Critical tools enabling analysis of biomolecular condensates in microglial signaling and function in aging and Alzheimer Disease

项目摘要： 遗传学研究已经鉴定了几种基因中的序列变异，这些基因主要表达于 小胶质细胞，并与阿尔茨海默病（AD）的风险或恢复力增强有关。我们 初步实验表明，这些基因（如TREM2，PLCG2，ABI3）编码的蛋白质 可能作为TREM2受体下游的细胞内信号传导途径的组分起作用。这 级联调节小胶质细胞功能以响应TREM2激活。重要的是，这些蛋白质 (e.g. ABI3）或其相互作用蛋白（例如BLNK，与PLCG 2的相互作用物）含有低复杂性， 本质上无序的图案这些基序是相分离形成的蛋白质的特征 生物分子凝聚物。我们的初步实验证实，这些蛋白质确实相 分离以形成两个生物分子缩合物。一个含有PLCG 2。另一个是ABI3。两者都是 翻译后修饰（PTMs）。至关重要的是，AD相关突变改变了这些 冷凝物调节PTM和改变小胶质细胞迁移和吞噬作用。我们的观察表明， 这两种缩合物在调节与AD和衰老相关的小胶质细胞功能中起中心作用。因此，委员会认为， 这种信号通路和其中的生物分子凝聚物可能含有未被识别的 作为生物标志物和治疗剂的分子靶点，以管理衰老和AD中的小胶质细胞功能障碍。 这项建议将开发两个关键的有利资源，作为今后工作的基础。 首先，我们将产生表达mEmerald + SPOT和mScarlet + HA标签的人iPSC衍生的小胶质细胞。 CRISPR被工程化到内源性ABI3和PLCG 2基因中。该工具将允许同时 研究活细胞中两种凝聚物的生物物理学和细胞生物学。的多能性 iPSC将允许在其他细胞类型中对这些冷凝物进行未来分析，而无需额外的资源。第二、 我们将开发新的蛋白质纯化和质谱工作流程，以获得更完整的 这些浓缩物中的蛋白质清单。我们将首先关注简单的免疫纯化 鉴定稳定的相互作用蛋白的方法。我们将利用HA/SPOT标签和强大的，好- 这些标签的特征抗体与coIP PLCG 2和ABI 3及其相互作用伴侣，这将是 通过LC-MS/MS鉴定。作为原理证明，约10种粘合剂将被鉴定为真实的冷凝物 在人培养的小胶质细胞和脑切片中使用相互coIP和共定位研究来分析这些组分。 经过认证的凝析油合作伙伴本质上是有用的。然而，经验证的工作流程 这里将支持未来在各种条件下对人类iPSC衍生小胶质细胞进行大规模研究（TREM 2 活化、老化、AD相关序列变体）。这些资源将支持联合国环境规划署 该领域旨在了解凝聚物如何调节小胶质细胞功能，并发现分子靶点 用于精确的生物标志物和治疗，以管理衰老和AD中的小胶质细胞功能障碍。

Project Summary: Genetic studies have identified sequence variants in several genes that are predominantly expressed in microglia and are associated with either enhanced risk or resilience to Alzheimer Disease (AD). Our preliminary experiments reveal that the proteins encoded by some of these genes (e.g. TREM2, PLCG2, ABI3) likely function as components of an intracellular signaling pathway downstream of the TREM2 receptor. This cascade regulates microglial function in response to TREM2 activation. Crucially, several of these proteins (e.g. ABI3) or their interacting proteins (e.g. BLNK, an interactor with PLCG2) contain low complexity and intrinsically disordered motifs. These motifs are characteristic of proteins that phase separate to form biomolecular condensates. Our preliminary experiments confirm that these proteins do indeed phase separate to form two biomolecular condensates. One contains PLCG2. The other contains ABI3. Both are regulated by posttranslational modifications (PTMs). Crucially, AD-associated mutations alter these condensate-regulating PTMs and alter microglial migration and phagocytosis. Our observations suggest that both condensates play a central role in regulating microglial functions relevant to AD and aging. Consequently, this signaling pathway, and the biomolecular condensates within it, are likely to contain unrecognised molecular targets for biomarkers and therapeutics to manage microglial dysfunction in aging and AD. This proposal will develop two critical enabling resources that will underpin future work. First, we will create human iPSC-derived microglial expressing mEmerald + SPOT and mScarlet + HA tags CRISPR engineered into the endogenous ABI3 and PLCG2 genes. This tool will allow simultaneous investigation of the biophysics and cell biology of both condensates in living cells. The pluripotent nature of iPSCs will allow future analysis of these condensates in other cell types without additional resources. Second, we will develop novel protein purification and mass spectrometry workflows to obtain a more complete inventory of the proteins within these condensates. We will initially focus on simple immunopurification protocols to identify stable interacting proteins. We will exploit the HA/SPOT tags and robust, well- characterized antibodies to these tags to coIP PLCG2 and ABI3 with their interacting partners, which will be identified by LC-MS/MS. As proof of principle, ~10 binders will be authenticated as real condensate components using reciprocal coIP and colocalisation studies in human cultured microglia and brain sections. The authenticated condensate partners will be intrinsically useful. However, the validated workflows developed here will support future, large-scale studies in human iPSC derived microglia under various conditions (TREM2 activation, aging, AD-associated sequence variants). These resources will underpin future work by the field to understand how the condensates regulate microglial function, and uncover molecular targets for precision biomarkers and therapies to manage microglial dysfunction in aging and in AD.