Involvement of a Caspase‐3‐Like Cysteine Protease in 1‐Methyl‐4‐Phenylpyridinium‐Mediated Apoptosis of Cultured Cerebellar Granule Neurons

Abstract

Exposure of various neuronal cells or cell lines to high concentrations of 1‐methyl‐4‐phenylpyridinium (MPP⁺), the active metabolite of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), results in cell death. Recently, it has been reported that low concentrations of MPP⁺ induce apoptosis in susceptible neurons. We have further characterized MPP⁺‐mediated toxicity of cultured cerebellar granule neurons (CGNs) and found that exposure of CGNs to relatively low concentrations of MPP⁺ results in apoptosis, whereas higher concentrations result in necrosis. Cotreatment of CGNs with MPP⁺ and the tetrapeptide inhibitor of caspase‐3‐like proteases, acetyl‐DEVD‐CHO, markedly attenuates apoptotic but not necrotic death of these neurons. The more specific inhibitor of caspase‐1‐like proteases, acetyl‐YVAD‐CHO, however, was ineffective against MPP⁺ neurotoxicity. Moreover, cytoplasmic extracts prepared from MPP⁺‐treated CGNs contain markedly increased protease activity that cleaves the caspase‐3 substrate acetyl‐DEVD‐p‐nitroaniline. Finally, the cytoplasmic concentration of the apoptogenic protein cytochrome c was increased in a time‐dependent fashion in MPP⁺‐treated CGNs before the onset of apoptosis. Our data confirm that the neurotoxicity of MPP⁺ is due to both necrosis and apoptosis and suggest that the latter is mediated by activation of a caspase‐3‐like protease.