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Parkinson's disease (PD) is associated with loss of total glutathione (GSH) which may contribute to progressive cell death. Peripheral GSH administration has been used clinically with reported benefits. Despite this, there is little specific information to characterize its cellular uptake or clearance, brain elevation with peripheral delivery or neuroprotective efficacy in PD models. The current study was carried out to provide this information using in vitro and in vivo approaches. In rat mesencephalic culture, the monoethyl-ester of GSH (GEE), but not GSH (1-10mM, 24hr) produced a dosedependent elevation in GSH. The half-life for clearance was 10.14hr and was not different in cells depleted of GSH prior to loading. Elevation of GSH with GEE protected neurons from oxidative stress with H 2 O 2 or metabolic stress with the complex I and II inhibitors, MPP + and malonate, respectively. To determine if peripheral administration of GEE could elevate brain GSH levels, rats were administered 0.1-50mg/kg/d GEE via osmotic minipump either subcutaneously (sc) or via a cannula placed into the left cerebral ventricle (icv) for 28d. Only central delivery of GEE resulted in significant elevations of brain GSH. Elevation of brain GSH by icv infusion of GEE was examined for its neuroprotective effects against chronic central delivery of MPP + . Infusion of 0.142mg/kg/d MPP + for 28d caused a selective ipsilateral loss of striatal dopamine. Co-infusion of MPP + with 10mg/kg/d GEE significantly protected against striatal dopamine loss. These findings show that the ethyl ester of GSH but not GSH per se can elevate intracellular GSH, that brain elevation of GSH requires central delivery of the ethyl-ester and that this elevation provides neuroprotection against oxidative stress or chronic mitochondrial impairment. Keywords oxidative stress; glutathione ethyl ester; neurodegeneration; neuroprotectionThe underlying etiology of sporadic Parkinson's disease (PD) remains elusive, although much evidence implicates oxidative stress in the process. The substantia nigra (SN) from autopsied brain material has been shown to contain increased levels of protein carbonyls (Alam et al. 1997), malondialdehyde or 4-hydroxynonenol (Dexter et al. 1994a;Yoritaka et al. 1996) and 8-hydroxy-2-deoxyguanosine (Sanchez-Ramos et al. 1994), indicative of oxidative damage to proteins, lipids and DNA, respectively. One of the earliest biochemical derangements observed in the SN of patients with PD is loss of total glutathione levels (Perry & Yong 1986; Corresponding Author: Gail D. Zeevalk, zeevalgd@umdnj.edu, Department of Neurology, UMDNJ-Robert Wood Johnson Medical School, Building UBHC, Rm 405D, 675, Hoes Lane, Piscataway, N.J. 08854, Phone: 732 235-3494, Fax: 732 235-5295 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting p...

Acute excitotoxicity in embryonic chick retina and the ability of Cl- channel blockers to prevent toxicity were evaluated by measurement of endogenous amino acid release and histology. Treatment of retina with kainate, quisqualate, or N-methyl-D-aspartate resulted in a large dose-dependent release of gamma-aminobutyric acid and taurine, moderate release of glutamine and alanine, and no measurable release of glutamate or aspartate. Concentrations inducing maximal gamma-aminobutyric acid release were 50 microM quisquaalate, 100 microM kainate, and 100 microM N-methyl-D-aspartate. Treatment with 1 mM glutamate resulted in significant gamma-aminobutyric acid release, as well as an elevation in medium aspartate levels. Typical excitotoxic retinal lesions were produced by the agonists and, at the lower concentrations tested, revealed a regional sensitivity. There was a positive correlation between the amount of gamma-aminobutyric acid release and the extent of tissue swelling, suggesting that release may be secondary to toxic cellular events. Omission of Cl- completely blocked cytotoxic effects due to kainate or glutamate. Likewise, addition of the Cl-/bicarbonate anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate at 600 microM protected retina from cytotoxic damage from all excitotoxic analogs and restored amino acid levels to baseline values. Furosemide, which blocks Na+/K+/2Cl- cotransport, was only minimally effective in reducing amino acid release induced by the agonists. Consistent with the latter, histological examination showed the continued presence of the lesion but with general reduction of cellular edema. These results indicate that although influx of Cl- is a central component of the acute excitotoxic phenomenon, mechanisms other than passive Cl- flux may be involved.

Reproductive success depends on a robust and appropriately timed preovulatory LH surge. The LH surge, in turn, requires ovarian steroid modulation of GnRH neuron activation by the neuropeptide kisspeptin and glutamate and gamma-aminobutyric acid (GABA) neurotransmission in the medial preoptic area (mPOA). Middle-aged females exhibit reduced excitation of GnRH neurons and attenuated LH surges under estrogen-positive feedback conditions, in part, due to increased GABA and decreased glutamate neurotransmission in the mPOA. This study tested the hypothesis that altered kisspeptin regulation by ovarian steroids plays a role in age-related LH surge dysfunction. We demonstrate that middle-aged rats exhibiting delayed and attenuated LH surges have reduced levels of Kiss1 mRNA in the anterior hypothalamus under estrogen-positive feedback conditions. Kisspeptin application directly into the mPOA rescues total LH release and the LH surge amplitude in middle-aged rats and increases glutamate and decreases GABA release to levels seen in the mPOA of young females. Moreover, the N-methyl-D-aspartate receptor antagonist MK801 blocks kisspeptin reinstatement of the LH surge. These observations suggest that age-related LH surge dysfunction results, in part, from reduced kisspeptin drive under estrogen-positive feedback conditions and that kisspeptin regulates GnRH/LH release, in part, through modulation of mPOA glutamate and GABA release.

