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Astrocytes release small molecules to protect neurons from oxidative stress Wang, Xue-Feng


The aim of the present experiments is to explore the mechanisms by which astrocytes may protect neurons from oxidative stress with a focus on small molecules. A non-contact astrocyte-neuron co-culturing method was first developed. It was observed that neuronal survival was promoted substantially when neurons were cocultured with a confluent astrocyte feeder layer. The mechanisms by which a stable cysteine level is maintained by astrocytes have been explored in recent years. However, it is still unclear how cysteine is released. In my experiments, cysteine, glutathione, and related compounds in astrocyte conditioned medium were analyzed with HPLC methods. My data suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione. Neuron conditioned medium, CSF, and plasma of the carotid artery and internal jugular vein were also analyzed. The results indicate that cystine, rather than cysteine, is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Though cysteine plays a critical role in regulating intracellular levels of glutathione, its cytotoxicity has also been noted, particularly in neurons. In the experiments of this thesis, I found that copper substantially accelerates the autoxidation rate of cysteine even at submicromolar levels, whereas iron and other transition metal ions, including manganese, chromium, and zinc, are less efficient. In tissue culture tests, it was found that cysteine toxicity depends highly on its autoxidation rate and on the total amount of cysteine being oxidized, suggesting that the toxicity can be attributed to the free radicals produced from cysteine autoxidation, but not to cysteine itself. The in vivo mechanisms that protect against cysteine toxicity were further explored. Catalase and pyruvate were each found to inhibit the production of hydroxyl radicals generated by cysteine autoxidation. In tissue culture, they both protected primary neurons against cysteine toxicity catalyzed by copper. Pyruvate, but not catalase or glutathione peroxidase, was detected in astrocyte conditioned medium and CSF. Herein, a concerted mechanism of neuroprotection by astrocytes is outlined.

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