Abstract:Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione ϩ cystine^cysteine ϩ cysteineglutathione disulfide. Conditioned medium from neuronenriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS. Key Words: AstrocytesNeurons-Glutathione -Conditioned medium-Oxidative stress-Apoptosis. J. Neurochem. 74, 1434Neurochem. 74, -1442Neurochem. 74, (2000.Glutathione (GSH) is the major cellular antioxidant and as such plays an important neuroprotective role. Cellular GSH levels are closely correlated with cell survival under adverse conditions (Meister and Anderson, 1983;Ratan et al., 1994;Drukarch et al., 1997). GSH is synthesized from glutamate, cysteine (CSH), and glycine. CSH is the rate-limiting precursor of GSH synthesis (Beutler, 1989). In vitro experiments have shown that the extracellular abundance of thiol-containing compounds substantially influences intracellular GSH levels (Meister, 1989). Adding CSH to a neuronal culture that has been temporarily deprived of amino acids can increase GSH content, whereas cystine (CSSC), the oxidized form of CSH, cannot (Kranich et al., 1996). However, CSH is very unstable owing to autoxidation under aerobic conditions.The mechanisms by which a stable CSH level is maintained by astrocytes have been explored in recent years. Astrocytes have been shown to have profound neurosupportive effects in neuronal culture experiments (Banker, 1980;McCaffery et al., 1984;Vernadakis, 1988;Yuzaki et al., 1993;Wang and Cynader, 1999). Release of thiols by astrocytes has been reported and considered to play an important role to increase neuronal GSH synthesis (Yudkoff et al., 1990;Sagara et al., 1993Sagara et al., , 1996Dringen et ...

We have found previously that astrocytes can provide cysteine to neurons. However, cysteine has been reported to be neurotoxic although it plays a pivotal role in regulating intracellular levels of glutathione, the major cellular antioxidant. Here, we show that cysteine toxicity is a result of hydroxyl radicals generated during cysteine autoxidation. Transition metal ions are candidates to catalyze this process. 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. The autoxidation rate of cysteine in rat CSF is equal to that observed in the presence of ϳ0.2 M copper. In tissue culture tests, we 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.We have also explored the in vivo mechanisms that protect against cysteine toxicity. 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. This protection is attributed to their ability to react with hydrogen peroxide, preventing the formation of hydroxyl radicals. Pyruvate, but not catalase or glutathione peroxidase, was detected in astrocyte-conditioned medium and CSF. Our data therefore suggest that astrocytes can prevent cysteine toxicity by releasing pyruvate.

SUMMARY Glucagon-like peptide-1 (GLP-1) and its analogs act as appetite suppressants and have been proven to be clinically efficacious in reducing body weight in obese individuals. Central GLP-1 is expressed in a small population of brainstem cells located in the nucleus tractus solitarius (NTS), which project to a wide range of brain areas. However, it remains unclear how endogenous GLP-1 released in the brain contributes to appetite regulation. By using chemogenetic tools, we discovered that central GLP-1 acts on the midbrain ventral tegmental area (VTA) and suppresses high-fat food intake. We used integrated pathway tracing and synaptic physiology to further demonstrate that activation of GLP-1 receptors specifically reduces the excitatory synaptic strength of dopamine (DA) neurons within the VTA that project to the nucleus accumbens (NAc) medial shell. These data suggest that GLP-1 released from NTS neurons can reduce highly palatable food intake by suppressing mesolimbic DA signaling.

A simple fluorescence lifetime imaging system using a gated micro-channel plate (MCP) image intensifier coupled to a CCD camera has been developed. Nanosecond-level time-resolved fluorescence images of a sample under a pulsed light excitation can be detected directly. With a rapid lifetime determination method for multigate detection, fluorescence lifetime imaging can be promptly performed. In the present system, laser excitation of sample and shutter action of an image intensifier are fully synchronized by means of an optical fiber delay line. In order to compensate for fluctuations in the excitation source, a simple intensity monitor circuit was developed. Details of the instrumental system and verification measurements on two component samples are presented.