Toll-Like Receptor-Mediated Photoreceptor Mitochondrial DNA Damage

Toll 样受体介导的光感受器线粒体 DNA 损伤

• 批准号: 7633114
• 负责人: NARSING A RAO
• 金额: $ 40.75万
• 依托单位: DOHENY EYE INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7633114
• 关键词: Adaptor Signaling Protein; Address; Age related macular degeneration; Animals; Apoptosis; Apoptotic; Autoimmune Diseases; Blindness; Bone Marrow; Caspase; Cells; Chimera organism; DNA Damage; Demyelinations; Development; Distant; Event; Generations; Genes; Genus Mycobacterium; Heating; Immune; Immune response; Immunity; Infiltration; Inflammation; Inflammatory; Inner Nuclear Layer; Knock-out; Mediating; Microglia; Mitochondria; Mitochondrial DNA; Modeling; Mus; Myelogenous; Natural Immunity; Neuraxis; Neurons; Organ; Outcome; Oxidative Stress; Oxidative Stress Induction; Pathogenesis; Pathologic Processes; Pathology; Pathway interactions; Photoreceptors; Play; Predisposition; Receptor Activation; Reperfusion Injury; Research Methodology; Retina; Retinal; Retinal Photoreceptors; Role; Site; Stress; Systemic disease; T-Lymphocyte; TLR4 gene; Testing; Therapeutic Intervention; Tissues; Toll-like receptors; Tumor Necrosis Factor Receptor; Up-Regulation; Uveitis; base; chemokine; cytokine; design; ganglion cell; in vivo; killings; knockout animal; neuron apoptosis; novel; public health relevance; receptor; receptor expression; receptor upregulation; retinal damage; transcription factor

DESCRIPTION (provided by applicant): Tissue damage in autoimmune disease has previously been attributed to T-cell and inflammatory cell infiltration and to the cytokines released thereafter. Activation of Toll-like receptors (TLRs) in innate immunity generates similar proinflammatory cytokines as those of adaptive immunity and inflict mitochondrial DNA damage. Such TLRs pathology has been overlooked until recently. Among various systemic diseases, TLRs activation has been implicated in the pathogenesis of CNS demyelination, ischemia/reperfusion injury of the organs, and age-related macular degeneration. In this application, TLR4 induced by heat-killed mycobacteria injected at distant sites initiates neuronal cell oxidative stress and mitochondrial DNA damage in the retina. The TLR4 upregulation was also found to associate primarily with the retinal microglia. Based on these preliminary findings, the following specific aims are proposed: 1. Determine the expression and localization of TLR and TNF-1 and its receptors in the mouse retina with an innate immune response. 2. Determine the in-vivo effects of TLR upregulation and interaction with MyD88 on mitochondrial oxidative stress, using various TLRs, MyD88, and other adaptor proteins knockout animals. 3. Determine the role of bone marrow derived retinal microglia TLR upregulation in the retinal neuronal cell mitochondrial oxidative stress, using bone marrow chimeras that express MyD88 limited to the microglia. 4. Localize oxidative stress-mediated mitochondrial DNA damage and caspases in the retina with upregulation of TLRs and demonstrate augmentation of retinal damage with T-cell-mediated adaptive immune response. The research methods designed are closely adhered to these specific aims. In innate immunity, TLRs, transcription factors (such as NFkB), cytokines (such as TNF-1), and iNOS are upregulated (specific aim 1) and are likely to co-localize to retinal microglia (specific aim 1). However, co-localization with other retinal immune cells will be also tested (specific aim 1). The association of TLRs to microglia will be further substantiated by bone marrow chimeric mice in which the retinal microglia will be bone marrow derived (Specific aim 3). The mitochondrial DNA damage mediated by TLRs activation will be tested by using various wild types and knockouts, including adaptor protein knockouts (specific aim 2). Further, the site of DNA damage and caspases will be sought in various retinal cells (specific aim 4). Once clarified, these results on initial events of TLR activation might have a global implications in autoimmune diseases and specific DNA damage in neuronal cells from TLR4 activation. PUBLIC HEALTH RELEVANCE: Intraocular inflammation, or uveitis, is a leading cause of blindness from retinal photoreceptor cell degeneration. This application deals with the previously overlooked aspect of early photoreceptor damage initiated by the innate immunity alone. By clarifying the mechanism of retinal damage rendered by Toll-like receptor activation, we can advance the design of therapeutic intervention to achieve the desired outcome.

描述（由申请人提供）：自身免疫性疾病中的组织损伤先前被归因于T细胞和炎症细胞浸润以及随后释放的细胞因子。先天免疫中 Toll 样受体 (TLR) 的激活会产生与适应性免疫相似的促炎细胞因子，并造成线粒体 DNA 损伤。直到最近，这种 TLR 病理学一直被忽视。在各种全身性疾病中，TLRs的激活与中枢神经系统脱髓鞘、器官缺血/再灌注损伤和年龄相关性黄斑变性的发病机制有关。在此应用中，由远处注射的热灭活分枝杆菌诱导的 TLR4 会引发视网膜中的神经元细胞氧化应激和线粒体 DNA 损伤。还发现 TLR4 上调主要与视网膜小胶质细胞有关。基于这些初步发现，提出以下具体目标： 1. 确定具有先天免疫反应的小鼠视网膜中 TLR 和 TNF-1 及其受体的表达和定位。 2. 使用各种 TLR、MyD88 和其他接头蛋白敲除动物，确定 TLR 上调以及与 MyD88 相互作用对线粒体氧化应激的体内影响。 3.使用仅限于小胶质细胞表达MyD88的骨髓嵌合体，确定骨髓来源的视网膜小胶质细胞TLR上调在视网膜神经元细胞线粒体氧化应激中的作用。 4. 通过上调 TLR 来定位视网膜中氧化应激介导的线粒体 DNA 损伤和半胱天冬酶，并证明 T 细胞介导的适应性免疫反应会增强视网膜损伤。设计的研究方法紧密贴合这些具体目标。在先天免疫中，TLR、转录因子（例如 NFkB）、细胞因子（例如 TNF-1）和 iNOS 上调（具体目标 1），并且可能共定位于视网膜小胶质细胞（具体目标 1）。然而，还将测试与其他视网膜免疫细胞的共定位（具体目标 1）。 TLR 与小胶质细胞的关联将通过骨髓嵌合小鼠得到进一步证实，其中视网膜小胶质细胞将来自骨髓（具体目标 3）。 TLR 激活介导的线粒体 DNA 损伤将通过使用各种野生型和敲除型（包括接头蛋白敲除型）进行测试（具体目标 2）。此外，将在各种视网膜细胞中寻找 DNA 损伤和半胱天冬酶的位点（具体目标 4）。一旦澄清，这些关于 TLR 激活初始事件的结果可能对自身免疫性疾病和 TLR4 激活引起的神经元细胞特异性 DNA 损伤产生全球影响。公众健康相关性：眼内炎症或葡萄膜炎是视网膜感光细胞变性导致失明的主要原因。该应用涉及先前被忽视的仅由先天免疫引起的早期感光器损伤的方面。通过阐明 Toll 样受体激活引起的视网膜损伤的机制，我们可以推进治疗干预的设计，以达到预期的结果。