MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia

Abstract

MEK1 is a dual-specificity kinase that phosphorylates extracellular-signal-responsive kinase (ERK)1͞2 on threonine and tyrosine residues and activates these mitogen-activated protein (MAP) kinases (1). The ERK MAP kinases are phosphorylated in the hippocampus in response to global brain ischemia (2, 3). The use of general tyrosine kinase inhibitors, such as genistein, decreases ERK2 phosphorylation in this model and is associated with protection against neuronal cell damage (2). These data, however, do not establish a direct relationship between ERK2 phosphorylation and cell injury. Tyrosine kinase inhibitors, such as genistein, also inhibit protein kinaseindependent systems, such as fatty acid synthesis, mitochondrial oxidative phosphorylation, and lactate transport (4). It has been shown that inhibition of MEK1 protects hippocampal neurons in a cell-culture model of seizure (5). In this study, we show that inhibition of the MEK1͞ERK pathway leads to neuroprotection from brain injury resulting from occlusion of the middle cerebral artery (MCA). MethodsIschemia Model. Adult male SV-129 mice (18-22 g, Taconic Farms, Germantown, NY) were housed under diurnal lighting conditions and allowed food and water ad libitum. Animals were anesthetized with 1.5% halothane and maintained in 1.0% halothane in 70% N 2 O and 30% O 2 by using a Fluotec 3 vaporizer (Colonial Medical, Amherst, NH). Ischemia was induced with a 8.0 nylon monofilament coated with silicone resin͞hardener mixture (Xantopren and Elastomer Activator, Bayer Dental, Osaka, Japan) as described previously (6).Two microliters of PD98059, SB203580, or DMSO was injected intracerebroventricularly (bregma Ϫ0.9 mm lateral, Ϫ0.1 mm posterior, Ϫ3.1 mm deep) 30 min before ischemia by using a Hamilton injection syringe. The concentrations used are stated in Results.Immunohistochemistry. Mice were deeply anesthetized with an overdose of sodium pentobarbital (100 mg͞kg i.p.) and then transcardially perfused with 0.9% saline solution, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. The brains were quickly removed and stored in the same fresh buffer containing 20% sucrose. Brains were cut with a freezing microtome into coronal sections 40 m thick. The sections were processed by the free-floating method as described previously (7).Western Blot Analysis. Postischemic and nonischemic brains were Dounce homogenized in 1 ml of potassium phosphate buffer [10 mM potassium phosphate, pH 7.05͞1 mM EDTA͞5 mM EGTA͞10 mM magnesium chloride͞50 mM ␤-glycerophosphate͞1 mM sodium vanadate͞1 mM dithiothreitol (DTT)͞ 0.5% Nonidet P-40͞0.1% Brij-35]. Lysates were clarified by centrifugation at 14,000 ϫ g for 10 min. The protein concentration in the supernatant was determined by the Bradford assay (Bio-Rad). To test for phosphorylation of ERK1͞2, 40 g of total cell lysate was subjected to electrophoresis on an SDS͞10% polyacrylamide gel and transferred onto Immobilon-P membrane (Millipore). Western blotting was performed with phospho-specific p44͞42 MAP kinas...

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MAPK signaling in neurodegeneration: influences of flavonoids and of nitric oxide

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