Trophic deprivation mediated neuronal death is important during development, acute brain or nerve trauma, and neurodegeneration. Serum deprivation (SD) approximates trophic deprivation in vitro, and an in vivo model is neuronal death in the mouse dorsal lateral geniculate nucleus (LGNd) after ablation of the visual cortex (VCA). Oxidant-induced intracellular Zn 2+ release, ([Zn 2+ ] i ), from metallothionein-3 (MT-III), mitochondria, or "protein Zn 2+ " was implicated in trophic deprivation neurotoxicity. We previously showed that neurotoxicity of extracellular Zn 2+ required entry, elevation in [Zn 2+ ] i , reduction of NAD + and ATP levels causing inhibition of glycolysis and cellular metabolism. Exogenous NAD + and sirtuin inhibition attenuated Zn 2+ neurotoxicity. Here we show that: 1) Zn 2+ is released intracellularly after oxidant and SD injuries, and sensitivity to these injuries is proportional to neuronal Zn 2+ content; 2) NAD + loss is involved; restoration of NAD + using exogenous NAD + , pyruvate, or nicotinamide attenuated these injuries, and potentiation of NAD + loss potentiated injury; 3) Neurons from genetically modified mouse strains which reduce intracellular Zn 2+ content (MT-III knockout), reduce NAD + catabolism (PARP-1 knockout), or increase expression of an NAD + synthetic enzyme (Wld s ) each had attenuated SD and oxidant neurotoxicities; 4) Sirtuin inhibitors attenuated, and sirtuin activators potentiated these neurotoxicities; 5) VCA induces Zn 2+ staining and death only in ipsilateral LGNd neurons, and a 1ppm Zn 2+ diet attenuated injury; 6) Finally, NAD + synthesis and levels are involved because LGNd neuronal death after VCA was dramatically reduced in Wld s animals, and by intraperitoneal pyruvate or nicotinamide. Zn 2+ toxicity is involved in serum and trophic deprivation induced neuronal death. Keywordsvisual cortex ablation; mouse; pyruvate; sirtuin; dorsal lateral geniculate nucleus Target deprivation mediated neuronal death plays a large role during development, trauma, and neurodegeneration. In the developing nervous system, 20-80% of all neurons produced during embryogenesis die before reaching adulthood as a result of competition between neurons for innervation of their targets. This results in matching of the size of the target cell population with the number of innervating neurons (Oppenheim, 1991;Purves et al., 1988). Target deprivation mediated neuronal death is apoptotic and occurs by programmed cell death (PCD) (Deshmukh & Johnson, 1997;Martin et al., 1998). PCD is required for the * corresponding author, FAX: (504) 568-5801, telephone: (504) 599-0880, csheli@lsuhsc.edu. NIH Public Access Author ManuscriptEur J Neurosci. Author manuscript; available in PMC 2011 September 1. Serum Deprivation Models Target Deprivation, and Both Induce an Oxidative InjuryWe and others have shown that serum deprivation (SD) induces substantial oxidative stress leading to partial ATP depletion, K + loss involving inhibition of the Na + /K + ATPase, and the apoptotic cascade . We...

Background: Inhibition of mitochondrial function occurs in many neurodegenerative diseases, and inhibitors of mitochondrial complexes I and II are used to model them. The complex II inhibitor, 3-nitroproprionic acid (3-NPA), kills the striatal neurons susceptible in Huntington’s disease. The complex I inhibitor N-methyl-4-phenylpyridium (MPP⁺) and 6-hydroxydopamine (6-OHDA) are used to model Parkinson’s disease. Zinc (Zn²⁺⁾ accumulates after 3-NPA, 6-OHDA and MPP⁺ in situ or in vivo. Objective: We will investigate the role of Zn²⁺ neurotoxicity in 3-NPA, 6-OHDA and MPP⁺. Methods: Murine striatal/midbrain tyrosine hydroxylase positive, or near-pure cortical neuronal cultures, or animals were exposed to 3-NPA or MPP⁺ and 6-OHDA with or without neuroprotective compounds. Intracellular zinc ([Zn²⁺]_i), nicotinamide adenine dinucleotide (NAD⁺), NADH, glycolytic intermediates and neurotoxicity were measured. Results: We showed that compounds or genetics which restore NAD⁺ and attenuate Zn²⁺ neurotoxicity (pyruvate, nicotinamide, NAD⁺, increased NAD⁺ synthesis, sirtuin inhibition or Zn²⁺ chelation) attenuated the neuronal death induced by these toxins. The increase in [Zn²⁺]_i preceded a reduction in the NAD⁺/NADH ratio that caused a reversible glycolytic inhibition. Pyruvate, nicotinamide and NAD⁺ reversed the reductions in the NAD⁺/NADH ratio, glycolysis and neuronal death after challenge with 3-NPA, 6-OHDA or MPP⁺, as was previously shown for exogenous Zn²⁺. To test efficacy in vivo, we injected 3-NPA into the striatum of rats and systemically into mice, with or without pyruvate. We observed early striatal Zn²⁺ fluorescence, and pyruvate significantly attenuated the 3-NPA-induced lesion and restored behavioral scores. Conclusions: Together, these studies suggest that Zn²⁺ accumulation caused by MPP⁺ and 3-NPA is a novel preventable mechanism of the resultant neurotoxicity.

Pancreatic zinc (Zn(2+)) concentrations are linked to diabetes and pancreatic dysfunction, but Zn(2+) is also required for insulin processing and packaging. Zn(2+) released with insulin increases β-cell pancreatic death after streptozotocin toxin exposure in vitro and in vivo. Triosephosphate accumulation, caused by NAD(+) loss and glycolytic enzyme dysfunction, occur in type-1 diabetics (T1DM) and animal models. We previously showed these mechanisms are also involved in Zn(2+) neurotoxicity and are attenuated by nicotinamide- or pyruvate-induced restoration of NAD(+) concentrations, Zn(2+) restriction, or inhibition of Sir2 proteins. We tested the hypothesis that similar Zn(2+)- and NAD(+)-mediated mechanisms are involved in β-cell toxicity in models of ongoing T1DM using mouse insulinoma cells, islets, and nonobese diabetic (NOD) mice. Zn(2+), streptozotocin, and cytokines caused NAD(+) loss and death in insulinoma cells and islets, which were attenuated by Zn(2+) restriction, pyruvate, nicotinamide, NAD(+), and inhibitors of Sir2 proteins. We measured diabetes incidence and mortality in NOD mice and demonstrated that pyruvate supplementation, or genetic or dietary Zn(2+) reduction, attenuated these measures. T-lymphocyte infiltration, punctate Zn(2+) staining, and β-cell loss increased with time in islets of NOD mice. Dietary Zn(2+) restriction or Zn(2+) transporter 5 knockout reduced pancreatic Zn(2+) staining and increased β-cell mass, glucose homeostasis, and survival in NOD mice, whereas Zn(2+) supplementation had the opposite effects. Pancreatic Zn(2+) reduction or NAD(+) restoration (pyruvate or nicotinamide supplementation) are suggested as novel targets for attenuating T1DM.

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