APOE and the PPP: Glucose Metabolism and Oxidative Stress in Alzheimer's Disease

摘要 代谢功能障碍可能导致几种与年龄有关的疾病，包括 阿尔茨海默病（AD）。载脂蛋白E（APOE）基因编码人类中的三种主要同种型。 人口：E2、E3和E4。E4是散发性AD最重要的遗传危险因素，而E2是 保护性的AD患者和认知正常E4个体的一个未充分研究的标志是大脑 葡萄糖代谢低下与E4相关的葡萄糖摄取减少在认知障碍之前开始数十年 然而，其发生的机制及其与AD风险的相关性仍然未知。的 大脑主要代谢葡萄糖，其中大量的葡萄糖被分流到磷酸戊糖 在神经元和星形胶质细胞中均存在PPP通路。PPP产生抗氧化还原因子，如 NADPH和谷胱甘肽，PPP活性降低，增加氧化应激和细胞死亡。有趣的是， 我们的新的初步数据描述了一个小鼠模型与人类apoE，重演了E4相关的 葡萄糖代谢减少，还记录了多种PPP代谢物的减少。因此，中央 这一建议的假设是APOE通过亚型特异性地影响神经元功能和存活。 葡萄糖代谢的变化。具体来说，我们假设E4有助于认知障碍， 通过代谢重编程，其中葡萄糖摄取减少，并通过PPP进行氧化还原管理 减少了。我们在小鼠中的初步数据显示，脑葡萄糖摄取逐步减少（E2>E3>E4）， 体外结果表明这些差异是由于星形胶质细胞通过GLUT-1摄取的变化。因此在 第一个目的是，我们将检验E4通过下调脑葡萄糖摄取的假设。 应用闪烁邻近分析结合apoE靶向操作检测星形胶质细胞葡萄糖转运蛋白GLUT-1 同种型、总蛋白浓度和葡萄糖转运蛋白。为了验证E4减少的假设， 葡萄糖进入PPP，我们将定量跟踪葡萄糖进入和代谢细胞通过 由稳定同位素解析代谢组学（SIRM）提供的独特的同位素产物“追踪”，并将其转化为 我们通过对人脑组织的分析得出的结果。最后，我们将检验E4加剧 由于PPP介导的氧化应激管理的减少而引起的氧化损伤和细胞死亡。这将 在体外通过PPP酶的药理学操作和在体内通过评估 认知功能，AD病理学和氧化损伤，使用氧化还原蛋白质组学分析脑组织， 用PPP刺激剂处理的人apoE小鼠。如果成功的话，这一提议将提供新的治疗方法， 目标是使高风险个体的葡萄糖代谢正常化。通过增加大脑代谢， 葡萄糖摄取和进入PPP可能对预防或延迟AD的发作有很大影响。

ABSTRACT Metabolic dysfunction may contribute to the development of several age-related diseases, including Alzheimer's disease (AD). The gene Apolipoprotein E (APOE) encodes three major isoforms in the human population: E2, E3, and E4. E4 is the most significant genetic risk factor for sporadic AD, while E2 is protective. An understudied hallmark of AD patients – and of cognitively normal E4 individuals – is cerebral glucose hypometabolism. E4-associated reductions in glucose uptake begin decades prior to cognitive impairment, however the mechanism by which it occurs and its relevance to AD risk remain unknown. The brain predominantly metabolizes glucose, a substantial amount of which is shunted to the pentose phosphate pathway (PPP) in both neurons and astrocytes. The PPP generates antioxidant reducing factors such as NADPH and glutathione, and decreased PPP activity increases oxidative stress and cell death. Interestingly, our novel preliminary data describe a murine model with human apoE that recapitulates an E4-associated decrease in glucose metabolism and also documents decreases in multiple PPP metabolites. Thus, the central hypothesis of this proposal is that APOE influences neuronal function and survival through isoform-specific changes in glucose metabolism. Specifically, we hypothesize that E4 contributes to cognitive impairment through metabolic reprogramming in which glucose uptake is decreased and redox management via the PPP is reduced. Our preliminary data in mice show a stepwise decrease in brain glucose uptake (E2>E3>E4), and in vitro results suggest these differences are due to changes in astrocytic uptake via GLUT-1. Therefore, in the first Aim, we will test the hypothesis that E4 decreases cerebral glucose uptake through downregulation of the astrocytic glucose transporter GLUT-1 using a scintillation proximity assay with targeted manipulation of apoE isoforms, total protein concentrations and glucose transporters. To test the hypothesis that E4 decreases glucose entry into the PPP, we will quantitatively track glucose entry and metabolism in the cell through the unique precursor-product “tracing” afforded by Stable Isotope Resolved Metabolomics (SIRM), and translate our results through analysis of human brain tissue. Finally, we will test the hypothesis that E4 exacerbates oxidative damage and cell death due to a reduction in PPP-mediated management of oxidative stress. This will be accomplished in vitro through pharmacological manipulation of PPP enzymes and in vivo by assessing cognitive function, AD pathology, and oxidative damage using redox proteomics analysis of brain tissue from human apoE mice treated with a PPP stimulant. If successful, this proposal will provide novel therapeutic targets to normalize glucose metabolism in high-risk individuals. Enhancing cerebral metabolism by increasing glucose uptake and entry into the PPP could have great impact in preventing or delaying the onset of AD.