2D NMR of Energy Rescue from PARP in Brain Slice Hypoxia

描述（由申请人提供）： 多核高分辨率NMR光谱学用于研究PARP（聚ADP-核糖）聚合酶）过度激活导致能量衰竭后灌流的P7脑皮层切片中的代谢拯救。DNA损伤激活PARP，PARP将NAD切割成烟酰胺和ADP-核糖，然后将ADPribose聚合物附着在核蛋白上。聚ADP-核糖基化被PARG（聚ADP-核糖糖水解酶）去除。PARP单独或与PARG一起过度活化可增加NAD消耗。在特定目标#1中，将使用与为细胞培养物开发的范例类似的范例，其中来自MNNG诱导的DNA损伤的过度PARP/PARG活性耗尽NAD，关闭糖酵解，并且是致命的，除非通过PARP/PARG抑制剂或给予TCA循环底物（如丙酮酸、酮戊二酸和谷氨酰胺）提供拯救。在用一种或多种碳-13标记的底物进行切片灌注建立稳态标记后，将进行PARP活化（有和无补救），并将根据用高氯酸提取的代谢产物中发现的同位素异构体组成评价代谢途径。将使用具有冷冻探针的14.1特斯拉系统来获得2D [1H-13 C] HSQC（异质单量子相干）光谱，其间接地检测碳-13（激发碳，检测）旋子，其具有更高的灵敏度。丙酮酸脱氢酶和丙酮酸羧化酶从丙酮酸进入TCA循环的流量的比较也用于评估神经元-胶质细胞在代谢损伤和恢复中的差异。将确定最佳代谢补救方案。将采用免疫组织化学、蛋白质印迹和荧光显微镜评估细胞凋亡和坏死。将使用1D 31 P NMR光谱和常规测定法监测NAD、NTP、NDP和PCr的消耗和回收。在特定目标#2中，PARP在我们先前发现的缺氧-复氧范例中被激活，以显示由一氧化氮和氧衍生的自由基引起的ATP损失、损伤和损害。具体目标#3研究保护免受氧化应激。将优化谷胱甘肽的代谢增强，使用2D NMR从谷胱甘肽中结合的相同氨基酸的同位素异构体中分离游离谷氨酸、甘氨酸和半胱氨酸的同位素异构体。所获得的知识将测试代谢机制的假设，并提供对抗PARP/PARG能量消耗的见解，以增加大脑的缺血耐受性。

DESCRIPTION (provided by applicant): Multinuclear high resolution NMR spectroscopy is used to study metabolic rescue in superfused P7 cerebrocortical slices after energy failure from overactivation of PARP (poly(ADP-ribose)polymerase). DNA damage activates PARP, which cleaves NAD into nicotinamide and ADP-ribose, and then attaches ADPribose polymers onto nuclear proteins. Poly-ADP-ribosylation is removed by PARG (poly-ADP-ribose glycohydrolase). Excessive activation of PARP, alone or together with PARG, can increase NAD consumption. In Specific Aim #1 a paradigm similar to one developed for cell cultures will be used where excessive PARP/PARG activity from MNNG-induced DNA damage depletes NAD, shuts glycolysis, and is fatal except when rescue is provided by PARP/PARG inhibitors or administration of TCA-cycle substrates, such as pyruvate, ketoglutarate, and glutamine. After slice superfusion with one or more carbon-13 labeled substrates establishes steady state labeling, PARP activation with and without rescue will be done, and metabolic pathways will be evaluated from isotopomer compositions found in metabolic products extracted with perchloric acid. A 14.1 Tesla system with a cryoprobe will be used to obtain 2D [1 H-13C] HSQC (Heteronuclear Single Quantum Coherence) spectra that detects carbon-13 indirectly (excite carbons, detect )rotons, which have greater sensitivity). Comparisons of pyruvate dehydrogenase and pyruvate carboxylase fluxes from pyruvate into the TCA cycle are also used to assess neuron-glial differences in metabolic injury and recovery. Optimum metabolic rescue regimens will be identified. Apoptosis and necrosis will be assessed with immunohistochemistry, Western blots, and fluorescence microscopy. Depletion and recovery of NAD, NTP, NDP and PCr will be monitored with 1D 31P NMR spectroscopy and conventional assays. In Specific Aim #2 PARP is activated in a hypoxia-reoxygenation paradigm previously found by us to show ATP loss, injury and damage by radicals derived from nitric oxide and oxygen. Specific Aim #3 studies protection from oxidative stress. Metabolic augmentation of glutathione will be optimized, using 2D NMR to resolve isotopomers of free glutamate, glycine, and cysteine from isotopomers of the same amino acids bound in glutathione. Knowledge obtained will test hypotheses of metabolic mechanisms and provide insights for fighting PARP/PARG energy depletion so as to increase the brain's ischemic tolerance.