenesis

描述(申请人提供)：阿尔茨海默病(AD)是一种毁灭性的神经疾病，影响着全球数百万人。这种疾病的病理特征是β-淀粉样蛋白(A-β)积聚到细胞外斑块中，微管相关蛋白tau在细胞内积聚。炎症也被证明在病理学中发挥了重要作用，这一理论得到了最近在AD患者中发现的免疫调节蛋白TREM2突变的支持。虽然已知TREM2在抑制炎症和促进吞噬功能方面很重要，但它在AD中的确切作用尚不清楚。我们的初步数据表明，TREM2与Hsp60结合，Hsp60是一种促炎症免疫调节因子，在细胞应激时会增加。由于Hsp60在炎症方面的相反作用，我们预测Hsp60偏向TREM2与下游成分相互作用，从而阻止其抗炎功能。我们还表明，在阿尔茨海默病小鼠模型中，神经元内Aβ水平升高的神经元中HSP60水平增加。我们预测，HSP60下调细胞应激高的区域的TREM2活性，从而在特定时间和发生细胞应激的特定位置促进炎症。如果没有TREM2，我们预测炎症的平衡将倾向于促炎反应，炎症将不再局限于细胞应激区。为了确定TREM2在AD中的作用，我们将首先研究Hsp60-TREM2信号在体外的作用。使用从WT或缺乏TREM2的转基因小鼠培养的小胶质细胞，我们将能够通过qRT-PCR和吞噬作用，使用荧光微球摄取试验和流式细胞术来确定Hsp60处理对细胞因子产生的TREM2依赖的影响。我们还旨在利用免疫组织化学、Western blotting和流式细胞术来确定5xFAD小鼠模型中TREM2和Hsp60在病理发展过程中的表达和定位的变化。我们预计，在病理早期，HSP60的表达将在神经元中增加，TREM2将在全球范围内上调，以重新调节炎症的平衡。最后，我们将观察TREM2的结构性丢失对AD小鼠病理的影响。我们预计，这些小鼠将增加促炎细胞因子的产生，并降低吞噬能力。因此，当用硫代巴比妥酸染色检测斑块数量和Neun染色检测神经元丢失来确定这些小鼠的病理时，TREM2-/-5xFAD小鼠将表现出更早和更严重的变化。我们还预测，在新对象识别、放射状手臂水迷宫和恐惧条件反射等测试中缺乏TREM2的AD小鼠的认知能力会更早下降。通过这些研究，我们希望更好地了解TREM2在AD中的作用，并为AD患者的治疗干预提供一条新的途径。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a devastating neurological disease that affects millions of people across the globe. The pathology of this disease is characterized by the accumulation of the beta amyloid (Aβ) protein into extracellular plaques and the intracellular accumulation of the microtubule associated protein tau. Inflammation has also been shown to play an important role in pathology, a theory supported by the recent discovery of mutations in the immune regulating protein TREM2 in AD patients. While it is known that TREM2 is important in suppressing inflammation and promoting phagocytosis, its exact role in AD is unclear. Our preliminary data suggests that TREM2 binds to Hsp60, a pro-inflammatory immune regulator that is increased in response to cellular stress. Because of their opposing actions on inflammation, we predict that Hsp60 biases TREM2 to interact with downstream components that block its anti-inflammatory function. We also show that Hsp60 levels are increased in neurons containing elevated levels of intraneuronal Aβ in a mouse model of AD. We predict that Hsp60 downregulates TREM2 activity in areas where cellular stress is high, thereby promoting inflammation at the specific time and in the specific location where cellular stress is occurring. Without TREM2, we predict that the balance in inflammation will tip toward proinflammatory responses and that inflammation will no longer be localized to areas of cellular stress. In order to determine the role of TREM2 in AD, we will first examine the role of Hsp60-TREM2 signaling in vitro. Using microglia cultured from WT or transgenic mice lacking TREM2, we will be able to determine the TREM2-dependent effects of Hsp60 treatment on cytokine production using qRT-PCR and phagocytosis, using a fluorescent microsphere uptake assay coupled with flow cytometry. We also aim to determine the changes in TREM2 and Hsp60 expression and localization during the progression of pathology in the 5xFAD AD mouse model using immunohistochemistry, Western blotting, and flow cytometry. We expect that expression of Hsp60 will increase in neurons early in pathology, and that TREM2 will be upregulated globally to re-set the balance of inflammation. Finally, we will look at the effect of the constitutive loss of TREM2 on pathology in AD mice. We expect that these mice will have increased production of pro- inflammatory cytokines and decreased phagocytic capacity. Consequently, when pathology in these mice is determined using ThioS staining for plaque number, and NeuN staining to assay neuronal loss, TREM2-/- 5xFAD mice will show earlier and more severe changes. We also predict earlier cognitive decline in AD mice which lack TREM2 in tests such as novel object recognition, radial arm water maze and fear conditioning. Through these studies, we hope to gain a better understanding of the role of TREM2 in AD and provide a novel avenue for the development of therapeutic interventions for AD patients.