The neuropathology of Amnesic Shellfish Poisoning, one of the shellfish poisoning syndromes in the United States, caused by the marine toxin domoic acid (DOM) is not yet fully understood. Most research into the neurotoxicity of DOM, a glutamate analog, has focused on its direct excitotoxicity to neuronal tissue. Recent studies however have shown that glial tissue, namely astrocytes and microglia appear to play a role in DOM's toxicity to the nervous system. Our working hypothesis, which we have published ((58) and Appendix # 2), is that """"""""...exposure to the marine toxin domoic acid activates neonatal brain microglia and causes subsequent release of neurotoxic matrix metalloproteinases and cytokines"""""""". Pathological activation of microglia by DOM and concomitant release of inflammatory matrix metalloproteinases and cytokines are thus hypothesized to contribute to excitotoxic neuronal death. Over the past two years we have begun to test our hypothesis. Our recently published results provide initial experimental support for our working hypothesis, because we observed release of the cytokine TNF- alpha and the matrix metalloproteinase MMP-9 after a 1-6 hour exposure of rat neonatal microglia to DOM in vitro. The proposal's specific aims describe a series of experiments designed to expand the testing of our working hypothesis by extending the length of in vitro exposure of rat neonatal microglia to DOM, as well as investigating the potential activation of additional matrix metalloproteinases and cytokines by means of cDNA array technology. Furthermore, if pilot studies determine that DOM also activates the immortalized human embryonic microglial CHME cell line in vitro to release both inflammatory cytokines and matrix metalloproteinases, we will extend our studies to this human microglia cell line. In summary, the proposal's specific aims will focus on the long-term (6-48 hours) effect of DOM on neonatal brain microglia activation and concomitant generation of 2 classes of potentially inflammatory and neurotoxic mediators, namely cytokines and matrix metalloproteinases, that the PI and his collaborators have shown are generated when rat neonatal microglia are exposed to a short-term in vitro treatment with DOM. ? Specific Aim 1: To determine the long-term (6-48 hours) effect of DOM on the generation of matrix metalloproteinases by rat neonatal microglia at both the functional and molecular level. ? Specific Aim 2: To investigate the long-term (6-48 hours) effect of DOM on the generation of TNF-alpha and additional cytokines by rat neonatal microglia at both the functional and molecular level ? Specific Aim 3: To assess the effect of DOM on the generation of cytokines and matrix metalloproteinases by immortalized human embryonic microglial CHME cell line at both the functional and molecular level. ? Completion of this research will increase our understanding of the role activated microglia cells and their mediators play in DOM-induced excitotoxic neuronal injury and thus contribute to potential novel approaches for the therapy of the neuropathology associated with Amnesic Shellfish Poisoning in humans ? ?
