Abstract
Oxidative stress can trigger neuronal cell death and has been implicated in several chronic neurological diseases and in acute neurological injury. Oxidative toxicity can be induced by glutamate treatment in cells that lack ionotrophic glutamate receptors, such as the immortalized HT22 hippocampal cell line and immature primary cortical neurons. Previously, we found that neuroprotective effects of geldanamycin, a benzoquinone ansamycin, in HT22 cells were associated with a down-regulation of c-Raf-1, an upstream activator of the extracellular signal-regulated protein kinases (ERKs). ERK activation, although often attributed strictly to neuronal cell survival and proliferation, can also be associated with neuronal cell death that occurs in response to specific insults. In this report we show that delayed and persistent activation of ERKs is associated with glutamate-induced oxidative toxicity in HT22 cells and immature primary cortical neuron cultures. Furthermore, we find that U0126, a specific inhibitor of the ERK-activating kinase, MEK-1/2, protects both HT22 cells and immature primary cortical neuron cultures from glutamate toxicity. Glutamate-induced ERK activation requires the production of specific arachidonic acid metabolites and appears to be downstream of a burst of reactive oxygen species (ROS) accumulation characteristic of oxidative stress in HT22 cells. However, inhibition of ERK activation reduces glutamate-induced intracellular Ca(2+) accumulation. We hypothesize that the precise kinetics and duration of ERK activation may determine whether downstream targets are mobilized to enhance neuronal cell survival or ensure cellular demise.
Highlights
Oxidative stress can trigger neuronal cell death and has been implicated in several chronic neurological diseases and in acute neurological injury
Glutamate Activation of extracellular signal-regulated protein kinases (ERKs) in HT22 Cells—Previously, we showed that down-regulation of c-Raf-1 was associated with neuroprotective effects of GA in HT22 cells following exposure to toxic levels of glutamate [34]. c-Raf-1 is an upstream activator of the ERK pathway through its phosphorylation and activation of the ERK kinases, MEK-1/2 [38]
Oxidative stress is associated with a number of neurodegenerative diseases and has been found to induce apoptosis in neuronal cell lines [33, 52] and cultured primary neurons [11], and it contributes to neuronal cell damage that results from various ischemic insults [53, 54]
Summary
Antibodies and Reagents—The anti-phospho-p44/42 MAP Kinase mouse monoclonal antibody was purchased from New England Biolabs (Beverly, MA), and the anti-MAP kinase 1/2 rabbit polyclonal antibody was from Upstate Biotechnology (Lake Placid, NY). Cells were maintained for 24 h in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum prior to treatment with glutamate and/or baicalein or U0126. As shown previously [10], glutamate toxicity in similar primary cultures of rat embryonic cortical neurons is limited to neurons and is due to oxidative stress that results from cystine depletion. Cell Extracts and Western Blotting—Following indicated treatments, HT22 cells or primary cortical neuron cultures were washed twice with ice-cold phosphate-buffered saline and harvested in 1 ml of phosphatebuffered saline containing 10 mM Tris-Cl, pH 7.5, and 1 mM EDTA. HT22 cells, or primary cortical neuronal cultures, were seeded onto 96-well plates at 5,000 cells per well in growth medium (see above) and grown overnight prior to the initiation of any experimental treatments. A similar quantitation was carried out for Fura-2 AM by averaging the ratio of fluorescent signals acquired at 340 and 380 nm
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