Glutathione depletion in nigrostriatal slice cultures: GABA loss, dopamine resistance and protection by the tetrahydrobiopterin precursor sepiapterin

Abstract

Dopaminergic neurons in culture are preferentially resistant to the toxicity of glutathione (GSH) depletion. This effect may be due to high intrinsic levels of tetrahydrobiopterin (BH 4). Here we studied the effects of manipulating GSH and/or BH 4 levels on selective neurotoxicity in organotypic nigrostriatal slice cultures. Following treatments with l-buthionine sulfoximine (BSO, 10–100 μM, 2 days exposure, 2 days recovery), either alone or in combination with the BH 4 precursor l-sepiapterin (SEP, 20 μM), or the BH 4 synthesis inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP, 5 mM), toxic effects were assessed by HPLC analysis of medium and tissues, cellular propidium iodide (PI) uptake, lactate dehydrogenase (LDH) efflux, as well as stereological counting of tyrosine-hydroxylase (TH) positive cells. Thirty micromolar BSO produced 91% GSH and 81% GABA depletion and general cell death, but no significant effect on medium homovanillic acid (HVA) or tissue dopamine (DA) levels. SEP prevented or delayed GABA depletion, PI uptake and LDH efflux by BSO, whereas DAHP in combination with BSO caused (almost) complete loss of medium HVA, tissue DA and TH positive cells. We suggest that under pathological conditions with reduced GSH, impaired synthesis of BH 4 may accelerate nigral cell loss, whereas increasing intracellular BH 4 may provide protection to both DA and GABA neurons.