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; 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.

项目摘要： 遗传研究已经确定了几种主要表达的几种基因中的序列变异 小胶质细胞，与对阿尔茨海默氏病（AD）的风险或韧性增强有关。我们的 初步实验表明，这些基因中的某些基因编码的蛋白质（例如Trem2，plcg2，abi3） 可能是TREM2受体下游细胞内信号通路的组成部分。这 级联反应于TREM2激活，调节小胶质细胞功能。至关重要的是，其中几种蛋白质 （例如ABI3）或它们的相互作用蛋白（例如Blnk，与PLCG2的相互作用者）的复杂性低，并且 本质上无序的主题。这些基序是蛋白质的特征，该相位分开形成 生物分子冷凝物。我们的初步实验证实了这些蛋白质确实相位 分开形成两种生物分子冷凝物。一个包含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，与广告相关的突变改变了这些 对PTM的冷凝物调节并改变小胶质细胞迁移和吞噬作用。我们的观察表明 两种凝结物在调节与AD和衰老相关的小胶质细胞功能方面都起着核心作用。最后， 这种信号通路以及其中的生物分子冷凝物可能包含未识别的 生物标志物和治疗剂的分子靶标，用于管理衰老和AD中的小胶质细胞功能障碍。 该提案将开发两个关键的促进资源，以支持未来的工作。 首先，我们将创建人类IPSC衍生的小胶质表达纪念物 + spot + spot + scarlet + ha标签 CRISPR设计为内源性ABI3和PLCG2基因。该工具将允许同时 研究活细胞中两种冷凝水的生物物理学和细胞生物学的研究。多元化的本质 IPSC将允许在没有其他资源的情况下对其他细胞类型中的这些冷凝物进行分析。第二， 我们将开发新型的蛋白质纯化和质谱工作流程，以获得更完整的 这些冷凝水中蛋白质的清单。我们最初将专注于简单的免疫治疗 识别稳定相互作用蛋白的协议。我们将利用HA/点标签和强大的，很好 表征了这些标签与COIP PLCG2和ABI3及其相互作用伙伴的抗体，这将是 由LC-MS/MS确定。作为原理的证明，将〜10个粘合剂作为真实冷凝物进行身份验证 在人培养的小胶质细胞和脑切片中使用相互共同定位研究的组件。 身份验证的冷凝水合作伙伴将在本质上有用。但是，经过验证的工作流程开发了 这里将支持在各种疾病下人类IPSC衍生的小胶质细胞中未来的大规模研究（TREM2 激活，衰老，与广告相关的序列变体）。这些资源将支持未来的工作 了解凝结物如何调节小胶质细胞功能并发现分子靶标的场 用于精确的生物标志物和疗法，以管理衰老和AD中的小胶质细胞功能障碍。