Reprogramming proinflammatory microglia by restoring mitochondrial function

创伤性脑损伤(TBI)是导致长期残疾和痴呆的主要原因。长寿的小胶质细胞 大脑的免疫细胞，激活到多种反应状态，对损伤作出反应。促炎的程度 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。这项拟议的研究非常有意义，因为它将评估一种临床安全的干预措施 改善颅脑损伤诱导的可能导致痴呆和其他慢性疾病的有害小胶质细胞激活 颅脑损伤后的神经功能障碍。我们的数据表明，代谢从氧化磷酸化转变为 促炎症激活过程中的糖酵解涉及对受损的溶酶体周转的损害 线粒体发生有丝分裂，随后是复合体I亚基降解。艾地苯酮恢复氧气 损伤线粒体的消耗和减轻促炎的一氧化氮和白介素1β 制作。艾地苯酮恢复耗氧量可降低细胞内氧浓度。 我们发现，通过在低氧条件下孵育细胞来降低氧浓度，可以防止复合体I 降解和吞丝分裂功能受损。出乎意料的是，抗氧化剂未能产生类似的救援效果，这表明 氧的作用与氧化应激无关。利用蛋白质组学，我们发现了一种显著的积累 线粒体/溶酶体富集细胞组分中的脯氨基3-羟基酶2(P3H2)。P3H2基因在细胞内的表达 大脑是小胶质细胞特异性的，我们的初步数据显示，小鼠挫伤周围皮质中P3H2升高 在TBI之后。P3H2酶以氧为底物对靶蛋白进行脯氨酸羟化反应。艾地苯酮可 通过降低氧气利用率抑制P3H2的活性，阻止P3H2的翻译后 修饰参与质量控制的线粒体或溶酶体蛋白。这项研究将考验中央 艾地苯酮抑制脑损伤诱导的小胶质细胞激活、慢性神经变性和 通过逆转P3H2依赖的丝裂原吞噬抑制而导致的认知缺陷。以下具体目标使用最先进的小鼠模型来监控小胶质细胞线粒体的周转，并包括新的方法来分类和 基于线粒体功能的小胶质细胞体外实验研究。目标1中的实验将检验预测 艾地苯农通过减少细胞内氧含量挽救炎性小胶质细胞的有丝分裂 浓度，从而抑制P3H2活性。同时使用雄性和雌性小鼠，并将性视为一种 变量，目标2的实验将检验艾地苯酮或基因P3H2基因敲除改善的预测 脑外伤通过挽救小胶质细胞有丝分裂而导致促炎性小胶质细胞堆积和神经功能障碍。 积极的结果将支持艾地苯酮治疗脑损伤引起的痴呆的翻译研究， 有潜力帮助数以百万计的男女生活在脑损伤的破坏性后果中。

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.