Zinc-induced cortical neuronal death: Contribution of energy failure attributable to loss of NAD+ and inhibition of glycolysis

Abstract

Excessive zinc influx may contribute to neuronal death after certain insults, including transient global ischemia. In light of evidence that levels of intracellular free Zn2+ associated with neurotoxicity may be sufficient to inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), experiments were performed looking for reduced glycolysis and energy failure in cultured mouse cortical neurons subjected to lethal Zn2+ exposure. As predicted, cultures exposed for 3-22 hr to 40 μM Zn2+ developed an early increase in levels of dihydroxyacetone phosphate (DHAP) and fructose 1,6- bisphosphate (FBP) and a progressive loss of ATP levels, followed by neuronal cell death; furthermore, addition of the downstream glycolytic substrate pyruvate to the bathing medium attenuated the fall in ATP and neuronal death. However, an alternative to direct Zn2+ inhibition of GAPDH was raised by the observation that Zn2+ exposure also induced an early decrease in nicotinamide-adenine dinucleotide (NAD+) levels, an event itself capable of inhibiting GAPDH. Favoring this indirect mechanism of GAPDH inhibition, the neuroprotective effects of pyruvate addition were associated with normalization of cellular levels of NAD+, DHAP and FBP. Zn2+-induced neuronal death was also attenuated by addition of the energy substrate oxaloacetate, the activator of pyruvate dehydrogenase, dichloroacetate, or the inhibitors of NAD+ catabolism, niacinamide or benzamide. Acetyl carnitine, α-keto butyrate, lactate, and β-hydroxy-butyrate did not attenuate Zn2+-induced neurotoxicity, perhaps because they could not regenerate NAD+ or be used for energy production in the presence of glucose.