PDF(Pubmed)
Abstract：
Cell metabolism reprogramming to sustain energy production, while reducing oxygen and energy consuming processes is crucially important for the adaptation to hypoxia/ischemia. Adaptive metabolic rewiring is controlled by hypoxia-inducible factors (HIFs). Accumulating experimental evidence indicates that timely activation of HIF in brain-resident cells improves the outcome from acute ischemic stroke. However, the underlying molecular mechanisms are still incompletely understood. Thus, we investigated whether HIF-dependent metabolic reprogramming affects the vulnerability of brain-resident cells towards ischemic stress. Methods: We used genetic and pharmacological approaches to activate HIF in the murine brain in vivo and in primary neurons and astrocytes in vitro. Numerous metabolomic approaches and molecular biological techniques were applied to elucidate potential HIF-dependent effects on the central carbon metabolism of brain cells. In animal and cell models of ischemic stroke, we analysed whether HIF-dependent metabolic reprogramming influences the susceptibility to ischemic injury. Results: Neuron-specific gene ablation of prolyl-4-hydroxylase domain 2 (PHD2) protein, negatively regulating the protein stability of HIF-α in an oxygen dependent manner, reduced brain injury and functional impairment of mice after acute stroke in a HIF-dependent manner. Accordingly, PHD2 deficient neurons showed an improved tolerance towards ischemic stress in vitro, which was accompanied by enhanced HIF-1-mediated glycolytic lactate production through pyruvate dehydrogenase kinase-mediated inhibition of the pyruvate dehydrogenase. Systemic treatment of mice with roxadustat, a low-molecular weight pan-PHD inhibitor, not only increased the abundance of numerous metabolites of the central carbon and amino acid metabolism in murine brain, but also ameliorated cerebral tissue damage and sensorimotor dysfunction after acute ischemic stroke. In neurons and astrocytes roxadustat provoked a HIF-1-dependent glucose metabolism reprogramming including elevation of glucose uptake, glycogen synthesis, glycolytic capacity, lactate production and lactate release, which enhanced the ischemic tolerance of astrocytes, but not neurons. We found that strong activation of HIF-1 in neurons by non-selective inhibition of all PHD isoenzymes caused a HIF-1-dependent upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 redirecting glucose-6-phosphate from pentose phosphate pathway (PPP) to the glycolysis pathway. This was accompanied by a reduction of NADPH production in the PPP, which further decreased the low intrinsic antioxidant reserve of neurons, making them more susceptible to ischemic stress. Nonetheless, in organotypic hippocampal cultures with preserved neuronal-glial interactions roxadustat decreased the neuronal susceptibility to ischemic stress, which was largely prevented by restricting glycolytic energy production through lactate transport blockade. Conclusion: Collectively, our results indicate that HIF-1-mediated metabolic reprogramming alleviates the intrinsic vulnerability of brain-resident cells to ischemic stress.
摘要：
细胞代谢重新编程以维持能量生产，而减少氧气和能量消耗过程对于适应缺氧/缺血至关重要。适应性代谢重新布线受缺氧诱导因子（HIF）控制。越来越多的实验证据表明，及时激活大脑驻留细胞中的HIF可改善急性缺血性中风的预后。然而,潜在的分子机制仍未完全理解。因此，我们调查了HIF依赖性代谢重编程是否影响脑驻留细胞对缺血性应激的脆弱性.方法：我们使用遗传和药理学方法激活体内鼠脑中以及体外原代神经元和星形胶质细胞中的HIF。许多代谢组学方法和分子生物学技术被用于阐明对脑细胞中心碳代谢的潜在HIF依赖性作用。在缺血性中风的动物和细胞模型中，我们分析了HIF依赖性代谢重编程是否影响缺血性损伤的易感性.结果：脯氨酸-4-羟化酶结构域2（PHD2）蛋白的神经元特异性基因消融，以氧依赖的方式负调节HIF-α的蛋白质稳定性，以HIF依赖性方式减少急性中风后小鼠的脑损伤和功能损害。因此，PHD2缺陷型神经元在体外表现出改善的对缺血应激的耐受性，伴随着通过丙酮酸脱氢酶激酶介导的丙酮酸脱氢酶抑制HIF-1介导的糖酵解乳酸产生的增强。用罗沙司他对小鼠进行系统治疗，一种低分子量的泛PHD抑制剂，不仅增加了许多代谢产物的中央碳和氨基酸代谢鼠脑的丰度，而且还可以改善急性缺血性卒中后的脑组织损伤和感觉运动功能障碍。在神经元和星形胶质细胞中，roxadustat引起HIF-1依赖性葡萄糖代谢重编程，包括葡萄糖摄取升高，糖原合成，糖酵解能力,乳酸产生和乳酸释放，增强了星形胶质细胞的缺血耐受性，但不是神经元。我们发现，通过非选择性抑制所有PHD同工酶对神经元中HIF-1的强烈激活导致6-磷酸果糖-2-激酶/果糖-2,6-双磷酸酶-3的HIF-1依赖性上调，将葡萄糖-6-磷酸从戊糖磷酸途径（PPP）重定向到糖酵解途径。这伴随着PPP中NADPH产量的减少，这进一步降低了神经元的内在抗氧化储备，让他们更容易受到缺血性压力的影响.尽管如此，在保留神经元-神经胶质相互作用的器官型海马培养物中，roxadustat降低了神经元对缺血性应激的易感性，通过乳酸转运阻滞限制糖酵解能量的产生在很大程度上阻止了这种情况。结论：集体，我们的结果表明,HIF-1介导的代谢重编程减轻了脑内细胞对缺血性应激的内在脆弱性.
