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The Journal of Neuroscience, January 1, 2000, 20(1):179-186
Ribozyme-Mediated Inhibition of Caspase-3 Protects Cerebellar Granule Cells from Apoptosis Induced by Serum-Potassium Deprivation
Basil A. Eldadah, Renée F. Ren, and Alan I. Faden Georgetown Institute for Cognitive and Computation Sciences, Departments of Neuroscience, Neurology, and Pharmacology, Interdisciplinary Program in Neuroscience, Georgetown University Medical Center, Washington, DC 20007
Apoptosis is an important mechanism of physiological and pathological cell death. It is regulated by several gene products, including caspases and the bcl-2-like proteins, whose roles have been demonstrated in numerous systems. One of these is a model of cerebellar granule cells (CGCs) in which apoptosis is induced by acute removal of serum and depolarizing concentrations of potassium. Previous work by several authors showed that benzyloxycarbonyl-DEVD-fluoromethylketone, a somewhat selective caspase inhibitor, significantly protected CGCs from apoptosis; however, because this molecule targets multiple caspases, it is not known whether a single caspase is primarily responsible for effecting cell death in this model. We attempted to answer this question by cotransfecting CGCs with green fluorescent protein reporter and a hammerhead ribozyme directed against caspase-3 mRNA. Maximal protection by this ribozyme was observed after 24 hr of deprivation, at which time apoptosis was 18 ± 0.7% compared with 32 ± 2% in control cells. Significant protection was also observed with human inhibitor of apoptosis (IAP)-like protein-X-linked IAP, a specific inhibitor of caspase-3, -7, and -9, and with p35, a general caspase inhibitor. Overexpression of bcl-2 produced almost complete protection from apoptosis after 24 hr of serum-K+ deprivation (5 ± 2 vs 44 ± 2% in control cells). These results confirm that caspases play an important role in CGC apoptosis and indicate that caspase-3 itself is a significant mediator of this process.
Key words: apoptosis; caspase; cerebellar granule cells; ribozyme; IAP; transfection
Copyright © 2000 Society for Neuroscience 0270-6474/0/201179-08$05.00/0
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