Activation of Calpain I Converts Excitotoxic Neuron Death into a Caspase-independent Cell Death

doi:10.1074/jbc.275.22.17064

Activation of Calpain I Converts Excitotoxic Neuron Death into a Caspase-independent Cell Death*

From the ^‡Interdisciplinary Center for Clinical Research (IZKF), Research Group “Apoptosis and Cell Death,” the ^‖Department of Pharmacology and Toxicology, Westphalian Wilhelms University, D-48149 Münster, Germany, ^§GRECC, Sepulvda Veterans Affairs Medical Center, UCLA, Sepulvda, California 91343, and ^¶RIKEN Brain Science Institute, Saitama 351-0198, Japan

Abstract

Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-d-aspartate (300 μm, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nm) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the N-methyl-d-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca²⁺-activated neutral protease calpain I was readily detectable after the exposure toN-methyl-d-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochromec to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure toN-methyl-d-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.

Footnotes

↵* This work was supported by Grant BMBF 01 KS 9604/0 from IZKF Universität Münster (to J. H. M. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵** To whom correspondence should be addressed: Interdisciplinary Center for Clinical Research (IZKF), Research Group “Apoptosis and Cell Death,” Faculty of Medicine, Westphalian Wilhelms University, Röntgenstrasse 21, D-48149 Münster, Germany. Tel.: 49-251-83-52251; Fax: 49-251-83-52250; E-mail: prehn@uni-muenster.de.
↵2 S. Lankiewicz, C. M. Luetjens, N. T. Bui, A. J. Krohn, M. Poppe, G. M. Cole, T. C. Saido, and J. H. M. Prehn, unpublished observation.
Abbreviations:

NMDA

N-methyl-d-aspartate

Ac-DEVD-AMC

acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin

CI-1

calpain inhibitor I

Me₂SO

dimethyl sulfoxide

HBS

Hepes-buffered saline

Z-IETD-AFC

benzyloxylcarbonyl-Ile-Glu-Thr-Asp-7-amido-4-(trifluoromethyl)coumarin

R-123

rhodamine-123

STS

staurosporine

Z-VDVAD-AFC

benzyloxylcarbonyl-Val-Asp-Val-Ala-Asp-7-amido-4-(trifluoromethyl)coumarin

PAGE

polyacrylamide gel electrophoresis

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

PIPES

1,4-piperazinediethanesulfonic acid
- Received December 23, 1999.
- Revision received February 28, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.