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.
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