Glutamate receptor- and Ca2+-mediated neurotoxicity was the focus of study during past grant periods. Recently, we have begun to examine a related form of neurotoxicity, also enhanced by glutamate receptor activation but mediated by Zn2+ rather than Ca2+. Zn2+-mediated neurotoxicity likely contributes to central neuronal death after certain insults, such as transient global ischemia. Our Central Hypothesis is that extracellular Zn2+ can kill neurons by: 1) entering across the plasma membrane, largely through voltage-gated Ca2+ channels (VGCCs) in depolarized neurons; 2) increasing intracellular free Zn2+ ([Zn2+]i); 3) interfering with glycolysis, causing ATP levels to fall; 4) triggering apoptosis (at lower Zn2+ levels). The proposed experiments will test aspects of this central hypothesis in cultured murine cortical neurons, delineating mechanisms underlying Zn2+-induced neuronal death to advance efforts to develop therapeutic countermeasures that might be used to reduce brain damage after cardiac arrest. Cultured neurons will be exposed to varying concentrations of extracellular zinc for brief (""""""""fast toxicity"""""""") or prolonged (""""""""slow toxicity"""""""") time periods. We plan to define the relationships linking transmembrane Zn2+ influx (measured with patch-clamp and radio-isotope flux techniques), [Zn2+]I (measured with dye videomacroscopy), cellular Zn2+ content (measured with atomic absorption spectroscopy or inductively-coupled plasma spectroscopy), and cellular apoptosis (v.s. necrosis). We will also measure resultant neuronal levels of ATP, NAD+, NADH and glycolytic intermediates, mitochondrial transmembrane potential, and cytoplasmic reactive oxygen species (measured with dihydroethidium dye). Finally, we will test genetic perturbations of cellular Zn2+ homeostasis, specifically increased or decreased expression of the key plasma membrane Zn2+ transporter, ZnT-1, or the major neuronal intracellular Zn2+ binding protein, metallothionein-III, will produce the changes in vulnerability to Zn2+ neurotoxicity predicted by the central hypothesis.
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