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

项目总结 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病，影响 全球50岁或以上的人中，每10万人中就有3人。SALS的病理特征是 RNA/DNA结合蛋白反式反应DNA的超磷酸化、不溶性聚集体 结合蛋白(TDP-43)。众所周知，聚集的TDP-43会导致整个大脑功能障碍 中枢神经系统(CNS)，最终导致神经细胞死亡和症状发作。然而， 聚集的TDP-43产生神经毒性的确切分子机制(S)尚未确定。 最近的证据表明，先天性免疫细胞，特别是浸润性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。这种神经炎性反应 因此，预计会损害神经元的完整性，并导致神经退化。此外，以及 适应性免疫细胞，T和B淋巴细胞，已在ALS脑和 然而，它们在病理中的机制作用仍不清楚。这既牵涉到先天的也牵涉到 获得性免疫反应在sals发病中的作用。最近的发展也表明，小胶质细胞促进 蛋白质聚集体刺激时的次级T细胞反应。这种T细胞的激活可能会导致 神经元直接死亡。因此，我假设聚集的TDP-43会导致促炎， 神经毒性由先天和获得性免疫反应介导的神经退行性级联反应 在萨尔斯。在目标1中，我将用聚集的哺乳动物来源的TDP-43来处理小胶质细胞，并监测对 吞噬小体完整性和炎症激活。用小胶质细胞模型处理的初步数据 TDP-43聚集体显示出强健的聚集摄取和吞噬小体破裂。此外，质量 经TDP-43处理的巨噬细胞培养的光谱分析显示，促炎和 免疫激活因子和吞噬细胞功能障碍的标志物。在目标2中，我将评估TDP-43的能力- 治疗后的小胶质细胞通过T细胞活化促进运动神经元死亡。我会确定TDP-43是否- 经处理的小胶质细胞在培养中参与并促进CD4+和CD8+T细胞的激活。最后，我会确定是否 使用SALS患者来源的T细胞和激活的细胞毒性CD8+T细胞促进运动神经元死亡 诱导多能干细胞(IPSC)来源的运动神经元。总之，这项工作将使用新的体外模型来 阐明TDP-43聚集导致运动神经元死亡和SALS发病的未知机制。 这一知识有助于生物标记物和治疗开发，以改进诊断和治疗 SALS患者的治疗策略。

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.