In this study, the importance of the Mg2+ blockade of the N-methyl-D-aspartate (NMDA) receptor during metabolic stress was examined in embryonic day 13 chick retina. Retina exposed to mild conditions of metabolic stress (i.e., blockade of glycolysis with 1 mM iodoacetate for 30 min) underwent acute histological somal and neuritic swelling and an increase in gamma-aminobutyric acid (GABA) release into the medium. These acute signs of metabolic stress were eliminated by NMDA antagonists present during pharmacological blockade of glycolysis, occurred in the absence of a net increase in extracellular glutamate or aspartate, and were not affected by the presence or absence of Ca2+ in the incubation medium. One possible explanation for the activation of NMDA receptors in the absence of an increase in extracellular ligand is that NMDA sensitivity during metabolic stress may be governed at the receptor level. Depolarization of membrane potential during metabolic stress may result in the loss of the Mg2+ blockade from the NMDA receptor channel, resulting in an increased potency for glutamate. To test this, the dose-response characteristics for NMDA, glutamate, and kainate in the presence or absence of extracellular Mg2+ and the effects of Mg2+ on metabolic inhibition were examined. The potency for NMDA- or glutamate-mediated acute toxicity was enhanced two- to fivefold in the absence of Mg2+. Omission of Mg2+ greatly decreased the minimal concentration of agonist needed to produce acute excitotoxicity; 25 versus 5 microM for NMDA and 300 versus 10 microM for glutamate in 1.2 or zero Mg2+, respectively. Elevating external Mg2+ to 20 mM completely protected against NMDA-mediated acute toxic effects. In contrast, varying external Mg2+ had no effect on kainate-induced toxicity. Acute toxicity caused by inhibition of metabolism was not potentiated in the absence of Mg2+ but was attenuated by elevating extracellular Mg2+. The protective effect of Mg2+ during metabolic inhibition was not additive with NMDA antagonists, suggesting that the action of Mg2+ was at the level of the NMDA receptor. These findings are consistent with the hypothesis that the Mg2+ block is lifted during metabolic inhibition and may be the primary event resulting in NMDA receptor activation.

Previous in vitro studies in our laboratory have shown that mancozeb (MZ) and maneb (MB), both widely used EBDC fungicides, are equipotent neurotoxicants that produce cell loss in mesencephalic dopaminergic and GABAergic cells after an acute 24 h exposure. Mitochondrial uncoupling and inhibition were associated with fungicide exposure. Inhibition of mitochondrial respiration is known to increase free radical production. Here the mechanism(s) of neuronal damage associated with MZ exposure was further explored by determining the role that reactive oxygen species (ROS) played in toxicity. Damage to mesencephalic dopamine and GABA cell populations were significantly attenuated when carried out in the presence of ascorbate or SOD indicative of a free radical mediated contribution to toxicity. ROS generation monitored by H 2 O 2 production using Amplex Red increased in a dose-dependent manner in response to MZ. Inhibition of intracellular catalase with aminotriazole had little effect on H 2 O 2 generation, whereas exogenously added catalase significantly reduced H 2 O 2 production demonstrating a large extracellular contribution to ROS generation. Conversely, cells preloaded with the ROS indicator dye DCF showed significant MZ-induced ROS production, demonstrating an increase in intracellular ROS. Both the organic backbone of MZ as well as its associated Mn ion, but not Zn ion were responsible and required for H 2 O 2 generation. The functionally diverse NADPH oxidase inhibitors, diphenylene iodonium chloride, apocynin, and 4-(2-aminoethyl)benzene-sulfonyl fluoride hydrochloride significantly attenuated H 2 O 2 production by MZ. In growth medium lacking cells, MZ produced little H 2 O 2 , but enhanced H 2 O 2 generation when added with xanthine plus xanthine oxidase whereas, in cultured cells, allopurinol partially attenuated H 2 O 2 production by MZ. Minocycline, an inhibitor of microglial activation, modestly reduced H 2 O 2 formation in mesencephalic cells. In contrast, neuronal enriched cultures or cultures treated with MAC-1-SAP to kill microglia, did not show an attenuation of ROS production. These findings demonstrate that Mn-containing EBDC fungicides such as MZ and MB can produce robust ROS generation that likely occurs via redox cycling with extracellular and intracellular oxidases. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeurotoxicology. Author manuscript; available in PMC 2008 November 1. The findings further show that microglia may contribute to but are not required for ROS production by MZ.