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Delayed antagonism of calpain reduces excitotoxicity in cultured neurons
J R Brorson1, C J Marcuccilli, R J Miller
1Department of Neurology, University of Chicago Ill 60637, USA.
Background And Purpose:
Glutamate receptor antagonists can produce protection against the neurotoxicity of excessive glutamate stimulation. However, antagonism of the postreceptor processes that produce cell damage may provide a longer window of opportunity for protecting neurons after the initiation of excitotoxic injury. Among various processes that have been thought to mediate the toxic effects of glutamate are activation of the Ca(2+)-dependent proteases calpain I and II and the activation of nitric oxide synthase. We tested the potential for neuroprotection by delayed application of calpain antagonists after excitotoxic treatment.
Methods:
Primary cultures of cerebellar and hippocampal neurons were exposed to the glutamate receptor agonists kainate and N-methyl-D-aspartate (NMDA) for 20-minute periods, and survival was examined by fluorescent assay after 24 hours. Enzyme antagonists were applied at various time points during this interval.
Results:
The neurotoxic effects of NMDA in cultured hippocampal neurons and of kainate in cultured cerebellar neurons have been previously shown to be Ca2+ dependent. Here we show that in both of these examples of glutamate receptor-mediated toxicity, activation of a calpainlike proteolytic activity occurred, which was blocked by the calpain inhibitor MDL-28170. This inhibitor also limited the toxicity, even when applied at times up to 1 hour after the onset of the toxic exposure. Another protease inhibitor, E-64, also blocked the proteolysis and toxicity produced by kainate in cerebellar neurons. Blocking nitric oxide synthase activity after 1 hour with the antagonist NG-nitro-L-arginine was also protective of cerebellar and hippocampal neurons, as was the combination of MDL-28170 and NG-nitro-L-arginine.
Conclusions:
The activation of calpain is among several enzymatic processes that contribute to the toxicity of glutamate receptor stimulation, and blocking these postreceptor mechanisms can be effective in protecting neurons from excitotoxicity at delayed time points.
Insights
Delayed application of calpain antagonists effectively protects neurons from glutamate excitotoxicity by targeting postreceptor processes. This approach offers a prolonged therapeutic window for neuroprotection after injury initiation.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Glutamate receptor antagonists offer neuroprotection against excitotoxicity.
- Targeting postreceptor pathways may extend the therapeutic window for neuronal protection.
- Calpain activation and nitric oxide synthase are implicated in glutamate-induced neurotoxicity.
Purpose of the Study:
- To investigate the neuroprotective potential of delayed calpain antagonist application following excitotoxic treatment.
- To determine if blocking postreceptor mechanisms offers a therapeutic window for neuronal protection.
Main Methods:
- Primary cultures of cerebellar and hippocampal neurons were exposed to kainate or N-methyl-D-aspartate (NMDA).
- Neuronal survival was assessed using fluorescent assays 24 hours post-exposure.
- Calpain and nitric oxide synthase antagonists were applied at various time points after excitotoxic insult.
Main Results:
- Glutamate receptor-mediated toxicity involved calpain-like proteolytic activity, inhibited by MDL-28170 and E-64.
- MDL-28170 provided neuroprotection even when applied up to 1 hour after toxic exposure.
- Inhibition of nitric oxide synthase with NG-nitro-L-arginine also conferred protection, as did the combination of both inhibitors.
Conclusions:
- Calpain activation is a key enzymatic process contributing to glutamate excitotoxicity.
- Blocking postreceptor mechanisms, such as calpain activation, provides effective neuroprotection at delayed time points.