Acetate Mediates Alcohol Excitotoxicity in Dopaminergic-like PC12 Cells

Abstract

Neuronal excitotoxicity is the major cause of alcohol-related brain damage, yet the underlying mechanism remains poorly understood. Using dopaminergic-like PC12 cells, we evaluated the effect of N-methyl-d-aspartate receptors (NMDAR) on acetate-induced changes in PC12 cells: cell death, cytosolic calcium, and expression levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Treatment of PC12 cells with increasing concentrations of acetate for 4 h caused a dose-dependent increase in the percentage of cells staining positive for cell death using propidium iodide (PI) exclusion and cytosolic reactive oxygen species (ROS) using cell ROX detection analyzed via flow cytometry. The EC50 value for acetate was calculated and found to be 4.40 mM for PI and 1.81 mM for ROS. Ethanol up to 100 mM had no apparent changes in the percent of cells staining positive for PI or ROS. Acetate (6 mM) treatment caused an increase in cytosolic calcium measured in real-time with Fluo-4AM, which was abolished by coapplication with the NMDAR blocker memantine (10 μM). Furthermore, cells treated with acetate (6 mM) for 4 h had increased expression levels of TNFα relative to control, which was abolished by coapplication of memantine (10 μM). Co-application of acetate (6 mM) and memantine had no apparent reduction in acetate-induced cell death. These findings suggest that acetate is capable of increasing cytosolic calcium concentrations and expression levels of the pro-inflammatory cytokine TNFα through an NMDAR-dependent mechanism. Cell death from acetate was not reduced through NMDAR blockade, suggesting alternative pathways independent of NMDAR activation for excitotoxicity.

Neuroinflammatory processes are augmented in mice overexpressing human heat-shock protein B1 following ethanol-induced brain injury

“…Primary cell cultures were treated with ethanol in order to investigate the effect of alcohol on different cell types of the brain. Ethanol can induce inflammatory processes in the brain directly by itself and also by its metabolites [ 33 , 34 ], and it can also contribute to cerebral inflammation indirectly by disrupting the integrity of the blood-brain barrier, subsequently promoting the infiltration of peripheral immune cells to the brain [ 35 ]. Considering this affiliation of ethanol treatment with inflammation, we also treated our model with pro-inflammatory cytokines in order to model the inflammatory milieu connected with the presence of ethanol.…”

Gut Microbiota and Short Chain Fatty Acids: Influence on the Autonomic Nervous System

“…The difference in effects may be attributed to the various transporters/receptors with which SCFAs interact: monocarboxylate transporter (MCTs), G protein-coupled receptors (GPCRs), or olfactory receptors [8,10]. The expression and localization of these receptors found in specific neurons and/or various regions of the body, or perhaps their concentrations may influence the receptor response and phenotypic output [8,[10][11][12]. Comprehension of the downstream effects of SCFA-receptor activation and the influence on the autonomic nervous system will lead to novel discoveries in pathophysiology and lead to new approaches in therapeutics and medicine.…”

“…When acetate activates N-methyl-D-aspartate receptors (NMDARs) in the central nucleus of the amygdala, sympathetic activity increases resulting in a rise in ABP [11]. In a dose-dependent manner acetate has been shown to induce excitotoxicity in dopaminergic PC12 cells [12] by either binding to NMDARs or entering the cell presumably though MCTs. The opening of NMDARs channels increases cytosolic Ca 2?…”

Local Metabolism of Ethanol to Acetic Acid/Acetate in the Central Nucleus of Amygdala Elicits Sympathoexcitatory Responses through Activation of NMDAR in Sprague Dawley Rats

“…The RVLM as we and others have previously reported 13,37,38 and is also similar to cytotoxic doses we observed in dopaminergic-like PC12 cells. 14 Our Emax value of 37.5 mM was utilized to elicit maximum responses to both intrinsic excitability as well as NMDAR mediated inward currents. A one-way ANOVA with Bonferroni post-hoc test revealed differences (F(4,28) = 6.41, p=0.0009) in firing frequency observed between control vs 7.5 mM, 37.5 mM acetate and control vs 75 mM acetate.…”

“…82 Fyn can also be directly activated via nitric oxide (NO) and other free radicals, 83 leading to gain of function of NMDAR. 80,81 Previous work out of our lab exploring cellular effects of acetate have revealed an acetate induced increase in cytosolic reactive oxygen species 14 which in addition to being cytotoxic may also play a role in Fyn regulation of NMDAR function. Our finding of memantine only being slightly effective at reducing acetate induced toxicity in cell culture, 14 combined with the above mentioned potential contributing mechanisms suggest multiple signaling cascades evoked by acetate.…”