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Published on: May 12, 2013
A Bcl-2 antisense oligonucleotide increases alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) toxicity
1Department of Neurology, University of Pittsburgh School of Medicine, PA 15261, USA.
Abstract:
Both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated neurotoxicity and the induction of death-regulatory genes have been implicated in the pathophysiology of delayed ischemic neuronal injury. To assess the role of the antiapoptotic gene Bcl-2 in the modulation of AMPA toxicity, we exposed neuron-enriched cultures from rat cerebral cortex to AMPA, in the absence or presence of an antisense oligodeoxynucleotide (ODN) directed against Bcl-2. AMPA produced concentration-dependent toxicity detected by a decrease in fluorescence of the redox indicator Alamar blue and by an increase in lactic acid dehydrogenase release. This effect was accompanied by the induction of Bcl-2 protein expression, with maximal induction at 100 microM AMPA. A phosphorothioate antisense ODN against Bcl-2 reduced the AMPA-stimulated induction of Bcl-2 protein levels, detected by western blotting, by about 70%. In the presence of the antisense ODN, but not sense or scrambled ODNs, the toxicity of 100 microM AMPA was increased by about 60%. These findings suggest that induction of Bcl-2 expression by AMPA may have a protective role to limit AMPA receptor-mediated neuronal damage and that modifying Bcl-2 expression could have therapeutic potential in ischemia.
Insights
Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor activation increases the antiapoptotic gene Bcl-2, offering neuroprotection. Inhibiting Bcl-2 with antisense oligodeoxynucleotides exacerbates AMPA-induced neuronal injury.
Area of Science:
- Neuroscience
- Molecular Biology
Background:
- Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated neurotoxicity contributes to ischemic brain injury.
- The role of antiapoptotic genes, such as Bcl-2, in mitigating this damage is not fully understood.
Purpose of the Study:
- To investigate the involvement of the antiapoptotic gene Bcl-2 in modulating AMPA receptor-mediated neurotoxicity.
- To determine if Bcl-2 induction plays a protective role against AMPA toxicity.
Main Methods:
- Primary neuron-enriched cultures from rat cerebral cortex were exposed to AMPA.
- Antisense oligodeoxynucleotides (ODNs) targeting Bcl-2 were used to inhibit its expression.
- Neuronal toxicity was assessed using Alamar blue fluorescence and lactate dehydrogenase release.
- Bcl-2 protein levels were measured by Western blotting.
Main Results:
- AMPA induced concentration-dependent neurotoxicity.
- AMPA exposure led to increased Bcl-2 protein expression.
- Antisense ODN against Bcl-2 significantly reduced AMPA-induced Bcl-2 levels.
- Inhibition of Bcl-2 with antisense ODN potentiated AMPA-induced neuronal toxicity.
Conclusions:
- AMPA receptor activation induces Bcl-2 expression, suggesting a protective mechanism against AMPA-mediated neuronal damage.
- Modulating Bcl-2 expression may offer a therapeutic strategy for ischemic neuronal injury.
