Astrocyte Control of Toxin-Mediated Neuron Death: Role of the Gamma-Glutamyl Path

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DESCRIPTION (provided by principal investigator): Background Toxin-mediated oxidative stress (OS) is a trigger that ultimately commits neurons to apoptotic death. The event can be associated with decreased cell glutathione (GSH) and cell death is mitigated by augmenting neuron GSH. Astrocytes protect neurons from toxin-related OS and subsequent apoptosis via the ?-glutamyl cycle, which maintains neuron GSH homeostasis. An understanding of this neuroprotective pathway, especially its regulatory components and species comparisons, could ultimately lead to novel clinical interventions for devastating neurodegenerative disorders as well as establish accurate model systems. Hypothesis: We hypothesize that within the astrocyte-neuron axis, there is a highly effective, regulated pathway consisting of components which can enhance neuron GSH homeostasis in response to EtOH (E) and Parkinson's Disease-producing environmental toxins, thereby minimizing death of neurons. However, there are exposure patterns to these environmental cross-stressors which damage components of the pathways, thereby impairing its neuroprotective capacity. Specific Aim 1: Aim one will utilize cultures of neurons and astrocytes, alone and in co-culture, to address the direct impact of rotenone, paraquat, and E on astrocyte and neuron components of the ?-glutamyl cycle. Experimental parameters will be effects of the environmental toxins, alone or in combination, on inward transport of key precursors of GSH, GSH enzymatic synthesis, and components of this machinery at which neuroprotection occurs. Specific Aim 2: Aim two will address regulation of three essential components of the neuroprotective pathway. To be determined will be basic mechanisms underlying regulatory and damaging responses to the toxins. These will be regulation of GSH efflux via multidrug resistance protein(s) (Mrp), and two plasma membrane ectopeptidases, ?-glutamyl transpeptidase (GGT) and aminopeptidase N (ApN). Specific Aim 3: Aim 3 will use an in vivo model to extend the in vitro findings to the intact animal. Experiments will utilize two-photon excitation microscopy to determine toxin effects specifically in cortical astrocytes and neurons in the living brain. These will be time-lapse determinations of expressions of GGT and ApN, GSH content and apoptosis-related events during toxin exposure. They will utilize transgenic mice deficient in Mrpl, Mrp4, or GGT. General Description: The proposal addresses a new system by which glial cells protect neurons from the toxic effects of three environmental toxins. These compounds can cause neurons to die as a result of oxidative damage and cells called astrocytes prevent this by maintaining neuron antioxidants. We will determine how the toxins damage it (to prevent this from happening), and how this system is normally controlled.