Divergent roles for the ERK1/2 signaling pathway in neuronal oxidative stress

In the mouse HT22 hippocampal cell line and immature primary cortical neurons, excessive glutamate treatment results in intracellular cysteine depletion, subsequent glutathione loss and the steady accumulation of reactive oxygen species (ROS). This form of oxidative stress ultimately leads to cell death. Previous data from our laboratory had shown that delayed and persistent activation of extracellular signal-regulated kinases-1/2 (ERK1/2) is associated with glutamate induced oxidative toxicity in HT22 cells and immature primary neurons. In addition, U0126, a specific inhibitor of the ERK-activating kinase, MEK-1/2, inhibits ERK activation and prevents cells death induced by glutamate. However the mechanisms responsible for this chronic activation of ERK during oxidative stress have not been well characterized. Results from this thesis demonstrated that overexpression of a dominant negative mutant of MEK1 blocked glutamate toxicity in transfected HT22 cells. These data confirmed previous results and illustrated that ERK1/2 activation is necessary for oxidative toxicity. However overexpression of a constitutively active MEK1ERK chimera (LA-MEK1ERK2) that induced robust ERK activation and translocation into nucleus did not trigger toxicity in HT22 cells. Thus, ERK1/2 phosphorylation and activation is not sufficient for glutamate induced cell oxidative toxicity. Activation of ERK1/2 in HT22 cells has a distinct kinetic profile with an initial peak occurring between 30 minutes and 1 hour of glutamate treatment and a second peak typically emerging after 6 hours. I demonstrate here that the initial phase of ERK1/2 induction is due to activation of metabotropic glutamate receptor type I (mGluRI). ERK1/2 activation by mGluRI contributes to an HT22 cell adaptive response to oxidative stress as glutamate induced toxicity is enhanced upon pharmacological inhibition of mGluRI. The protective effect of ERK1/2 activation at early times after glutamate treatment is mediated by a restoration of glutathione (GSH) levels that are reduced due to depletion of intracellular cysteine pools. Additional results suggest that mGluRI may be involved in regulating mRNA and protein levels of glutamate-cysteine ligase (GCL), which would lead to enhanced glutathione synthesis. Thus, ERK1/2 activation by mGluRI protects HT22 cells from oxidative toxicity through upregulation of GCL transcription and translation, and subsequent enhancement of GSH levels