Human Bcl-2 protects against AMPA receptor-mediated apoptosis.
Dysfunctions of the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L-glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor-mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl-2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl-2 gene, the influence of Bcl-2 on AMPA receptor-mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1-100 microM), and cellular injury was analyzed 48 h after insult using phase-contrast microscopy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay, and DNA staining with 4,6-diamidino-2-phenylindole and Sytox Green. AMPA produced a concentration-dependent increase in cell death that was significantly attenuated by human Bcl-2. AMPA (3 microM) increased the number of apoptotic nuclei to 60% of control in wild-type cultures, and human Bcl-2 significantly decreased the number of apoptotic nuclei to 30% of AMPA-treated cultures. Human Bcl-2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1-10 nM) and H2O2 (0.1-30 microM) and where neuronal death was by apoptosis, but not against H2O2-induced necrosis. Our findings indicate that overexpression of Bcl-2 in primary cultured neurons protects in an insult-dependent manner against AMPA receptor-mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.
Cheung
N S
NS
Department of Pharmacology, Monash University, Clayton, Victoria, Australia.
Beart
P M
PM
Pascoe
C J
CJ
John
C A
CA
Bernard
O
O
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005838:Genotype; D018698:Glutamic Acid / Q000502:physiology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009336:Necrosis; D019579:Neocortex / Q000166:cytology; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D009498:Neurotoxins / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism; D018091:Receptors, AMPA / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D009498:Neurotoxins; D016877:Oxidants; D019253:Proto-Oncogene Proteins c-bcl-2; D018091:Receptors, AMPA; D018698:Glutamic Acid; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D004247:DNA; D006861:Hydrogen Peroxide; D019311:Staurosporine