Search

Parkinson disease is a specific form of neurodegeneration characterized by a loss of nigra-striatal dopaminergic neurons in the midbrain of humans. The disease is also characterized by an increase in oxidative stress and a loss of glutathione in the midbrain region. A potential link between all these factors is the oxidation of dopamine to dopaminochrome (DAC). Using the murine mesencephalic cell line MN9D, we have shown that DAC [50-250 microM] leads to cell death in a concentration-dependent manner, whereas oxidized l-dopa, dopachrome [50-250 microM], is only toxic at the highest concentration used. Furthermore, chronic exposure of MN9D cells to low concentrations of DAC [50-100 microM] is cytotoxic between 48 and 96 h. DAC also increases superoxide production within MN9D cells as indicated by dihydroethidium fluorescence, that can be prevented by co-administration with the antioxidant, N-acetylcysteine [5 mM]. Moreover, the cytotoxicity induced by DAC can also be prevented by administration of N-acetylcysteine [1-5mM]. Finally, depletion of reduced glutathione in MN9D cells by buthionine sulfoximine [50-100 microM] administration significantly enhances the cytotoxic effect of low concentrations of DAC [50-100 microM] and DAC [175 microM] itself reduces the proportion of oxidized glutathione in total glutathione within 30 min of administration in MN9D cells. Overall, we have shown that DAC causes MN9D cell death in an oxidatively dependent manner that appears closely linked with a rapid loss of reduced glutathione. These findings have implications for understanding the pathogenesis of neurodegenerative pathways in Parkinson disease.

The major cholesterol metabolite in brain, 24(S)-hydroxycholesterol (24S-HC), serves as a vehicle for cholesterol removal. Its effects on neuronal function, however, have only recently begun to be investigated. Here, we review that nascent work. Our own studies have demonstrated that 24S-HC has potent positive modulatory effects on N-methyl-d-aspartate (NMDA) receptor (NMDAR) function. This could have implications not only for brain plasticity but also for pathological NMDAR overuse. Other work has demonstrated effects of 24S-HC on neuronal survival and as a possible biomarker of neurodegenerative disease. Depending on circumstances, both upregulation/mimicry of 24S-HC signaling and down-regulation/antagonism may have therapeutic potential. We are interested in the possibility that synthetic analogues of 24S-HC with positive effects at NMDARs may hold neurotherapeutic promise, given the role of NMDA receptor hypofunction in certain neuropsychiatric disorders.

Oxysterols have emerged as important biomarkers in disease and as signaling molecules. We recently showed that the oxysterol 24(S)-hydroxycholesterol, the major brain cholesterol metabolite, potently and selectively enhances NMDA receptor function at a site distinct from other modulators. Here we further characterize the pharmacological mechanisms of 24(S)-hydroxycholesterol and its synthetic analogue SGE201. We describe an oxysterol antagonist of this positive allosteric modulation, 25-hydroxycholesterol. We found that 24(S)-hydroxycholesterol and SGE201 primarily increased the efficacy of NMDAR agonists but did not directly gate the channel or increase functional receptor number. Rather than binding to a direct aqueous-accessible site, oxysterols may partition into the plasma membrane to access the NMDAR, likely explaining slow onset and offset kinetics of modulation. Interestingly, oxysterols were ineffective when applied to the cytosolic face of inside-out membrane patches or through a whole-cell pipette solution, suggesting a non-intracellular site. We also found that another natural oxysterol, 25-hydroxycholesterol, although exhibiting slight potentiation on its own, non-competitively and enantioselectively antagonized the effects of 24(S)-hydroxycholesterol analogues. In summary, we suggest two novel allosteric sites on NMDARs that separately modulate channel gating, but together oppose each other.