Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity

Abstract

Excessive release of glutamate and the subsequent inf lux of calcium are associated with a number of neurological insults that result in neuronal death. The calcium-activated intracellular signaling pathways responsible for this excitotoxic injury are largely unknown. Here, we report that PD098059, a selective inhibitor of the calcium-activated p44͞42 mitogen-activated protein kinase (MAP kinase) pathway, reduces neuronal death in a cell-culture model of seizure activity. Dissociated hippocampal neurons grown chronically in the presence of kynurenate, a broad spectrum glutamatereceptor antagonist, and elevated amounts of magnesium exhibit intense seizure-like activity after the removal of these blockers of excitatory synaptic transmission. A 30-min removal of the blockers produced extensive neuronal death within 24 h as assayed by the uptake of trypan blue and the release of lactate dehydrogenase. Phospho-p44͞42 MAP kinase immunoreactivity after 30 min of seizure-like activity was present in many neuronal somata and dendrites as well as some synaptic terminals, consistent with both the presynaptic and postsynaptic effects of this pathway. The addition of PD098059 (40 M; EC 50 ‫؍‬ 10 M) during a 30-min washout of synaptic blockers inhibited the phosphorylation of p44͞42 MAP kinase and reduced both the trypan-blue staining (n ‫؍‬ 13) and the release of lactate dehydrogenase (n ‫؍‬ 16) by 73% ؎ 18% and 75% ؎ 19% (mean ؎ SD), respectively. The observed neuroprotection could be caused by an effect of PD098059 on seizure-like events or on downstream signaling pathways activated by the seizure-like events. Either possibility suggests a heretofore unknown function for the p44͞42 MAP kinase pathway in neurons.Stimulation of numerous cell-surface receptors leads to the activation of kinase cascades that integrate and amplify extracellular signals and transmit them to intracellular targets (1). Mitogen-activated protein kinases (MAP kinases), a family of serine͞threonine kinases, are activated by phosphorylation on threonine and tyrosine (2, 3). One subfamily of MAP kinases, known as extracellular signal-regulated kinase (ERK), is activated strongly by mitogens, including growth factors, leading to many cellular responses including proliferation and differentiation (4, 5). Two ERK isoforms, ERK1 (p44) and ERK2 (p42), are highly expressed in the brain; p42 is particularly enriched in the dendrites and somata of discrete neuronal populations including the hippocampus (4, 6). The function of p44͞42 MAP kinase in postmitotic, terminally differentiated neurons is unclear, although recent reports implicate these kinases in synaptic plasticity (7,8).Stimulation of glutamate receptors and influx of Ca 2ϩ are associated with excitotoxic injury (9, 10) and lead to the phosphorylation of p44͞42 MAP kinase in neurons (11-16).Several neurological insults that produce an excessive release of glutamate and neuronal death, including hypoglycemia, ischemia, and kainate-or bicuculline-induced seizures, also result in the phosphoryla...

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