The University of Georgia College of Veterinary Medicine

Georgia Veterinary Scholar Program

2003 Scholars

Georgia Veterinary Scholar	Faculty Mentor

Tonya McNinch University of Georgia Class of 2005	Dr. Thomas Murray

Brevetoxin Exposure Produces Differential Alterations in the Phosphorylation of Stress Kinases P38 and JNK in Murine Cerebral Cortical and Cerebellar Granular Neurons (CGN’s)

The neurotoxin brevetoxin associated with red tide, PbTx-2, is produced by the marine dinoflagellate Karenia brevis. Brevetoxin produces neurotoxic changes by opening voltage-gated sodium channels, thereby depolarizing excitable cells and causing glutamate release. Glutamate acts on the post-synaptic neurons, partly through N-methyl D-aspartate channels, to cause a massive calcium influx. Mitogen-activated protein kinases (MAPK’s) are important mediators of cell signaling in response to extracellular stimuli and/or cell stress. We chose to investigate alterations in phosphorylation of two “stress kinases”, p38 and JNK, upon exposure of murine neocortical neurons and cerebellar granular neurons (CGN’s) to different brevetoxin concentrations. These two kinases, upon phosphorylation, promote cell death. We hypothesize that brevetoxin exposure will increase the phosphorylation of both kinases in cerebellar granule neurons, suggesting decreased cell viability after toxin exposure. Cerebral cortical neurons are expected to be more tolerant to the toxin, showing little to no increase in phosphorylation of these proteins.

To test this hypothesis, we harvested neocortical cells from fetal mouse pups at day 17 of gestation, and CGN’s were harvested from 8 day-old rat pups. The neurons were plated onto 12 well plates. The neurons were exposed to 100nM or 1?M concentrations of brevetoxin for varying time intervals after 10-15 days in culture. After exposure, we extracted 30?g of protein from each sample, electrophoresed on a SDS-PAGE gel, and transferred to a nitrocellulose membrane. We exposed the membranes to primary and then secondary antibodies for the two phosphorylated kinases and examined the bands to determine increases or decreases in phosphorylation as compared to controls. We found that there is a concentration-dependent increase in the phosphorylation of both kinases in CGN’s, with 1?M brevetoxin causing a greater increase in phosphorylation as compared to 100nM. Phosphorylation of JNK decreased as compared to controls at 15 minutes of exposure to 100 nM brevetoxin in cortical neurons. These results suggest that, similar to the toxicity, brevetoxin increases JNK activation in a concentration-dependent manner in CGN’s. The decrease in JNK phosphorylation in cortical neurons upon exposure to 100nM brevetoxin for 15 minutes suggests that lower doses of brevetoxin exposure may, in contrast, have a neuroprotective effect on cerebral cortical cells. We hope to validate the concentration dependence of brevetoxin toxicity and, most importantly, we hope to investigate the implied neuroprotective effect of low doses of brevetoxin in cerebrocortical neurons.