Abstract
Earlier, we reported that gestational ethanol (E) can dysregulate neuron glutathione (GSH) homeostasis partially via impairing the EAAC1-mediated inward transport of Cysteine (Cys) and this can affect fetal brain development. In this study, we investigated if there is a role for the transulfuration pathway (TSP), a critical bio-synthetic point to supply Cys in E-induced dysregulation of GSH homeostasis. These studies utilized an in utero E binge model where the pregnant Sprague–Dawley (SD) rat dams received five doses of E at 3.5 g/kg by gastric intubation beginning embryonic day (ED) 17 until ED19 separated by 12 h. The postnatal day 7 (PN7) alcohol model employed an oral dosing of 4 g/kg body weight split into 2 feedings at 2 h interval and an iso-caloric and iso-volumic equivalent maltose-dextrin milk solution served as controls. The in vitro model consisted of cerebral cortical neuron cultures from embryonic day (ED) 16–17 fetus from SD rats and differentiated neurons from ED18 rat cerebral cortical neuroblasts. E concentrations were 4 mg/mL. E induced an accumulation of cystathionine in primary cortical neurons (PCNs), 2nd trimester equivalent in utero binge, and 3rd trimester equivalent PN7 model suggesting that breakdown of cystathionine, a required process for Cys supply is impaired. This was associated with a significant reduction in cystathionine γ-lyase (CSE) protein expression in PCN (p < 0.05) and in fetal cerebral cortex in utero (53%, p < 0.05) without a change in the expression of cystathionine β-synthase (CBS). Concomitantly, E decreased Cse mRNA expression in PCNs (by 32% within 6 h of exposure, p < 0.05) and in fetal brain (33%, p < 0.05). In parallel, knock down of CSE in differentiated rat cortical neuroblasts exaggerated the E-induced ROS, GSH loss with a pronounced caspase-3 activation and cell death. These studies illustrate the importance of TSP in CSE-related maintenance of GSH and the downstream events via Cys synthesis in neurons and fetal brain.
Highlights
The damaging effects of maternal ethanol (E) intake on the developing brain are extensively documented in animals and humans
We have shown that dysregulation of GSH homeostasis along with apoptotic death of neurons occur in fetal brain and fetal neuron culture systems exposed to ethanol, our understanding of the mechanisms responsible for the decrease in GSH remain incomplete
We have shown that this E-impaired GSH homeostasis and death of neurons still occurred in the presence of E-mediated upregulation of nuclear factor E2-related factor 2 (Nrf2), a redox-responsive transcription factor that is involved in regulating the genes involved in GSH de novo biosynthetic pathway [4]
Summary
The damaging effects of maternal ethanol (E) intake on the developing brain are extensively documented in animals and humans. Clinically relevant blood E levels alter biochemical activity and molecular expression of several genes thereby compromising neurons in the developing brain and accelerating their rate of apoptotic death [4,5,6,7,8,9,10,11] This enhanced apoptosis has been connected to the high sensitivity of fetal cells, especially a subpopulation of cells with the lowest GSH content to E-induced oxidative stress (OS) [1,2,3,4,5,6]. The transcription factor, nuclear factor E2-related factor 2 (Nrf2), is a master regulator of redox signaling that activates several antioxidant targets including but not limited to the genes governing GSH homeostasis The latter include glutamate-cysteine ligase (GCL) (rate limiting enzyme in GSH synthesis), glutathione reductase (generation of reduced/active GSH), and multiple components of the gamma glutamyl cycle. Our goal in the current study has been to determine if there is a dysregulation of CSE and if so, is the impaired supply of Cysteine (Cys) due to CSE deregulation via trans-sulfuration pathway a/the control point for alcohol-induced GSH loss and cell death
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