INTRACELLULAR MAGNESIUM AND EXCITOTOXICITY

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Mg2+ is an abundant intracellular cation that is critical for the control of a wide array of intracellular processes. However, relatively little is known about the homeostatic mechanisms that control intracellular free Mg2+ ([Mg2+]i, nor about mechanisms that might perturb (Mg2+]i. It has recently been shown that stimuli that are normally considered excitotoxic to neurons produce millimolar increases in [Mg2+]i in cultured neurons. The long term goal of this project is to understand the role of [Mg2+]i in processes associated with neuronal injury and to develop effective treatment strategies based on manipulation of Mg2+ homeostasis. This will be accomplished by the following specific aims: 1. characterization of mechanisms that alter [Mg2+]i. [Mg2+]i will be measured in cultured rat forebrain neurons using Mg2+-sensitive fluorescent dyes. The characteristics of the (Mg2+]i response to glutamate receptor activation will be established, and the hypothesis that the [Mg2+]i is proportional to Ca2+ will be tested. Fluorescence imaging techniques will be used to determine the source of the intracellular Mg2+ release. 2. Elucidation of the mechanisms of [Mg2+]i homeostasis. The role of a number of putative cellular Mg2+ buffering mechanisms will be established. In particular, the hypothesis that Na+/Mg2+ exchange contributes to neuronal Mg2+ buffering will be examined. 3. Determination of the role of changes in intracellular Mg2+ in excitotoxicity. Preliminary results show that excitotoxic stimuli are very effective at raising [Mg2+]i. The role of elevated [Mg2+]i as well as the effect of tissue Mg2+ loss in excitotoxicity will be investigated. This will establish the role of intracellular Mg2+ in excitotoxic cell death and highlight novel therapeutic approaches to ameliorating neuronal injury. 4. Regulation of neuronal Ca2+ homeostasis by intracellular Mg2+. There is very little information available about the role of intracellular Mg2+ in regulating neuronal function. The influence of [Mg2+]i changes on Ca2+ entry and Ca2+ homeostasis will be investigated, and will provide another link between [Mg2+]i changes and excitotoxicity. These studies will provide substantial new information about the role of a poorly understood cation in the regulation of neuronal function. More importantly, these studies will reveal the involvement of Mg2+ in excitotoxic neuronal injury and thereby highlight mechanism that contribute to neuronal death. We also anticipate that these studies will generate novel concepts about the treatment of neuronal injury based on manipulation of the Mg2+ environment and thereby positively impact the treatment of stroke and traumatic brain injury.