The role of macrophage metabolism and age in recovery from spinal cord injury

项目总结/摘要 在美国，脊髓损伤（SCI）的平均年龄已经增加到50.5岁。意见 来自几个独立实验室的研究表明，炎症，特别是持续的促炎症反应， 巨噬细胞活化，有助于与年龄相关的SCI缺陷。此外，最近发现， 免疫调节性SCI疗法的功效是年龄依赖性的。迫切需要了解 SCI中持续促炎性巨噬细胞活化的年龄依赖性机制。这样做的目的 一项建议是研究巨噬细胞生物能量学在年龄依赖性炎症和SCI中的作用 病理生理学核心假设是，巨噬细胞代谢中的年龄依赖性损伤驱动了 促炎性巨噬细胞活化，并有助于SCI后的继发性损伤。前提是 丙酮酸脱氢酶（PDH）途径是促炎或抗炎巨噬细胞活化的关键调节因子 作为糖酵解（促炎）或氧化磷酸化（OXPHOS，即TCA或 Krebs循环活性;抗炎）。PDH激酶（PDK）调节PDH，从而充当看门人 对于OXPHOS。待检验的假设是，PDK抑制将驱动代谢过程（即增加的代谢）。 OXPHOS）是修复性巨噬细胞活化和通过PDK改善SCI恢复所需的。因此，委员会认为， 三个独立的目的被设计为选择性地靶向巨噬细胞代谢机制，使用 与PDK敲除小鼠嵌合，以及使用新产生的脂质体药物治疗 二氯醋酸盐（DCA）制剂，一种PDK抑制剂。4和14个月大的小鼠将经历T9挫伤SCI 来模拟当前SCI的人口统计。目的1将确定PDH作为SCI巨噬细胞的瓶颈 使用新优化的纯化方法进行代谢，该方法允许在SCI后分离巨噬细胞 以及细胞外酸化（ECAR，即糖酵解）和氧消耗（OCR，即糖酵解）的评估。 OXPHOS）使用Seahorse生物分析仪测定。目的2将确定巨噬细胞的代谢靶点 使用最先进的技术，包括单细胞RNA测序和体内稳定 同位素解析代谢组学，以确定居民小胶质细胞和外周的代谢谱 衍生的巨噬细胞。目的3将通过评估确定PDK抑制作为治疗SCI的治疗靶点 解剖和功能的恢复。完成拟议的工作将确定如何利用 CNS巨噬细胞的修复功能，并通过完善转化治疗改善临床实践 包括年龄作为SCI治疗和恢复的潜在影响的策略。了解到 哪些代谢活性调节巨噬细胞功能将为年龄依赖性CNS提供深入了解 炎症，从而推进神经创伤，神经科学和衰老领域。巨噬细胞代谢 是许多炎症性疾病包括心血管疾病、糖尿病、癌症 等等，而建议目标的完成，将从多方面推动人类健康的研究。

PROJECT SUMMARY/ABSTRACT The average age at the time of spinal cord injury (SCI) has increased to 50.5 years old in the US. Observations from several independent laboratories demonstrate that inflammation, specifically sustained pro-inflammatory macrophage activation, contributes to age-related SCI deficits. In addition, it was recently discovered that the efficacy of immunomodulatory SCI therapies is age-dependent. There is an urgent need to understand the age-dependent mechanisms of sustained pro-inflammatory macrophage activation in SCI. The purpose of this proposal is to investigate the role of macrophage bioenergetics in age-dependent inflammation and SCI pathophysiology. The central hypothesis is that age-dependent impairments in macrophage metabolism drive pro-inflammatory macrophage activation and contribute to secondary injury after SCI. The premise is that the pyruvate dehydrogenase (PDH) pathway is a key regulator of pro- or anti-inflammatory macrophage activation as a connecting link between glycolysis (pro-inflammatory) or oxidative phosphorylation (OXPHOS, i.e. TCA or Krebs cycle activity; anti-inflammatory). PDH kinase (PDK) regulates PDH thereby serving as the gatekeeper for OXPHOS. The hypothesis to be tested is that PDK inhibition will drive metabolic processes (i.e. increased OXPHOS) required for reparative macrophage activation and improved SCI recovery by PDK. Accordingly, three independent Aims are designed to selectively target macrophage metabolism mechanistically, using chimerization with PDK knockout mice, and therapeutically, using a newly generated liposomal drug formulation of dichloroacetate (DCA), a PDK inhibitor. 4 and 14-month-old mice will undergo T9 contusion SCI to model the current SCI demographic. Aim 1 will identify PDH as a bottleneck for SCI macrophage metabolism using a newly optimized purification approach that allows for isolation of macrophages after SCI and assessment of extracellular acidification (ECAR, i.e. glycolysis) and oxygen consumption (OCR, i.e. OXPHOS) rates using the Seahorse bioanalyzer. Aim 2 will identify metabolic targets for macrophage dysfunction after SCI using state-of-the-art techniques including single-cell RNA-sequencing and in vivo Stable Isotope-Resolved Metabolomics to determine the metabolic profiles of resident microglia and peripherally derived macrophages. Aim 3 will identify PDK inhibition as a therapeutic target to treat SCI through evaluation of anatomic and functional recovery. Completion of the proposed work will identify ways to harness the reparative functions of CNS macrophages and improve clinical practice by refining translational treatment strategies including age as a potential influence in SCI treatment and recovery. Understanding the extent to which metabolic activity regulates macrophage function will provide insight into age-dependent CNS inflammation, thereby advancing the fields of neurotrauma, neuroscience, and aging. Macrophage metabolism is a contributing factor in a host of inflammatory conditions including cardiovascular disease, diabetes, cancer, etc., and the completion of the proposed aims will advance the study of human health on multiple fronts.