Investigation of the innate and adaptive immune responses to TDP-43 aggregates in Amyotrophic Lateral Sclerosis

肌萎缩侧索硬化症中 TDP-43 聚集体的先天性和适应性免疫反应的研究

• 批准号: 10556333
• 负责人: Baggio Angelo Evangelista
• 金额: $ 2.27万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10556333
• 关键词: ALS pathology; ALS patients; Affect; Amyotrophic Lateral Sclerosis; Antigen Presentation; Area; Atrophic; Autopsy; B-Lymphocytes; Biological Assay; Biological Models; Brain; CD8-Positive T-Lymphocytes; Caliber; Cell Death Signaling Process; Cell secretion; Cells; Central Nervous System; Cessation of life; Clinical; Coculture Techniques; Cytotoxic T-Lymphocytes; DNA-Binding Proteins; Data; Development; Diagnosis; Exposure to; Functional disorder; Human; Immune; Immune response; Immunoassay; Immunologic Factor Activation; Immunology; In Vitro; Individual; Infiltration; Inflammation; Inflammatory; Innate Immune Response; Interferon Type II; Investigation; Knowledge; Macrophage; Mass Spectrum Analysis; Mediating; Metabolic; Microglia; Modeling; Molecular; Monitor; Motor Neuron Disease; Motor Neurons; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurosciences; Neurosciences Research; Pathogenesis; Pathologic; Pathology; Patients; Peripheral; Phagocyte Bactericidal Dysfunction; Phagocytosis; Phagolysosome; Phagosomes; Phenotype; Proteomics; RNA; RNA analysis; Reporting; Role; Route; Rupture; Source; Spinal Cord; Surface; Survival Rate; Symptoms; System; T cell response; T-Cell Activation; T-Lymphocyte; TDP-43 aggregation; Testing; Translational Research; Vesicle; Work; adaptive immune response; biomarker development; cell killing; cell type; cytotoxic CD8 T cells; diagnostic strategy; imaging modality; immunological status; improved; in vitro Model; induced pluripotent stem cell; insight; interdisciplinary approach; live cell imaging; misfolded protein; neuroinflammation; neuron loss; neurotoxic; neurotoxicity; novel; protein TDP-43; protein aggregation; response; sporadic amyotrophic lateral sclerosis; superresolution imaging; therapeutic development; therapeutic target; training opportunity; transcriptome sequencing; transcriptomics; transmission process; treatment strategy; uptake

PROJECT SUMMARY Sporadic amyotrophic lateral sclerosis (sALS) is the most common progressive motor-neuron disease, affecting 3 in 100,000 individuals 50 years or older worldwide. sALS is pathologically characterized by the presence of hyper-phosphorylated, insoluble aggregates of the RNA/DNA binding protein Transactive Response DNA Binding Protein (TDP-43). It is well established that aggregated TDP-43 causes dysfunction throughout the central nervous system (CNS), ultimately resulting in neuronal cell death and symptom onset. However, the exact molecular mechanism(s) by which aggregated TDP-43 imparts neurotoxicity has not yet been determined. Recent evidence implicates innate immune cells, particularly infiltrating peripheral macrophages and microglia, as a source of inflammation when stimulated by misfolded protein aggregates. This neuroinflammatory response is therefore expected to compromise neuron integrity and contribute to neurodegeneration. Furthermore, and adaptive immune cells, T- and B-lymphocytes, have been reported in post-mortem analyses of ALS brain and spinal cord, however their mechanistic role in pathology remains unclear. This implicates both innate and adaptive immune responses in sALS pathogenesis. Recent developments also suggest that microglia promote secondary T-cell responses when stimulated by protein aggregates. This T-cell activation may then result in direct neuronal death. Therefore, I hypothesize that aggregated TDP-43 causes a pro-inflammatory, neurodegenerative cascade mediated by innate and adaptive immune responses that drives neurotoxicity in sALS. In Aim 1, I will treat microglia with aggregated mammalian-derived TDP-43 and monitor modulation of phagolysosomal integrity and inflammatory activation. Preliminary data using models of microglia treated with TDP-43 aggregates demonstrate robust aggregate uptake and phagosome rupture. Additionally, mass spectrometry analysis of TDP-43-treated macrophage cultures revealed an increase in pro-inflammatory and immune activation factors, and markers of phagocytic dysfunction. In Aim 2, I will assess the ability of TDP-43- treated microglia to promote motor-neuron death by way of T-cell activation. I will determine if TDP-43- treated microglia engage with and promote CD4+ and CD8+ T-cell activation in culture. Finally, I will determine if activated cytotoxic CD8+ T-cells prompt motor-neuron death in vitro using sALS patient-derived T-cells and induced pluripotent stem cell (iPSC)-derived motor-neurons. In all, this work will use novel in vitro models to elucidate the unknown mechanism by which TDP-43 aggregation leads to motor-neuron death and sALS onset. This knowledge lends itself to biomarker and therapeutic development to improve diagnostic and treatment strategies for sALS patients.

项目摘要 散发性肌萎缩侧索硬化症（sALS）是最常见的进行性运动神经元疾病，影响 全球50岁或以上人群中每10万人中有3人。sALS的病理特征是存在 RNA/DNA结合蛋白的过度磷酸化的不溶性聚集体 结合蛋白（TDP-43）。众所周知，聚集的TDP-43会导致整个组织功能障碍 中枢神经系统（CNS），最终导致神经元细胞死亡和症状发作。但 聚集的TDP-43赋予神经毒性的确切分子机制尚未确定。 最近的证据表明先天免疫细胞，特别是浸润的外周巨噬细胞和小胶质细胞， 当被错误折叠的蛋白质聚集体刺激时，作为炎症的来源。这种神经炎症反应 因此预期会损害神经元的完整性并导致神经变性。此外且 适应性免疫细胞，T淋巴细胞和B淋巴细胞，已经在ALS脑的死后分析中报道， 然而，它们在病理学中的机制作用仍不清楚。这意味着先天和 适应性免疫应答在sALS发病机制中的作用最近的发展也表明，小胶质细胞促进 当被蛋白质聚集体刺激时的继发性T细胞应答。这种T细胞活化可能导致 直接神经元死亡。因此，我假设聚集的TDP-43引起促炎， 先天性和适应性免疫反应介导的神经退行性级联反应导致神经毒性 在sALS。在目标1中，我将用聚集的拟南芥衍生的TDP-43治疗小胶质细胞，并监测小胶质细胞对 吞噬溶酶体完整性和炎症激活。使用经以下处理的小胶质细胞模型的初步数据： TDP-43聚集体表现出稳健的聚集体摄取和吞噬体破裂。此外，质量 TDP-43处理的巨噬细胞培养物的光谱分析揭示了促炎和 免疫激活因子和吞噬功能障碍的标志物。在目标2中，我将评估TDP-43的能力， 处理的小胶质细胞通过T细胞活化促进运动神经元死亡。我来决定TDP-43- 经处理的小胶质细胞参与并促进培养物中的CD 4+和CD 8 + T细胞活化。最后，我将决定， 活化的细胞毒性CD 8 + T细胞使用sALS患者来源的T细胞在体外促进运动神经元死亡， 诱导多能干细胞（iPSC）衍生的运动神经元。总之，这项工作将使用新的体外模型， 阐明TDP-43聚集导致运动神经元死亡和sALS发作的未知机制。 这些知识有助于生物标志物和治疗方法的发展，以改善诊断和治疗 sALS患者的治疗策略