DNA damage-induced inflammation and its brain-specific consequences

摘要 阿尔茨海默氏症和帕金森氏症等神经退行性疾病代表着一组不同的 条件。虽然脑细胞丢失是最突出的表型，但每种疾病也表现为 慢性炎症过程的证据。拟议中的工作将探索一种新认识的触发因素 中毒性神经炎性过程、胞浆DNA(细胞DNA)。先天免疫系统的细胞，使用 CGAS/STIN和TLR9，将细胞DNA的存在解释为入侵的病毒或细菌病原体和 积极回应。在衰老和神经退行性疾病中，细胞应激导致DNA的释放 从细胞自身的线粒体或核基因组中分离出来的片段进入细胞质，从而引发“不育” 炎症反应。干扰素相关基因和核因子κB系统被动员，两者都引起了巨大的 如果他们的反应变得习惯性，就会造成伤害。对无菌炎症的研究传统上集中在细胞上。 先天免疫系统(如小胶质细胞)，或附属细胞，如星形胶质细胞。我们建议采取更多的 更广泛的方法，通过分别测试大脑中的每种细胞类型在慢性脑损伤中的作用 在阿尔茨海默氏症和帕金森氏症中发现炎症。我们将跟踪核和 线粒体片段占细胞DNA总量。作为压力源，我们将使用ATM抑制来阻止DNA 修复，抑制TFAM以破坏线粒体，以及更多与疾病相关的刺激，如Aβ 阿尔茨海默病和帕金森氏病的α-突触核蛋白。我们预测线粒体的贡献 而细胞核DNA片段在不同的情况下会有所不同，从而产生独特的TLR9和cGAS/STIN 导致标志性分泌组的反应，有助于临床症状的多样性。要测试 对于每一种病理潜能的细胞DNA刺激的免疫反应，我们都会用不同的刺激来挑战 培养星形胶质细胞、神经元、少突胶质细胞和小胶质细胞。然后我们将收集他们的条件培养液 并在相同细胞类型的幼稚培养上进行测试，以分离出各种有毒和营养元素 从不同的细胞释放出来。受刺激细胞的转录本将由RNAseq定义；蛋白质 分泌体的组成将通过LC/MS确定，虽然我们的初步研究将在培养细胞中进行，但我们 将使用三种不同的神经退行性疾病的小鼠模型在体内验证体外发现： 阿尔茨海默氏症、帕金森氏症和共济失调毛细血管扩张症。最后，我们建议制定一项多管齐下的战略，以 阻断细胞DNA诱导的炎症的影响。我们将搜索阻碍出口的化合物 从细胞核中提取细胞DNA，或刺激其从细胞质中清除。这些发现的意义 源于这样一个事实，即了解无菌炎症的过程以及如何阻止它将提供新鲜的 改进我们对许多神经退行性疾病的方法的策略。

Abstract Neurodegenerative diseases such as Alzheimer's and Parkinson's disease represent a diverse group of conditions. While the loss of brain cells is the most prominent phenotype, each disease also presents with evidence of a chronic inflammatory process. The proposed work will explore a newly recognized trigger of this toxic neuroinflammatory process, cytoplasmic DNA (cytoDNA). The cells of the innate immune system, using cGAS/STING and TLR9, interpret the presence of cytoDNA as an invading viral or bacterial pathogen and respond vigorously. In aging and neurodegenerative diseases, cellular stresses lead to the release of DNA fragments from the cell's own mitochondrial or nuclear genome into the cytoplasm, triggering a "sterile" inflammatory response. Interferon related genes and the NFκB system are mobilized and both cause great harm if their responses become chronic. Studies of sterile inflammation traditionally focus on the cells of the innate immune system (e.g., microglia), or accessory cells such as astrocytes. We propose to take a much broader approach, by separately testing each cell type in the brain for its role in contributing to the chronic inflammation found in Alzheimer's and Parkinson's. We will track the relative contributions of nuclear and mitochondrial fragments to the total amount of cytoDNA. As stressors, we will use ATM inhibition, to block DNA repair, TFAM inhibition to damage mitochondria, as well as more disease-relevant stimuli such as Aβ for Alzheimer's disease and α-synuclein for Parkinson's disease. We predict that the contributions of mitochondrial and nuclear cytoDNA fragments will differ in different situations, resulting in unique TLR9 and cGAS/STING responses that result in a signature secretome that contributes to the diversity of clinical symptoms. To test the pathological potential of each cytoDNA-stimulated immune response, we will use different stimuli to challenge cultures of astrocytes, neurons, oligodendrocytes and microglia. We will then collect their conditioned medium and test it on naïve cultures of the same cell types to isolate the full range of toxic and trophic elements released from different cells. The transcriptomes of the stimulated cells will be defined by RNAseq; the protein composition of the secretome will be determined by LC/MS. While our initial studies will be in cultured cells, we will validate the in vitro findings in vivo using mouse models of three distinct neurodegenerative diseases: Alzheimer's, Parkinson's and ataxia-telangiectasia. Finally, we propose to develop a multi-pronged strategy to block the impact of cytoDNA-induced inflammation. We will search for compounds that block the export of cytoDNA from the nucleus or stimulate its elimination from the cytoplasm. The significance of the findings derives from the fact that understanding the process of sterile inflammation and how to block it will offer fresh strategies to improve our approach to many neurodegenerative diseases.