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

描述(由申请人提供)： 用多核高分辨核磁共振波谱研究了PARP(聚ADP-核糖聚合酶)过度激活导致能量衰竭后，超融合P7大脑皮层脑片的代谢救援。DNA损伤激活PARP，PARP将NAD裂解成烟酰胺和ADP-核糖，然后将ADPribose聚合物连接到核蛋白上。聚-ADP-核糖水解酶(PARG)去除多-ADP-核糖化。PARP的过度激活，单独或与PARG一起激活，会增加NAD的消费量。在具体目标#1中，将使用与为细胞培养开发的范例类似的范例，其中MNNG诱导的DNA损伤中过多的PARP/PARG活性会耗尽NAD，关闭糖酵解，并且是致命的，除非提供PARP/PARG抑制剂或给予三氯乙酸循环底物，如丙酮酸、酮戊二酸和谷氨酰胺。在用一个或多个碳-13标记底物进行切片灌流建立稳定状态标记后，将进行PARP激活，并根据用高氯酸提取的代谢产物中的同位素组成来评估代谢途径。14.1Tesla系统和低温探测器将被用来获得2D[1H-13C]HSQC(异核单量子相干)谱，它间接地探测(激发碳，探测)具有更高灵敏度的碳13旋子。丙酮酸脱氢酶和丙酮酸羧化酶从丙酮酸进入TCA循环的通量的比较也被用来评估代谢性损伤和恢复中神经元-神经胶质细胞的差异。将确定最佳的新陈代谢救援方案。用免疫组织化学、Western blotts和荧光显微镜检测细胞的凋亡和坏死。NAD、NTP、NDP和PCR的消耗和回收将通过一维31P核磁共振谱和常规分析进行监测。在特定的目标#2中，PARP在我们先前发现的缺氧-复氧范式中被激活，以显示由一氧化氮和氧产生的自由基导致的ATP丢失、损伤和损伤。具体目标3研究氧化应激的保护作用。谷胱甘肽的代谢增强将得到优化，使用2D核磁共振将游离谷氨酸、甘氨酸和半胱氨酸的同种异构体从与谷胱甘肽结合的相同氨基酸的同种异构体中分离出来。所获得的知识将检验代谢机制的假说，并为对抗PARP/PARG能量耗竭提供见解，从而提高大脑的缺血耐受性。

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.