Neuronal Apoptosis Induced by Pharmacological Concentrations of 3‐Hydroxykynurenine

Abstract

: We have studied neurotoxicity induced by pharmacological concentrations of 3‐hydroxykynurenine (3‐HK), an endogenous toxin implicated in certain neurodegenerative diseases, in cerebellar granule cells, PC12 pheochromocytoma cells, and GT1‐7 hypothalamic neurosecretory cells. In all three cell types, the toxicity was induced in a dose‐dependent manner by 3‐HK at high micromolar concentrations and had features characteristic of apoptosis, including chromatin condensation and internucleosomal DNA cleavage. In cerebellar granule cells, the 3‐HK neurotoxicity was unaffected by xanthine oxidase inhibitors but markedly potentiated by superoxide dismutase and its hemelike mimetic, MnTBAP [manganese(III) tetrakis(benzoic acid)porphyrin chloride]. Catalase blocked 3‐HK neurotoxicity in the absence and presence of superoxide dismutase or MnTBAP. The formation of H₂O₂ was demonstrated in PC12 and GT1‐7 cells treated with 3‐HK, by measuring the increase in the fluorescent product, 2′,7′‐dichlorofluorescein. In both PC12 and cerebellar granule cells, inhibitors of the neutral amino acid transporter that mediates the uptake of 3‐HK failed to block 3‐HK toxicity. However, their toxicity was slightly potentiated by the iron chelator, deferoxamine. Taken together, our results suggest that neurotoxicity induced by pharmacological concentrations of 3‐HK in these cell types is mediated primarily by H₂O₂, which is formed most likely by auto‐oxidation of 3‐HK in extracellular compartments. 3‐HK‐induced death of PC12 and GT1‐7 cells was protected by dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. The protection by dantrolene was associated with a marked increase in the protein level of Bcl‐2, a prominent antiapoptotic gene product. Moreover, overexpression of Bcl‐2 in GT1‐7 cells elicited by gene transfection suppressed 3‐HK toxicity. Thus, dantrolene may elicit its neuroprotective effects by mechanisms involving up‐regulation of the level and function of Bcl‐2 protein.