Age-related Decreases in NAD(P)H and Glutathione Cause Redox Declines Before ATP Loss During Glutamate Treatment of Hippocampal Neurons Mordhwaj S. Parihar, 1 Elizabeth A. Kunz, 1 and Gregory J. Brewer 1,2 * 1 Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois 2 Department of Neurology, Southern Illinois University School of Medicine, Springfield, Illinois Age-related glutamate excitotoxicity depends in an unknown manner on active mitochondria, which are key determinants of the cellular redox potential. Com- pared with embryonic and middle-aged neurons, old- aged rat hippocampal neurons have a lower resting reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and a lower redox ratio (NAD(P)H/flavin ade- nine nucleotide). Glutamate treatment resulted in an ini- tial increase in NAD(P)H concentrations in all ages, fol- lowed by a profound calcium-dependent, age-related decline in NAD(P)H concentration and redox ratio. With complex I of the electron transport chain inhibited by rotenone, treatment with glutamate or ionomycin only resulted in the increase in NAD(P)H fluorescence. High- performance liquid chromatography analysis of adenine nucleotides in brain extracts showed 50% less nicotin- amide adenine dinucleotide (NADH) and almost twice as much oxidized nicotinamide adenine dinucleotide, demonstrating a more oxidized ratio in old than middle- aged brain. Resting glutathione content also declined with age and further decreased with glutamate treat- ment without accompanying changes in adenosine tri- phosphate levels. We conclude that age does not affect production of NADH by dehydrogenases but that old- aged neurons consume more NADH and glutathione, leading to a catastrophic decline in redox ratio. V V C 2008 Wiley-Liss, Inc. Key words: aging; excitotoxicity; mitochondria; neuro- degenerative disease; redox potential Neurons rely heavily on mitochondrial function for generation of phosphoenergy in the form of adenosine triphosphate (ATP)/phosphocreatine and oxidation/ reduction power in the form of nicotinamide adenine dinucleotide (NADH)/glutathione to power membrane ionic pumps, anabolic biosynthesis, and catabolic oxida- tions and to sustain ionic homeostasis for synaptic trans- mission. In the human genome database, no fewer than 83 gene products use NADH as substrate, another 64 use oxidized nicotinamide adenine dinucleotide (NAD), and 106 use glutathione as substrates, not to mention all the proteins whose sulfhydryl oxidation status is con- trolled by the cellular redox potential related to NADH and NAD levels. Mitochondrial NADH has become an increasingly important measure of mitochondrial redox state because of the link with the regulation of DNA repair, transcription, metabolic activity, cellular resistance to stress or injury, and longevity (Pellerin and Magistretti, 2004). Nicotinamide adenine dinucleotide phosphate (NADPH) is an essential reducing equivalent for the regeneration of glutathione (GSH) and for the ac- tivity of the NADPH-dependent thioredoxin system (Chae et al., 1994). It is now clear that NADH and NADPH also function as antioxidants and endogenous inhibitors of opening the mitochondrial permeability tran- sition pore (Kirsch and de Groot, 2001). The balance between the consumption through electron transport (oxi- dation) and its generation by mitochondrial dehydroge- nases (reduction) maintains the steady-state NADH level and consequent redox level, NADH/NAD 1 , in the cell. Linkage of reduced nicotinamide adenine dinucleo- tide (phosphate) (NAD(P)H) and flavoprotein fluores- cence (expressed as flavin adenine nucleotide [FAD]) to the respiratory chain was extensively studied (Kunz et al., 1994; Wiedemann et al., 1998; Huang et al., 2002; Duchen et al., 2003; Chance, 2004; Rocheleau et al., 2004; Rothstein et al., 2005). To complement intrinsic measures of the reduced NADH, we have also Contract grant sponsor: Temple Award from the Alzheimer’s Association and the National Institute on Aging; Contract grant number: 2RO1 AG13435. *Correspondence to: Gregory J. Brewer, Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois Univer- sity School of Medicine, Springfield, IL 62794-9626. E-mail: gbrewer@siumed.edu Received 14 February 2006; Revised 31 October 2007; Accepted 9 January 2008 Published online 25 April 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21679 Journal of Neuroscience Research 86:2339–2352 (2008) ' 2008 Wiley-Liss, Inc.