Phospholipase A2, Oxidative Stress, and Neurodegeneration in Binge Ethanol-Treated Organotypic Slice Cultures of Developing Rat Brain
Article first published online: 1 AUG 2013
Copyright © 2013 by the Research Society on Alcoholism
Alcoholism: Clinical and Experimental Research
Volume 38, Issue 1, pages 161–169, January 2014
How to Cite
Moon, K.-H., Tajuddin, N., Brown, J., Neafsey, E. J., Kim, H.-Y. and Collins, M. A. (2014), Phospholipase A2, Oxidative Stress, and Neurodegeneration in Binge Ethanol-Treated Organotypic Slice Cultures of Developing Rat Brain. Alcoholism: Clinical and Experimental Research, 38: 161–169. doi: 10.1111/acer.12221
- Issue published online: 21 JAN 2014
- Article first published online: 1 AUG 2013
- Manuscript Accepted: 22 MAY 2013
- Manuscript Received: 13 FEB 2013
- National Institutes of Health National Institute of Alcohol Abuse and Alcoholism. Grant Numbers: U01 AA018279, T32 AA13527, R21 AA011543
- Loyola University Potts Foundation
- Arachidonic Acid;
- Monoacylglycerol Lipase
Brain neurodamage from chronic binge ethanol (EtOH) exposure is linked to neuroinflammation and associated oxidative stress. Using rat organotypic hippocampal–entorhinal cortical (HEC) slice cultures of developing brain age, we reported that binge EtOH promotes release of a neuroinflammatory instigator, arachidonic acid (AA), concomitant with neurodegeneration, and that mepacrine, a global inhibitor of phospholipase A2 (PLA2) enzymes mobilizing AA from phospholipids, is neuroprotective. Here, we sought with binge EtOH-treated HEC cultures to establish that PLA2 activity is responsible in part for significant oxidative stress and to ascertain the PLA2 families responsible for AA release and neurodegeneration.
HEC slices, prepared from 1-week-old rats and cultured 2 to 2.5 weeks, were exposed to 100 mM EtOH over 6 successive days, with 4 daytime “withdrawals” (no EtOH). Brain 3-nitrotyrosinated (3-NT)- and 4-hydroxy-2-nonenal (4-HNE)-adducted proteins, oxidative stress footprints, were immunoassayed on days 3 through 6, and mepacrine's effect was determined on day 6. The effects of specific PLA2 inhibitors on neurodegeneration (propidium iodide staining) and AA release (ELISA levels in media) in the cultures were then determined. Also, the effect of JZL184, an inhibitor of monoacylglycerol lipase (MAGL) which is reported to mobilize AA from endocannabinoids during neuroinflammatory insults, was examined.
3-NT- and 4-HNE-adducted proteins were significantly increased by the binge EtOH exposure, consistent with oxidative stress, and mepacrine prevented the increases. The PLA2 inhibitor results implicated secretory PLA2 (group II sPLA2) and to some extent Ca2+-independent cytosolic PLA2 (group VI iPLA2) in binge EtOH-induced neurotoxicity and in AA release, but surprisingly, Ca2+-dependent cytosolic PLA2 (group IV cPLA2) did not appear important. Furthermore, unlike PLA2 inhibition, MAGL inhibition failed to prevent the neurodegeneration.
In these developing HEC slice cultures, pro-oxidative signaling via sPLA2 and iPLA2, but not necessarily cPLA2 or MAGL, is involved in EtOH neurotoxicity. This study provides further insights into neuroinflammatory phospholipase signaling and oxidative stress underlying binge EtOH-induced neurodegeneration in developing (adolescent age) brain in vitro.