5-Lipoxygenase inhibitor MK-886 increases GluR1 phosphorylation in neuronal cultures in vitro and in the mouse cortex in vivo

Abstract

Modifications of AMPA glutamate receptor GluR1 phosphorylation are critical for neuroplastic mechanisms. Previous in vitro studies in brain slices employed MK-886, a functional inhibitor of the enzyme 5-lipoxygenase (5-LOX), and found increased GluR1 phosphorylation. Since slice preparations have accompanying postmortem phosphorylation changes, e.g., decreased GluR1 phosphorylation, it remains to be clarified whether MK-886 can affect GluR1 phosphorylation in intact neurons and in the brain in vivo. We used primary neuronal cultures prepared from embryonic mouse brain and in vivo drug administration to investigate the effects of MK-886 on GluR1 phosphorylation using quantitative Western immunoblotting assays. In vitro, MK-886 increased GluR1 phosphorylation at both serine 831 and serine 845. In vivo, repeated but not a single MK-886 injection increased GluR1 phosphorylation in the prefrontal cortex. These findings indicate that MK-886 has an intrinsic effect on neuronal phosphorylation both in vitro and in vivo and support the use of MK-886 as a pharmacological tool in studies of not only the 5-LOX pathway but also neuronal GluR1 functioning.

Introduction

MK-886 is an experimental drug typically used as inhibitor of the 5-lipoxygenase (5-LOX, 5-LO) pathway. 5-LOX is an enzyme involved in the synthesis of eicosanoids leukotrienes from arachidonic acid (Radmark and Samuelsson, 2005, Werz and Steinhilber, 2005). The relevance of 5-LOX for the central nervous system (CNS) functioning is indicated by reduced anxiety-like behavior (Uz et al., 2002) and reduced amyloid brain pathology (Firuzi et al., 2005) in 5-LOX-deficient mice. In normal human and rodent brain 5-LOX is expressed both in neurons and glia and its expression increases after traumatic or ischemic brain injury (Zhang et al., 2006, Zhou et al., 2006). Furthermore, 5-LOX expression and activity in the brain increase during aging (Uz et al., 1998, Qu et al., 2000, Chinnici et al., 2006).

The inhibitory effect of MK-886 on the 5-LOX pathway is believed to originate in the binding of this drug to the 5-LOX activating protein (FLAP), which enhances 5-LOX activity (Vickers, 1995). In primary neuronal cultures in vitro, MK-886 reduces neurotoxicity mediated by NMDA glutamate receptor-triggered activation of 5-LOX (Ge et al., 2006). In vivo, systemic administration of MK-886 reduced brain damage triggered in a model of focal cerebral ischemia (Ciceri et al., 2001). Applied to hippocampal rat slices in vitro, MK-886 significantly increased the phosphorylation of GluR1 but not GluR2 subunits of ionotropic glutamate/AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptors (Menard et al., 2005). The phosphorylation of the GluR1 subunit at Ser831 and Ser845 is crucial for the regulation of AMPA receptor membrane insertion (i.e., surface expression) and functioning (Palmer et al., 2005, Roche et al., 1994), and may contribute to learning, memory, and brain aging processes (Genoux et al., 2002). Surface expression and synaptic incorporation of GluR1 are increased by activation of D1 dopamine receptors (Gao et al., 2006) and GluR1 phosphorylation has been implicated in behavioral actions of cocaine (Boudreau and Wolf, 2005, Sun et al., 2005, Zhang et al., 2007). Interestingly, behavioral effects of cocaine are altered in mice deficient for 5-LOX (Kurtuncu et al., 2005) and 12-lipoxygenase (Walters et al., 2003).

To date, the increase of GluR1 phosphorylation by MK-886 has been demonstrated in postmortem brain slices only. Although useful, this preparation is characterized by time-dependent postmortem changes in the phosphorylation of GluR1. It has been shown that maintaining slices in vitro results in persistent dephosphorylation of GluR1 (Ho et al., 2004). Thus, the observed stimulatory effects of MK-886 on GluR1 phosphorylation in brain slices may be due to the prevention of postmortem dephosphorylation rather than an intrinsic drug-induced stimulation of GluR1 phosphorylation. To support these observations, the present study used primary neuronal cultures prepared from embryonic mouse brain and maintained in culture for at least 8 days prior to these experiments as well as in vivo systemic MK-886 administration. The effects of MK-886 on anxiety-like behavior were investigated in the elevated plus maze assay.