Elsevier

Neuroscience

Volume 167, Issue 4, 2 June 2010, Pages 1125-1137
Neuroscience

Clinical Neuroscience
Research Paper
Neuroprotection of ethanol against ischemia/reperfusion-induced brain injury through decreasing c-Jun N-terminal kinase 3 (JNK3) activation by enhancing GABA release

https://doi.org/10.1016/j.neuroscience.2010.02.018Get rights and content

Abstract

Our latest study indicated that ethanol could attenuate cerebral ischemia/reperfusion-induced brain injury through activating Ionotropic glutamate receptors Kainate Family (Gluk1)–kainate (KA) receptors and gamma-aminobutyric acid (GABA) receptors. However, the possible mechanism of the neuroprotective effects of ethanol remains unclear. In this study we report that ethanol shows neuroprotective effects against ischemic brain injury through enhancing GABA release and then decreasing c-Jun N-terminal kinase 3 (JNK3) activation. Electrophysiologic recording indicated that ethanol enhances GABA release from presynaptic neurons and the released GABA subsequently inhibits the KA receptor–mediated whole-cell currents. Moreover, our data show that ethanol can inhibit the increased assembly of the Gluk2–PSD-95–MLK3 (postsynaptic density protein-95, PSD-95 and mixed-lineage kinase 3, MLK3) module induced by cerebral ischemia and the activation of the MLK3-MKK4/7-JNK (mitogen-activated protein kinase kinase 4/7, MKK4/7) cascade. Pretreatment of the GABAA receptor antagonist bicuculline and antagonist of VGCC (a broad-spectrum blocker of the voltage-gated calcium channel [VGCC]) Chromic (CdCl2) can demolish the neuroprotective effects of ethanol. The results suggest that during ischemia-reperfusion, ethanol may activate presynaptic Gluk1-KA and facilitate Ca2+-dependent GABA release. The released GABA activates postsynaptic GABAA receptors, which suppress the ischemic depolarization and decrease the association of signaling module Gluk2–PSD-95–MLK3 induced by the activation of postsynaptic Gluk2-KA receptors. There is a raised possibility that ethanol inhibiting the JNK3 apoptotic pathway (MLK3/MKK4/7/JNK3/c-Jun/Fas-L) performs a neuroprotective function against ischemic brain injury.

Section snippets

Antibodies and reagents

The following primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA) were used: rabbit polyclonal anti-MLK3 (sc-13072), monoclonal anti-phospho-JNKs (sc-6254), monoclonal anti-JNK3, rabbit polyclonal anti-Fas-L (sc-6237), and rabbit polyclonal anti-c-Jun (sc-9164). Rabbit polyclonal anti-phospho-MLK3, anti-p-MKK7 (No. 4172), and anti-MKK7 (No. 4171) were obtained from Cell Signaling Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal anti-Gluk2 antibody (catalog No. 06-309)

Neuroprotective effects of ethanol against cerebral ischemia/reperfusion-induced apoptotic neuronal death in hippocampal CA1 neurons

To probe whether ethanol has neuroprotective effect against ischemic injury, we investigated the effects of ethanol on the neuronal survival of CA1 pyramidal neurons in rat hippocampus after 5 days of reperfusion. Cresyl Violet staining was used to examine the surviving cells in the CA1 region. Normal cells show round and pale-stained nuclei. The shrunken cells with pyknotic nuclei after reperfusion were counted as dead cells. Transient cerebral ischemia (15 minutes) followed by 5 days of

Discussion

In this study we reported for the first time that ethanol has a neuroprotective effect against ischemic brain injury by enhancing GABA release and decreasing JNK3 activation via suppression of the assembly of signaling module Gluk2PSD-95MLK3. Our experimental results indicate that ethanol has neuroprotective effects against ischemic brain injury in vivo (Fig. 1) through suppressing the phosphorylation of MLK3/MKK4/7/JNK3/c-Jun and the expression of Fas-L (Fig. 5, Fig. 6A, B). However, as an

Acknowledgments

Support was provided by the Great Research Project of the National Nature Science Foundation of China (grant 90608015), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (grants 08KJB180010 and 07KJA18030), and the Research Project of the National Youth Science Foundation of China (grant 30700117).

References (51)

  • S. McCrea et al.

    Acute ethanol administration and transient ischemia: a behavioral and neuropathological study

    Life Sci

    (2000)
  • M. Pascual et al.

    The GABAergic septohippocampal pathway in control and reeler mice: target specificity and termination onto reelin-expressing interneurons

    Mol Cell Neurosci

    (2004)
  • J.W. Phillis et al.

    Protective effects of the free radical scavengers, dimethyl sulfoxide and ethanol, in cerebral ischemia in gerbils

    Neurosci Lett

    (1998)
  • S.H. Qi et al.

    Neuroprotection of ethanol against cerebral ischemia/reperfusion-induced brain injury through GABA receptor activation

    Brain Res

    (2009)
  • P.H. Seeburg

    The molecular biology of mammalian glutamate receptor channels

    Trends Neurosci

    (1993)
  • R. Strong et al.

    Combination of low dose ethanol and caffeine protects brain from damage produced by focal ischemia in rats

    Neuropharmacology

    (2000)
  • H. Tian et al.

    Activation of c-Jun NH2-terminal kinase 3 is mediated by the GluR6PSD-95MLK3 signaling module following cerebral ischemia in rat hippocampus

    Brain Res

    (2005)
  • Q. Wang et al.

    Ethanol preconditioning protects against ischemia-reperfusion-induced brain damage: role of NADPH oxidase-derived ROS

    Free Radic Biol Med

    (2007)
  • J.L. Weiner et al.

    Ethanol modulation of GABAergic transmission: the view from the slice

    Pharmacol Ther

    (2006)
  • J. Xu et al.

    Additive neuroprotection of GABA A and GABA B receptor agonists in cerebral ischemic injury via PI-3K/Akt pathway inhibiting the ASK1-JNK cascade

    Neuropharmacology

    (2008)
  • J. Xu et al.

    Neuroprotection of GluR5-containing kainate receptor activation against ischemic brain injury through decreasing tyrosine phosphorylation of N-methyl-D-aspartate receptors mediated by Src kinase

    J Biol Chem

    (2008)
  • T. Yamaguchi et al.

    Late preconditioning by ethanol is initiated via an oxidant-dependent signaling pathway

    Free Radic Biol Med

    (2003)
  • F. Zhang et al.

    Activation of GABA receptors attenuates neuronal apoptosis through inhibiting the tyrosine phosphorylation of NR2A by Src after cerebral ischemia and reperfusion

    Neuroscience

    (2007)
  • M. Akiko et al.

    Single-dose ethanol administration downregulates expression of cytochrome p450 2E1 mRNA in aldehyde dehydrogenase2 knockout mice

    Alcohol

    (2007)
  • M.N. Ansari et al.

    Evaluation of antioxidant and neuroprotective effect of ethanolic extract of Embelia ribes Burm in focal cerebral ischemia/reperfusion-induced oxidative stress in rats

    Fundam Clin Pharmacol

    (2008)
  • Cited by (0)

    1

    S.-H. Qi and Y. Liu contributed equally to this work.

    View full text