Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia.
BACKGROUND
The excitatory amino acid domoic acid, a glutamate and kainic acid analog, is the causative agent of amnesic shellfish poisoning in humans. No studies to our knowledge have investigated the potential contribution to short-term neurotoxicity of the brain microglia, a cell type that constitutes circa 10% of the total glial population in the brain. We tested the hypothesis that a short-term in vitro exposure to domoic acid, might lead to the activation of rat neonatal microglia and the concomitant release of the putative neurotoxic mediators tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinases-2 and-9 (MMP-2 and -9) and superoxide anion (O2-).


RESULTS
In vitro, domoic acid [10 microM-1 mM] was significantly neurotoxic to primary cerebellar granule neurons. Although neonatal rat microglia expressed ionotropic glutamate GluR4 receptors, exposure during 6 hours to domoic acid [10 microM-1 mM] had no significant effect on viability. By four hours, LPS (10 ng/mL) stimulated an increase in TNF-alpha mRNA and a 2,233 % increase in TNF-alpha protein In contrast, domoic acid (1 mM) induced a slight rise in TNF-alpha expression and a 53 % increase (p < 0.01) of immunoreactive TNF-alpha protein. Furthermore, though less potent than LPS, a 4-hour treatment with domoic acid (1 mM) yielded a 757% (p < 0.01) increase in MMP-9 release, but had no effect on MMP-2. Finally, while PMA (phorbol 12-myristate 13-acetate) stimulated O2- generation was elevated in 6 hour LPS-primed microglia, a similar pretreatment with domoic acid (1 mM) did not prime O2- release.


CONCLUSIONS
To our knowledge this is the first experimental evidence that domoic acid, at in vitro concentrations that are toxic to neuronal cells, can trigger a release of statistically significant amounts of TNF-alpha and MMP-9 by brain microglia. These observations are of considerable pathophysiological significance because domoic acid activates rat microglia several days after in vivo administration.
Mayer
A M
AM
Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, Illinois 60515, USA. amayer@midwestern.edu
Hall
M
M
Fay
M J
MJ
Lamar
P
P
Pearson
C
C
Prozialeck
W C
WC
Lehmann
V K
VK
Jacobson
P B
PB
Romanic
A M
AM
Uz
T
T
Manev
H
H
D000818:Animals; D039481:CD11b Antigen / Q000032:analysis; D002470:Cell Survival / Q000187:drug effects; D002531:Cerebellum / Q000187:drug effects; D004347:Drug Interactions; D005455:Fluorescent Antibody Technique; D006801:Humans; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000633:toxicity*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008387:Marine Toxins / Q000633:toxicity; D013058:Mass Spectrometry; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D017628:Microglia / Q000187:drug effects* / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000032:analysis; D017470:Receptors, Glutamate / Q000032:analysis; D013481:Superoxides / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D039481:CD11b Antigen; D008070:Lipopolysaccharides; D008387:Marine Toxins; D012333:RNA, Messenger; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D014409:Tumor Necrosis Factor-alpha; C104724:glutamate receptor ionotropic, AMPA 4; D013481:Superoxides; D020780:Matrix Metalloproteinase 9; C012301:domoic acid; D007608:Kainic Acid