title: Prolonged glutamate excitotoxicity increases GluR1 immunoreactivity but decreases mRNA of GluR1 and associated regulatory proteins in dissociated rat retinae in vitro.
abstract: Glutamate excitotoxicity contributes to damage following injury to the central nervous system via mechanisms including changes in the expression of receptors, calcium overload, oxidative stress and cell death. Excitotoxicity is triggered by glutamate binding to receptors, including calcium permeable AMPA receptors, which can be upregulated under injury conditions. However, the transcriptional response of AMPA receptor regulatory proteins to excitotoxic conditions is unknown. Here, we use real-time quantitative PCR (RT-qPCR), to determine the effect of prolonged glutamate excitotoxicity on the expression of mRNA encoding for GluR1 and AMPA receptor regulatory proteins in dissociated rat retinal cultures that include neuronal (retinal ganglion cell (RGCs)) and glial (Müller) cell populations. mRNA levels of GluR1 and regulators of the GluR1 subunit of AMPA receptors, including Sap97, Cnih2 and Cnih3, decreased following 6, 24 and 48 h incubation with 5 mM glutamate: related regulators not associated with GluR1 were unaffected. In contrast, GluR1 protein, assessed immunohistochemically, was increased in both RGCs and Müller cells after 24 h glutamate exposure: western blotting analysis was inconclusive. Reductions in mRNA of GluR1 and associated regulators occurred prior to cell death, which was first detected at 24 h, and substantial by 48 h. Exposure to glutamate acutely increased the intracellular calcium concentration in the cultures and by 24 h, reactive oxygen species were increased. Our data suggest a negative feedback mechanism in retinal cells, that down-regulates transcription of genes encoding GluR1 regulatory proteins in response to glutamate exposure. Despite this mechanism, GluR1 protein levels remain increased, and are associated with increased reactive species and cell death. Therapeutic strategies targeting calcium permeable AMPA receptors should take into account that increases in GluR1 protein are not necessarily associated with increases in associated mRNA levels over time.
authors: Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, WA 6009, Australia; School of Chemistry and Biochemistry, The University of Western Australia, WA 6009, Australia.
authors: Michael
authors: M
authors: Challenor
authors: Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, WA 6009, Australia; School of Anatomy, Physiology and Human Biology, The University of Western Australia, WA 6009, Australia.
authors: Ryan
authors: R
authors: O'Hare Doig
authors: Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, WA 6009, Australia.
authors: Paula
authors: P
authors: Fuller
authors: Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, WA 6009, Australia.
authors: Marcus
authors: M
authors: Giacci
authors: Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, WA 6009, Australia.
authors: Carole
authors: C
authors: Bartlett
authors: School of Anatomy, Physiology and Human Biology, The University of Western Australia, WA 6009, Australia.
authors: Celeste H
authors: CH
authors: Wale
authors: School of Anatomy, Physiology and Human Biology, The University of Western Australia, WA 6009, Australia.
authors: Greg S
authors: GS
authors: Cozens
authors: School of Anatomy, Physiology and Human Biology, The University of Western Australia, WA 6009, Australia; Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia.
authors: Livia
authors: L
authors: Hool
authors: Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, WA 6009, Australia.
authors: Sarah
authors: S
authors: Dunlop
authors: School of Chemistry and Biochemistry, The University of Western Australia, WA 6009, Australia.
authors: K
authors: K
authors: Swaminathan Iyer
authors: Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, WA 6009, Australia.
authors: Jennifer
authors: J
authors: Rodger
authors: Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, WA 6009, Australia. Electronic address: Lindy.fitzgerald@uwa.edu.au.
authors: Melinda
authors: M
authors: Fitzgerald
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D015536:Down-Regulation / Q000187:drug effects*; D005136:Eye Proteins / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism; D051381:Rats; D018091:Receptors, AMPA / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D012164:Retinal Diseases / Q000378:metabolism* / Q000473:pathology; D009419:Nerve Tissue Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D005136:Eye Proteins; D018698:Glutamic Acid; D008565:Membrane Proteins; D018091:Receptors, AMPA; C539313:Cnih2 protein, rat; D009419:Nerve Tissue Proteins; C539314:Cnih3 protein, rat; C501119:Dlg1 protein, rat
keywords: AMPA receptor; Calcium; Cornichon; Glutamate excitotoxicity; Retina