Abstract
Central among the fetotoxic responses to in utero ethanol (E) exposure is redox-shift related glutathione (GSH) loss and apoptosis. Previously, we reported that despite an E-generated Nrf2 upregulation, fetal neurons still succumb. In this study, we investigate if the compromised GSH results from an impaired inward transport of cysteine (Cys), a precursor of GSH in association with dysregulated excitatory amino acid carrier1 (EAAC1), a cysteine transporter. In utero binge model involves administration of isocaloric dextrose or 20% E (3.5 g/kg)/ by gavage at 12 h intervals to pregnant Sprague Dawley (SD) rats, starting gestation day (gd) 17 with a final dose on gd19, 2 h prior to sacrifice. Primary cerebral cortical neurons (PCNs) from embryonic day 16–17 fetal SD rats were the in vitro model. E reduced both PCN and cerebral cortical GSH and Cys up to 50% and the abridged GSH could be blocked by administration of N-acetylcysteine. E reduced EAAC1 protein expression in utero and in PCNs (p < 0.05). This was accompanied by a 60–70% decrease in neuron surface expression of EAAC1 along with significant reductions of EAAC1/Slc1a1 mRNA (p < 0.05). In PCNs, EAAC1 knockdown significantly decreased GSH but not oxidized glutathione (GSSG) illustrating that while not the sole provider of Cys, EAAC1 plays an important role in neuron GSH homeostasis. These studies strongly support the concept that in both E exposed intact fetal brain and cultured PCNs a mechanism underlying E impairment of GSH homeostasis is reduction of import of external Cys which is mediated by perturbations of EAAC1 expression/function.
Highlights
Fetotoxic effects of maternal ethanol (E) intake are well documented in animals and humans
We have shown that this astrocyte-mediated pathway maintains cortical neuron GSH, protecting neurons from E-mediated apoptotic death [20,66], this is possible only after the emergence of astrocytes which occurs at or around a late second to early third trimester period of gestation
While such Slc1a1 regulating mechanism remains to be elucidated, our results suggest a deficiency of excitatory amino acid carrier1 (EAAC1) transport-dependent route of neuronal Cys uptake may be one of the pivotal reasons for the E-induced GSH loss-associated toxicity
Summary
Fetotoxic effects of maternal ethanol (E) intake are well documented in animals and humans. Since, during the early-E sensitive stages of brain development, neurons maintain GSH in the absence of mature astrocytes (AST) or oligodendroglia and given the causal association of OS with GSH loss in alcohol-induced neurodevelopmental toxicity, it is critical to determine the origin(s) of E-related GSH loss. Based on our Slc1a1 mRNA expression data, it appears that this alteration in Cys could occur due to the post-transcriptional targeting of Slc1a1/EAAC1 by miR 96-5p and miR 26a-5p besides transcriptional regulation that has been shown to dysregulate redox homeostasis and promote neurotoxicity [49,50,67,68] While such Slc1a1 regulating mechanism remains to be elucidated, our results suggest a deficiency of EAAC1 transport-dependent route of neuronal Cys uptake may be one of the pivotal reasons for the E-induced GSH loss-associated toxicity. The genetic loss- and gain-of-function studies are underway to delineate the role of EAAC1 in alcohol developmental neurotoxicity
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