Excitotoxic Cell Death Mediated by Ca2+-Permeable Ampa Receptors is Coupled to the JNK Signalling Pathway
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1
University of Coimbra, Center for Neuroscience and Cell Biology, Portugal
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2
University of Coimbra, Department of Zoology, Portugal
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3
Jonhs Hopkins University School of Medicine, Department of Neuroscience, Howard Hughes Medical Institute, United States
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4
University College London, Institute of Child Health, United Kingdom
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5
University of Coimbra, Faculty of Pharmacy, Portugal
In cerebral ischemia, excitotoxic cell death is due to overactivation of glutamate receptors following an excessive glutamate release and impairement in glutamate uptake. In this work, we investigated the specific contribution of Ca2+-permeable a-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) to excitotoxic cell death, using a HEK293 cell line constitutively expressing GluR4flip subunits of AMPARs (HEK- GluR4), and the excitotoxic signaling pathways induced by overactivation of this specific subtype of AMPARs. We hypothesized that c-Jun N-terminal kinase (JNK) could take part in this excitotoxic mechanism, since JNK is a mitogen-activated protein kinase (MAPK) activated in stress conditions, and was shown to be involved in several neuronal cell death paradigms. Stimulation of HEK-GluR4 cells with glutamate plus cyclothiazide for 1h decreased cell viability by 60%, as determined by MTT assay. The excitotoxic stimulation induced a biphasic JNK activation in a Ca2+-dependent manner. To determine the contribution of the JNK pathway to HEK-GluR4 excitotoxic death, cells were transduced with the JNK binding domain (JBD), which is a selective strategy to inhibit JNK signalling, or with a dominant negative form of c-Jun that inhibits the functional activity of this transcription factor. Both strategies conferred protection against Ca2+-permeable AMPAR-mediated cell death. Excitotoxic stimulation with glutamate increased c-Jun phosphorylation, and enhanced the JNK- dependent GluR4 phosphorylation. The GluR4 subunit is a newly characterized JNK substrate, which was shown to be specifically phosphorylated on T855 under physiological neuronal activity. The relevance of GluR4 T855 phosphorylation to the excitotoxic cell death mediated by Ca2+-permeable AMPARs is currently under evaluation using GluR4 subunits mutated at this site. Co-immunoprecipitation studies showed that GluR4 interacts with the JNK scaffold protein JIP-1, in HEK-GluR4 cells overexpressing this protein. Taken together, our results show that excitotoxicity mediated by Ca2+-permeable AMPARs is associated to the Ca2+-dependent activation of a cytotoxic JNK pathway. In vivo JIP-1 may be the molecular link between GluR4-containing AMPARs and the JNK signaling pathway.
Supported by Biogen Idec
Conference:
11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009.
Presentation Type:
Poster Presentation
Topic:
Neurodegenerative Disorders
Citation:
Vieira
M,
Fernandes
J,
Duarte
CB,
Huganir
R,
Ham
J,
Carvalho
AL and
Santos
A
(2009). Excitotoxic Cell Death Mediated by Ca2+-Permeable Ampa Receptors is Coupled to the JNK Signalling Pathway.
Front. Neurosci.
Conference Abstract:
11th Meeting of the Portuguese Society for Neuroscience.
doi: 10.3389/conf.neuro.01.2009.11.081
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Received:
07 Aug 2009;
Published Online:
07 Aug 2009.
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Correspondence:
Marta Vieira, University of Coimbra, Center for Neuroscience and Cell Biology, Alicante, Portugal, martavieira931@gmail.com