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Comparative Study
. 2010 Sep;32(6):894-904.
doi: 10.1111/j.1460-9568.2010.07372.x. Epub 2010 Aug 16.

Serum or target deprivation-induced neuronal death causes oxidative neuronal accumulation of Zn2+ and loss of NAD+

Christian T Sheline  1 , Ai-Li Cai, Julia Zhu, Chunxiao Shi
Affiliations
Comparative Study

Serum or target deprivation-induced neuronal death causes oxidative neuronal accumulation of Zn2+ and loss of NAD+

Christian T Sheline et al. Eur J Neurosci. 2010 Sep.

Abstract

Trophic deprivation-mediated neuronal death is important during development, after acute brain or nerve trauma, and in neurodegeneration. Serum deprivation (SD) approximates trophic deprivation in vitro, and an in vivo model is provided by 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 have previously shown that neurotoxicity of extracellular Zn(2+) required entry, increased [Zn(2+) ](i) , and 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 that 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) visual cortex ablation (VCA) induces Zn(2+) staining and death only in ipsilateral LGNd neurons, and a 1 mg/kg Zn(2+) diet attenuated injury; and finally (6) NAD(+) synthesis and levels are involved given that 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.

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Figures

Figure 1
Figure 1. Model of Zinc Neurotoxicity
The resulting increased [Zn2+]i may cause direct inhibition of mitochondria, or an indirect inhibition of GAPDH and mitochondria by a reduction in NAD+ levels induced by an unknown NAD+ catabolizing enzyme (sirtuins). Pyruvate and nicotinamide prevent NAD+ loss and enzyme inhibition. Black = Toxic, Gray = Therapeutic.
Figure 2
Figure 2. Zn2+ loading potentiated, and Zn2+ chelation attenuated SD, and oxidant mediated neurotoxicity
A. Near-pure neuronal cultures were cultured for 8 days - or + an additional 10 µM Zn2+ after which the neurons were stained for Zn2+ using TSQ and fluorescence photomicrographs taken. B. Near-pure cortical neuronal cultures were exposed chronically to the indicated conditions. Neuronal death was determined 24–36 hrs later by lactate dehydrogenase release to the medium scaled to the level associated with near complete death produced by exposure to 20 µM A23187 for 24 hrs, = 100 (mean ± SEM, n = 6–18 cultures per condition). * # $ & @ indicate difference from appropriate oxidant exposure in the absence of added Zn2+ at P<0.05
Figure 3
Figure 3. [Zn2+]i was increased by SD and oxidant exposures
Near-pure neuronal cultures were cultured for 7–8 days after which the neurons were loaded for 30 minutes with 2.5 µM FluoZin3-AM (Zn2+ fluorescent indicator). A. These cultures were then washed and exposed to SD and oxidants as indicated and identical exposure fluorescence photo-micrographs were taken at 200x magnification, bar represents 50 microns. B. The fluorescence intensity of the cells under these conditions were quantitated using Metamorph and are presented as the percentage of baseline fluorescence. The effects of therapeutic concentrations of compounds against ethacrynic acid induced increases in [Zn2+]i were also tested as indicated. * indicates difference from 1 hr control exposure at P<0.05; # indicates difference from 5 hr ethacrynic acid exposure at P<0.05
Figure 4
Figure 4. 3-AP potentiated SD and oxidant neurotoxicity, and increased [NAD+]i attenuated them; lactate was ineffective
Near-pure cortical neuronal cultures were exposed to the indicated conditions. Neuronal death was determined 24 hrs later by propidium iodide staining scaled to the level associated with near complete death produced by exposure to 20 µM A23187 for 24 hrs, = 100 (mean ± SEM, n = 9–18 cultures per condition). 3-AP indicates the addition of 50 µM 3-acetyl pyridine, NAD+ indicates the addition of 6 mM NAD+. Pyruvate, nicotinamide and lactate were present as indicated at 10 mM. * indicates difference from ethacrynic acid (ETH) exposure, # indicates difference from H2O2 exposure, $ indicates difference from glucose deprivation, and & indicates difference from serum deprivation at P < 0.05 by one-way ANOVA followed by a Bonferroni test.
Figure 5
Figure 5. Neuronal cultures from MT-III and PARP-1 knockouts and Wlds were differentially sensitive to SD and oxidant neurotoxicity
Near-pure cortical neuronal cultures from MT-III and PARP-1 knockouts, Wlds and control cultures were exposed to the indicated conditions. Neuronal death was determined 24 hrs later by lactate dehydrogenase release to the medium. * indicates difference between B6 control cultures and Wlds cultures after ETH exposure at P < 0.05; # indicates difference between control cultures and MT-III or PARP-1 knockouts after H2O2 exposure at P< 0.05. $ indicates difference between control cultures and MT-III or PARP-1 knockouts, or Wlds cultures after serum deprivation at P< 0.05. B6 is the appropriate control for MT-III −/− and Wlds, and 129 is the appropriate control for PARP-1 −/−.
Figure 6
Figure 6. Ethacrynic acid, H2O2, serum or glucose deprivation mediated neuronal injuries were attenuated by sirtinol or 2-hydroxynaphthaldehyde, and these injuries were potentiated by resveratrol or fisetin
Near-pure neuronal cultures were exposed to A) 25 µM ethacrynic acid (ETH), or 80 µM H2O2 for 24 hrs, or B) to 5 hrs of glucose deprivation, or continuous serum deprivation (36 hrs) and neuronal death was assessed by LDH release to the bathing medium (mean ± SEM, n = 6–18 cultures per condition). Fisetin and resveratrol exposures were a 3 hr pretreatment at 10 µM. Sirtinol and 2-hydroxynaphthaldehyde were present chronically as indicated at 10 µM. * indicates difference from 25 µM ETH; # indicates difference from 80 µM H2O2; $ indicates difference from 5 hours of glucose deprivation exposure; and & indicates difference from serum deprivation exposure at P < 0.05 by one-way ANOVA followed by a Bonferroni test.
Figure 7
Figure 7. Ipsilateral LGNd neurons die after visual cortex ablation; Wlds mice were resistant
Unilateral V1 ablation was performed and animals were sacrificed after 7–11 days, and the LGNd was stained for Zn2+ using ZP1, or death using FluoroJadeB. Identical exposure fluorescence photo-micrographs were taken at 100x magnification, bar represents 100 microns. Insets are the boxed area at 400x magnification, and the bar represents 25 microns.
Figure 8
Figure 8. Only ipsilateral LGNd neurons stain for Zn2+ and die after visual cortex ablation; pyruvate, or nicotinamide treated mice were resistant
Unilateral V1 ablation was performed and animals were sacrificed after 7–11 days, and the LGNd was stained for Zn2+ using ZP1, or death using FluoroJadeB. Identical exposure fluorescence photo-micrographs were taken at 100x magnification, bar represents 100 microns. Insets are the boxed area at 400x magnification, and the bar represents 25 microns.
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