Neuronal apoptosis induced by pharmacological concentrations of 3-hydroxykynurenine: Characterization and protection by dantrolene and Bcl-2 overexpression

We have studied neurotoxicity induced by pharmacological concentrations of 3-hydroxykynure-nine (3-HK), an endogenous toxin implicated in certain neur odegenerative 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 micro molar concentrations and had features characteristic of apoptosis, includin g chromatin condensation and internucleosomal DNA cleavage. In cerebellar g ranule cells, the 3-HK neurotoxicity was unaffected by xanthine oxidase inh ibitors but markedly potentiated by superoxide dismutase and its heme-like mimetic, MnTBAP [manganese(III) tetrakis(benzoic acid)porphyrin chloride]. Catalase blocked 3-HK neurotoxicity in the absence and presence of superoxi de 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 ce lls, inhibitors of the neutral amino acid transporter that mediates the upt ake of 3-HK failed to block 3-HK toxicity. However, their toxicity was slig htly potentiated by the iron chelator, deferoxamine. Taken together, our re sults 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-i nduced death of PC12 and GT1-7 cells was protected by dantrolene, an inhibi tor of calcium release from the endoplasmic reticulum. The protection by da ntrolene was associated with a marked increase in the protein level of Bcl- 2, a prominent antiapoptotic gene product. Moreover, overexpression of Bet- ii in GT1-7 cells elicited by gene transfection suppressed 3-HK toxicity. T hus, dantrolene may elicit its neuroprotective effects by mechanisms involv ing up-regulation of the level and function of Bcl-2 protein.