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

项目摘要/摘要 在美国，脊髓损伤(SCI)时的平均年龄已增加到50.5岁。观察 来自几个独立实验室的证据表明，炎症，特别是持续的促炎作用 巨噬细胞激活，导致与年龄相关的脊髓损伤缺陷。此外，最近发现， 免疫调节性脊髓损伤治疗的疗效与年龄有关。我们迫切需要了解 脊髓损伤中持续促炎巨噬细胞活化的年龄依赖性机制。这样做的目的是 建议研究巨噬细胞生物能量学在年龄依赖性炎症和脊髓损伤中的作用 病理生理学。中心假说是巨噬细胞代谢的年龄依赖性损伤驱动 促炎症巨噬细胞活化并参与脊髓损伤后的继发性损伤。前提是 丙酮酸脱氢酶(PDH)途径是促炎或抗炎巨噬细胞激活的关键调节因子 作为糖酵解(促炎)或氧化磷酸化(OXPHOS，即TCA或 克雷布斯循环活性；抗炎)。PDH激酶(PDK)调节PDH，从而起到把关作用 为了OXPHOS。有待检验的假设是，PDK抑制将推动代谢过程(即增加 OXPHOS)是修复性巨噬细胞激活和PDK促进脊髓损伤恢复所必需的。因此， 设计了三个独立的靶点来选择性地机械地针对巨噬细胞的新陈代谢，使用 利用PDK基因敲除小鼠进行嵌合化，并使用一种新产生的脂质体药物进行治疗 PDK抑制剂二氯乙酸酯(DCA)的配方。4个月和14个月龄小鼠将接受T9挫伤脊髓损伤 来模拟当前的SCI人口统计。目标1将确认PDH是脊髓损伤巨噬细胞的瓶颈 利用一种新的优化纯化方法分离脊髓损伤后的巨噬细胞进行代谢 以及细胞外酸化(ECAR，即糖酵解)和耗氧量(OCR，即 OXPHOS)使用海马生物分析仪的比率。AIM 2将确定巨噬细胞的代谢靶点 用包括单细胞RNA测序和体内稳定在内的最新技术研究脊髓损伤后的功能障碍 同位素分解代谢组学用于测定驻留小胶质细胞和外周血细胞的代谢谱 衍生的巨噬细胞。目的3通过评估确定PDK抑制作为治疗脊髓损伤的治疗靶点 解剖和功能恢复。拟议工作完成后，将确定如何利用 完善翻译治疗提高中枢神经系统巨噬细胞的修复功能 包括年龄在脊髓损伤治疗和康复中的潜在影响的策略。了解以下方面 哪种代谢活动调节巨噬细胞功能将有助于深入了解年龄依赖的中枢神经系统 炎症，从而推动了神经创伤、神经科学和老龄化领域的发展。巨噬细胞代谢 是心血管疾病、糖尿病、癌症等多种炎症性疾病的致病因素， 这些拟议目标的完成将在多个方面推进人类健康研究。

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.