Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons.
Bottom Line: Further, although depletion of NAD(P)H or GSH correlated linearly with neuron death, compared with GSH depletion, higher neurodegeneration was observed when NAD(P)H was extrapolated to zero, especially in old age, and in the 3xTg-AD neurons.Our data indicate that in aging and more so in AD-like neurons, NAD(P)H redox control is upstream of GSH and an oxidative redox shift that promotes neurodegeneration.Thus, NAD(P)H generation may be a more efficacious therapeutic target upstream of GSH and ROS.
Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, 62794-9626, USA.Show MeSH
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Mentions: Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme for NAD+ synthesis via the salvage pathway, while transhydrogenase and Kreb’s cycle dehydrogenases convert NAD+ to reduced NADH. If NAD+ is merely recycled without consumption, then inhibition of NAMPT will have no effect on NAD(P)H levels. However, if sirtuins and PARPs consume NAD+ to produce nicotinamide and O-acetyl-ADP-ribose, then replenishment of the NADH/NAD+ pool will require resynthesis through nicotinamide by NAMPT. To determine the importance of NAMPT to maintain NADH levels, we treated neurons for 15 h with different concentrations of the specific NAMPT inhibitor FK866 (Wang et al., 2011). In young 2-month non-Tg neurons (Fig. 1A), NAMPT inhibition decreased NAD(P)H by 24% from 59 to 45 μm at the highest 10 nm FK866 concentration, while 3xTg-AD neurons were inhibited 34% from 56 to 37 μm. These small decreases in young 2-month neurons suggest that most of the neuronal NAD+ is conserved and recycled through transhydrogenase and dehydrogenases. In 11-month neurons on the other hand (Fig. 1B), NAD(P)H levels start higher at 106 and 64 μm for non-Tg and 3xTg-AD neurons, respectively, with larger maximal inhibition of 51% to 52 μm and 64% to 23 μm. Similarly, large effects were observed in 21-month neurons (Fig. 1C), with maximal 53% inhibition of NAD(P)H levels to 35 μm for non-Tg and 50% to 17 μm for 3xTg-AD neurons. These large losses indicate greater consumption of the NAD+/NAD(P)H pool in 11- and 21-month than 2-month neurons. The increase in starting levels of NAD(P)H in middle age followed by the decline in old age indicates the importance of NAD(P)H for aging, as previously described and discussed by us in Ghosh et al. (2012). The larger genotype differences suggest increased consumption of NAD+ or impaired resynthesis in the 3xTg-AD neurons (Liu et al., 2009). Of final note, the IC50s in each case were about 1.5 μm FK866, indicating that neither age nor genotype affected the mechanism of inhibition. In summary, FK866 inhibition of NAMPT has large effects on NAD(P)H levels starting at middle age, suggesting neuron reliance on resynthesis through the salvage pathway. In practice, increases in inhibition of NAMPT by collective increments in FK866 increasingly depletes NAD(P)H in both genotypes which enabled its use in determination of the effects of NAD(P)H depletion on GSH levels and neurodegeneration.
Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, 62794-9626, USA.