Antioxidant N-acetylcysteine inhibits the activation of JNK3 mediated by the GluR6–PSD95–MLK3 signaling module during cerebral ischemia in rat hippocampus

doi:10.1016/j.neulet.2006.07.007

Neuroscience Letters

Volume 408, Issue 3, 20 November 2006, Pages 159-164

https://doi.org/10.1016/j.neulet.2006.07.007 Get rights and content

Abstract

Cerebral ischemia induces kainate receptor glutamate receptor 6 (GluR6) binding to the postsynaptic density protein 95 (PSD95), which in turn anchors mixed lineage kinase 3 (MLK3) via SH3 domain in rat brain. MLK3 subsequently activates c-Jun NH₂-terminal kinase (JNK) via MAP kinase kinases (MKKs). In this study, we investigated the association of PSD95 with GluR6 and MLK3, the autophosphorylation of MLK3, the combination of MLK3 with JNK3, and the phosphorylation of JNK3 during cerebral ischemia in rat hippocampus CA1. Our results indicate that the GluR6–PSD95–MLK3 complex quickly enhanced at 5 min of ischemia and peaked at 10 min of ischemia, and then gradually reduced with the prolonged time of ischemia. Interestingly, the combination of MLK3 and JNK3 gradually increased from 5 min to 30 min of ischemia. JNK3 phosphorylation first increased and then attenuated in cytosol, suggesting the translocation of activated JNK3 to nucleus during ischemia. To further investigate the possible mechanism of JNK3 activation, antioxidant N-acetylcysteine (NAC) was given to the rats 20 min prior to ischemia. Results indicate that NAC distinctly inhibited the association of PSD95 with GluR6 and MLK3, the autophosphorylation of MLK3, the combination of MLK3 with JNK3 and JNK3 activation. Taken together, these finding indicate that ischemic stimulation results in JNK3 activation through the GluR6–PSD95–MLK3 signaling module, and that the activation of JNK3 is closely related to oxidative stress.

Section snippets

Acknowledgement

This work was supported by a grant from the Key Project of the National Natural Science Foundation of China (No. 30330190).

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Stroke is a leading cause of adult morbidity and mortality with very limited treatment options. Evidence from preclinical models of ischemic stroke has demonstrated that the antioxidant N-acetylcysteine (NAC) effectively protects the brain from ischemic injury. Here, we evaluated a new pathway through which NAC exerted its neuroprotection in a transient cerebral ischemia animal model. Our results demonstrated that pretreatment with NAC increased protein levels of hypoxia-inducible factor-1α (HIF-1α), the regulatable subunit of HIF-1, and its target proteins erythropoietin (EPO) and glucose transporter (GLUT)-3, in the ipsilateral hemispheres of rodents subjected to 90 min middle cerebral artery occlusion (MCAO) and 24 h reperfusion. Interestingly, after NAC pretreatment and stroke, the contralateral hemisphere also demonstrated increased levels of HIF-1α, EPO, and GLUT-3, but to a lesser extent. Suppressing HIF-1 activity with two widely used pharmacological inhibitors, YC-1 and 2ME2, and specific knockout of neuronal HIF-1α abolished NAC’s neuroprotective effects. The results also showed that YC-1 and 2ME2 massively enlarged infarcts, indicating that their toxic effect was larger than just abolishing NAC's neuroprotective effects. Furthermore, we determined the mechanism of NAC-mediated HIF-1α induction. We observed that NAC pretreatment upregulated heat-shock protein 90 (Hsp90) expression and increased the interaction of Hsp90 with HIF-1α in ischemic brains. The enhanced association of Hsp90 with HIF-1α increased HIF-1α stability. Moreover, Hsp90 inhibition attenuated NAC-induced HIF-1α protein accumulation and diminished NAC-induced neuroprotection in the MCAO model. These results strongly indicate that HIF-1 plays an important role in NAC-mediated neuroprotection and provide a new molecular mechanism involved in the antioxidant’s neuroprotection in ischemic stroke.
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¹: The first two authors contributed equally to this work.

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Antioxidant N-acetylcysteine inhibits the activation of JNK3 mediated by the GluR6–PSD95–MLK3 signaling module during cerebral ischemia in rat hippocampus

Abstract

Section snippets

Acknowledgement

Glutathione monoethyl ester provides neuroprotection in a rat model of stroke

Neurosci. Lett.

Review: neurotransmitter receptors. II. AMPA and kainate receptors

Neuropharmacology

Reactive oxygen radicals in signaling and damage in the ischemic brain

J. Cereb. Blood Flow. Metab.

Multiple roles of glutathione in the central nervous system

Biol. Chem.

PDZ proteins organize synaptic signaling pathways

Cell

Mixed-lineage kinase control of JNK and p38 MAPK pathways

Nat. Rev. Mol. Cell Biol.

Identification and characterization of SPRK, a novel src-homology 3 domain-containing proline-rich kinase with serine/threonine kinase activity

J. Biol. Chem.

SAP90 binds and clusters kainate receptors causing incomplete desensitization

Neuron.

A critical role of neural-specific JNK3 for ischemic apoptosis

Proc. Natl. Acad. Sci. U.S.A.

The kinase activation loop is the key to mixed lineage kinase-3 activation via both autophosphorylation and hematopoietic progenitor kinase 1 phosphorylation

J. Biol. Chem.

Antioxidant therapy attenuates JNK activation and apoptosis in the remote noninfarcted myocardium after large myocardial infarction

Biochem. Biophys. Res. Commun.

Ischemic cell death in brain neurons

Physiol. Rev.

AMPA receptor-mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK, calpain and caspase 3

J. Neurochem.

Dopamine induces apoptosis through an oxidation-involved SAPK/JNK activation pathway

J. Biol. Chem.

Molecular mechanisms of the decision between life and death: regulation of apoptosis by apoptosis signal-regulating kinase 1

J. Biochem. (Tokyo)