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

能够分析小胶质细胞信号传导以及衰老和阿尔茨海默病功能中的生物分子凝聚物的关键工具

• 批准号: 10583982
• 负责人: Peter Henry St George-Hyslop
• 金额: $ 26.75万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10583982
• 关键词: Actins; Address; Aging; Alzheimer' s Disease; Antibodies; Antibody Affinity; Autopsy; BLNK gene; Biological Assay; Biological Markers; Biology; Biophysics; Brain; Cell Line; Cell model; Cells; Cellular biology; Characteristics; Client; Clustered Regularly Interspaced Short Palindromic Repeats; Cytoplasm; Cytoskeleton; Data; Disease; Engineering; Enzymes; Epitopes; Equipment and supply inventories; Fluorescence Recovery After Photobleaching; Functional disorder; Future; Genes; Genetic study; Human; Immunofluorescence Immunologic; In Situ; Investigation; Knowledge; Ligands; Ligation; Liquid substance; Location; Mass Spectrum Analysis; Mediating; Membrane; Microglia; Molecular; Molecular Target; Monitor; Mutation; Nature; Nucleoplasm; PLCG2 gene; Pathogenesis; Pathway interactions; Phagocytosis; Phase; Phase Transition; Phospho-Specific Antibodies; Physical condensation; Physiological; Play; Post-Translational Protein Processing; Proteins; Proteomics; Protocols documentation; Reagent; Resources; Risk; Role; Signal Pathway; Specificity; TREM2 gene; TYROBP gene; Testing; Therapeutic; Validation; Variant; Western Blotting; Work; abeta oligomer; brain tissue; cell type; design; experimental study; induced pluripotent stem cell; migration; neurotransmission; novel; potential biomarker; protein purification; receptor; resilience; response; scaffold; targeted biomarker; therapeutic target; tool

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.

项目概要： 遗传学研究已经确定了几个基因的序列变异，这些基因主要表达于 小胶质细胞与阿尔茨海默病（AD）的风险或恢复能力增加有关。我们的 初步实验表明，其中一些基因（例如 TREM2、PLCG2、ABI3）编码的蛋白质 可能作为 TREM2 受体下游细胞内信号通路的组成部分发挥作用。这 级联反应响应 TREM2 激活调节小胶质细胞功能。至关重要的是，其中一些蛋白质 （例如 ABI3）或其相互作用蛋白（例如 BLNK，与 PLCG2 的相互作用蛋白）复杂性低且 本质上是无序的图案。这些基序是相分离形成的蛋白质的特征 生物分子缩合物。我们的初步实验证实这些蛋白质确实相 分离形成两种生物分子缩合物。其中之一包含 PLCG2。另一个包含ABI3。两者都是 受翻译后修饰 (PTM) 调节。至关重要的是，AD 相关突变改变了这些 凝结物调节 PTM 并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发挥着核心作用。最后， 该信号传导途径及其内部的生物分子凝聚物可能包含未被识别的 控制衰老和 AD 中小胶质细胞功能障碍的生物标志物和疗法的分子靶点。 该提案将开发两个支持未来工作的关键支持资源。 首先，我们将创建表达 mEmerald + SPOT 和 mScarlet + HA 标签的人 iPSC 衍生小胶质细胞 CRISPR 工程化到内源 ABI3 和 PLCG2 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。的多能性质 iPSC 将允许未来在其他细胞类型中分析这些凝聚物，而无需额外的资源。第二， 我们将开发新颖的蛋白质纯化和质谱工作流程以获得更完整的 这些冷凝物中蛋白质的清单。我们首先将重点关注简单的免疫纯化 鉴定稳定相互作用蛋白质的方案。我们将利用 HA/SPOT 标签和强大的、良好的 将这些标签的抗体特征化为 coIP PLCG2 和 ABI3 及其相互作用的伙伴，这将是 通过LC-MS/MS鉴定。作为原理证明，约 10 个粘合剂将被认证为真正的凝结物 在人类培养的小胶质细胞和脑切片中使用相互 coIP 和共定位研究来确定成分。 经过验证的凝结物伙伴本质上是有用的。然而，经过验证的工作流程已开发出来 这里将支持未来在各种条件下对人类 iPSC 衍生的小胶质细胞进行大规模研究 (TREM2 激活、老化、AD 相关序列变异）。这些资源将支撑未来的工作 领域了解凝结物如何调节小胶质细胞功能并发现分子靶点 寻找精确的生物标志物和治疗方法来管理衰老和 AD 中的小胶质细胞功能障碍。