Zinc is a ubiquitous heavy metal that binds to proteins involved in critical cellular processes. Apart from its necessary role, excessive release of intracellular free zinc (Zn) is neurotoxic under stressed conditions characteristic of ischemic or epileptic neuronal injury. Our earlier results indicated that Zn-induced cell death is exacerbated in neurons compared to supporting neuroglia, suggesting that astrocytes have means to upregulate Zn buffering mechanisms, i.e. the Zn-binding protein metallothionein (MT). The first aim of this dissertation sought to address whether MT can effectively maintain Zn levels at a non-toxic minimum. From these studies, we have identified a dichotomous role for MT - protective as a Zn buffering agent and detrimental as an oxidant-labile source for toxic Zn. Previous studies demonstrated the role of Zn as a mitochondrial toxin. Although it has been widely speculated that Zn is taken up by the mitochondrial calcium uniporter, the evidence is not entirely convincing. In the second specific aim, we addressed the specific hypothesis that mitochondrial Zn uptake occurs though the uniporter. Using a novel model involving isolated mitochondria pre-incubated with a Zn-selective fluorophore and attached to glass coverslips, we demonstrated for the first time direct visualization of mitochondrial Zn transport. The third specific aim addresses the importance of mitochondria as dynamic intracellular ATP factories, whose intracellular trafficking is critical for neuronal viability. We hypothesized that elevated Zn would attenuate mitochondrial trafficking. Our results revealed that Zn inhibited mitochondrial movement at pathophysiological levels. Intriguingly, acute activation of phosphotidyl inositol 3-kinase was implicated in both Zn-mediated movement inhibition and toxicity, providing a novel role for this traditionally pro-survival signaling pathway.In summary, this dissertation identifies intracellular targets for Zn-mediated neurotoxicity. We specifically emphasize the relevance of mitochondria as a Zn target under two circumstances which are critically dependent on the Zn concentrations established - direct mitochondrial interactions that may involve Zn transport, and indirect mitochondrial interactions that affect intracellular mitochondrial trafficking
