Identification of Siglecs as molecular signatures of Disease-Associated Microglia during AD Progression

项目摘要 小胶质细胞参与阿尔茨海默病（AD）的发生和发展，使其调节成为阿尔茨海默病的一个重要因素。 治疗靶点小胶质细胞作为正效应细胞吞噬和清除毒性蛋白;作为负效应细胞 它们释放炎症介质。小胶质细胞功能的不平衡被认为有助于AD进展。 在与AD易感性相关的小胶质细胞调节蛋白中，Siglec 家族，唾液酸结合免疫球蛋白样凝集素。Siglecs成为AD研究的一个特殊焦点， 全基因组关联研究（GWAS）发现，Siglec家族成员CD 33（Siglec-3）， 与AD有关。Siglec-3过表达导致对AD的易感性增加，而消耗减少了对AD的易感性。 敏感性，支持Siglec介导的抑制限制小胶质细胞吞噬作用和 加重AD蛋白质病。由于多种抑制性Siglecs在人（和小鼠）小胶质细胞上表达， 了解Siglec通路的调节将使我们有机会调节小胶质细胞的功能， 预防或减缓AD的进展。转录组分析已被广泛用于揭示变化 在疾病中小胶质细胞的基因表达。然而，由于不同的小胶质细胞亚群显示出广泛的 反应和功能，单靠全转录组分析不能完全捕获依赖于上下文的 小胶质细胞在疾病期间的变化。最近的单细胞分析研究提供了一个高分辨率的视图， 小鼠CNS小胶质细胞亚型在发育和疾病过程中的转录景观。我们 初步的scRNA-seq和免疫细胞化学分析表明，Siglecs，特别是Siglec-F， SiglecG与在AD的不同阶段被激活的小胶质细胞的特定子集相关， 支持我们的假设，Siglec基因在特定阶段的小胶质细胞亚群中特异性表达 的AD。在这里，我们建议使用scRNA-seq和全转录组测序分析的组合， 了解AD模型中Siglecs信号传导与小胶质细胞表型之间的关系，以阐明 小胶质细胞亚型对AD进展的影响。我们开发了小鼠模型（Tau 4 R ΔK-AP小鼠）， 可以模拟AD发展的不同阶段，从症状前的早期阶段到AD的晚期阶段 伴随着广泛的神经元缺失和脑萎缩，这对当前的提议很有帮助。在两个目标中，我们 将利用Tau 4 R ΔK-AP小鼠和仅表达Aβ斑块的小鼠（APP;PS1小鼠）或仅表达Aβ斑块的小鼠（APP;PS1小鼠）。 tau缠结（Tau 4 R ΔK小鼠）以检验Siglecs在AD期间调节小胶质细胞功能的假设 两个目标的进步。目的1：确定Siglecs是否在以下亚组中特异性表达： 在疾病的不同阶段由AD病理激活的小胶质细胞。目标2：确定 作为DAM中的标记基因的Siglecs的调节。拟议研究的成功结果将有助于确定 Siglecs和其他小胶质细胞特异性基因在AD进展中的作用，并揭示了开发新的 AD治疗

PROJECT SUMMARY Microglia are implicated in the initiation and progression of Alzheimer's disease (AD), making their regulation a therapeutic target. As positive effectors, microglia phagocytose and clear toxic proteins; as negative effectors they release inflammatory mediators. Imbalance of microglial function is believed to contribute to AD progression. Among microglial regulatory proteins linked to AD susceptibility are immune inhibitory members of the Siglec family, sialic acid binding immunoglobulin-like lectins. Siglecs became a special focus of AD research when genome-wide association studies (GWAS) discovered that a Siglec family member, CD33 (Siglec-3), was associated with AD. Siglec-3 overexpression results in increased susceptibility to AD, and depletion decreases susceptibility, supporting the view that Siglec-mediated inhibition restricts microglial phagocytosis and exacerbates AD proteinopathy. As multiple inhibitory Siglecs are expressed on human (and mouse) microglia, understanding the regulation by Siglec pathways will give us opportunity to modulate the microglia function and prevent or slowdown the progression of AD. Transcriptome analyses has been widely used to revealed changes in gene expression of microglia in disease. However, since different subsets of microglia display a wide range of responses and functions, whole transcriptome analyses alone could not fully capture the context-dependent microglia variety during disease. Recent studies with single-cell analysis have provided a high-resolution view of the transcriptional landscape of microglia subtypes of the murine CNS during development and disease. Our preliminary scRNA-seq and immunocytochemical analysis demonstrated that Siglecs, especially Siglec-F, and SiglecG are associated with a specific subset of microglia that are activated at different stage of AD, which support our hypothesis that Siglec genes are specifically expressed in subsets of microglia at particular stages of AD. Here, we propose to use a combination of scRNA-seq and whole transcriptome sequencing analysis to understand the relationship between Siglecs signaling and the microglial phenotypes in AD models to elucidate the impact of microglia subtypes on AD progression. We have developed a mouse model (Tau4RΔK-AP mice) that could mimic different stages of AD development from pre-symptomatic early stage to advance stage of AD with widespread neuronal loss and brain atrophy, which is instrumental for the current proposal. In two aims, we will take advantage of Tau4RΔK-AP mice and mice expressing either only Aβ plaques (APP;PS1 mice) or only tau tangles (Tau4RΔK mice) to test the hypothesis that Siglecs regulate microglial function during AD progression in two Aims. Aim 1: To determine whether Siglecs are specifically expressed in the subset of microglia that are activated by AD pathologies at different stages of the disease. Aim 2: To determine the regulation of Siglecs as signature genes in DAMs. Successful outcome from proposed study will help define Siglecs and other microglia specific genes in AD progression, and reveal new strategies for development of new AD therapy.