Reprogramming proinflammatory microglia by restoring mitochondrial function

创伤性脑损伤（TBI）是长期残疾和痴呆的主要来源。小胶质细胞，长寿 大脑中的免疫细胞，激活多种反应状态以响应损伤。促炎程度 激活与神经损伤的严重程度相关，这表明未解决的激活是 致病的这项拟议中的研究非常重要，因为它将评估一种临床安全的干预措施， 改善有害的TBI诱导的小胶质细胞活化，这可能有助于痴呆和其他慢性 脑外伤后的神经功能缺损我们的数据表明，从氧化磷酸化代谢转变为 促炎活化过程中的糖酵解涉及对受损细胞的溶酶体周转的损害， 线粒体通过线粒体自噬，随后是复合物I亚基降解。艾地苯醌恢复氧气 被受损线粒体消耗并减弱促炎一氧化氮和白细胞介素-1 β 生产艾地苯醌恢复氧消耗降低细胞内氧浓度。 我们发现，简单地通过在缺氧条件下孵育细胞来降低氧浓度， 降解和线粒体自噬损伤。出乎意料的是，抗氧化剂未能产生类似的拯救作用，这表明 氧的作用是独立的氧化应激。利用蛋白质组学，我们发现了一个明显的积累 脯氨酰3-羟化酶2（P3 H2）在线粒体/溶酶体富集的细胞部分。P3 H2基因表达 大脑是小胶质细胞特异性，我们的初步数据显示小鼠挫伤周围皮层中P3 H2升高 在TBI之后P3 H2酶使用氧作为靶蛋白的脯氨酸羟基化的底物。艾地苯醌可 通过降低氧的利用率来抑制P3 H2的活性，防止P3 H2的后分解， 修饰涉及质量控制的线粒体或溶酶体蛋白。这项研究将测试中央 艾地苯醌抑制TBI诱导的小胶质细胞活化、慢性神经变性和 通过逆转P3 H2依赖的线粒体自噬抑制来改善认知缺陷。以下具体目标采用最先进的小鼠模型来监测小胶质细胞线粒体更新，并包括新的方法来分类和鉴定小胶质细胞线粒体。 基于线粒体功能离体研究小胶质细胞。目标1中的实验将检验以下预测： 艾地苯醌通过降低细胞内氧拯救促炎性小胶质细胞的线粒体自噬 浓度，从而抑制P3 H2活性。使用雄性和雌性小鼠，并将性别视为 变量，目标2中的实验将测试艾地苯醌或遗传P3 H2敲除改善 TBI通过挽救小胶质细胞线粒体自噬诱导促炎性小胶质细胞积聚和神经功能缺损 积极的结果将支持艾地苯醌治疗TBI诱导的痴呆症的转化研究， 有可能帮助数百万生活在创伤性脑损伤破坏性后果中的男女。

Traumatic brain injury (TBI) is a major source of long-term disability and dementia. Microglia, long-lived immune cells of the brain, activate to multiple reactive states in response to injury. The extent of pro-inflammatory activation correlates with the severity of neurological impairments, suggesting that unresolved activation is pathogenic. The proposed research is highly significant because it will evaluate a clinically safe intervention to ameliorate harmful TBI-induced microglial activation that may contribute to dementia and other chronic neurological deficits following TBI. Our data suggest that a metabolic shift from oxidative phosphorylation to glycolysis during pro-inflammatory activation involves impairment to the lysosomal turnover of damaged mitochondria by mitophagy, which is followed by Complex I subunit degradation. Idebenone restores oxygen consumption by damaged mitochondria and attenuates pro-inflammatory nitric oxide and interleukin-1beta production. The restoration of oxygen consumption by idebenone decreases intracellular oxygen concentration. We found that simply lowering oxygen concentration by incubating cells under hypoxia prevents Complex I degradation and mitophagy impairment. Unexpectedly, antioxidants failed to yield similar rescue, suggesting a role for oxygen that is independent of oxidative stress. Using proteomics, we discovered a marked accumulation of prolyl 3-hydroxylase 2 (P3H2) in a mitochondria/lysosome-enriched cell fraction. P3H2 gene expression within the brain is microglia-specific, and our preliminary data show elevated P3H2 in mouse peri-contusional cortex after TBI. P3H2 enzyme uses oxygen as a substrate for proline hydroxylation of target proteins. Idebenone may suppress the activity of P3H2 by decreasing oxygen availability, preventing P3H2 from post-translationally modifying mitochondria or lysosome proteins involved in quality control. This study will test the central hypothesis that idebenone suppresses TBI-induced microglial activation, chronic neurodegeneration, and cognitive deficits by reversing P3H2-dependent inhibition of mitophagy. The following specific aims employ state-of-the-art mouse models to monitor microglial mitochondrial turnover and include novel methods to sort and study microglia ex vivo based on mitochondrial function. The experiments in Aim 1 will test the prediction that idebenone rescues mitophagy in pro-inflammatory microglial cells by decreasing intracellular oxygen concentration, thereby inhibiting P3H2 activity. Using both male and female mice and considering sex as a variable, the experiments in Aim 2 will test the prediction that idebenone or genetic P3H2 knockout ameliorates TBI-induced pro-inflammatory microglia accumulation and neurological deficits by rescuing microglial mitophagy. Positive outcomes will support translational studies of idebenone to treat TBI-induced dementia, with the potential to help millions of men and women living with the devastating consequences of TBI.