Proteomic Analysis of Cerebral Spinal Fluid From California Sea Lions (Zalophus californianus) with Domoic Acid Toxicosis
IAAAM 2012
Benjamin A. Neely1; Jennifer L. Soper2; Frances M.D. Gulland2; John M. Arthur1,3; Michael G. Janech1,3
1Department of Medicine, Division of Nephrology, Medical University of South Carolina, Charleston, SC, USA; 2The Marine Mammal Center, Sausalito, CA, USA; 3Research Service, Ralph H. Johnson VA Medical Center, Charleston, SC, USA

Domoic acid toxicosis (DAT) is a major cause of California sea lion (Zalophus californianus) strandings along the western coast of the United States.1 Domoic acid is a potent neurotoxin produced by some marine diatoms, which causes hippocampal atrophy and necrosis as a result of excessive stimulation of AMPA-kainate and NMDA receptors.2 Assigning a diagnosis of DAT is difficult because the toxin is rapidly cleared from the body and is often not present at the time of stranding. Clinical signs that support a diagnosis of DAT are seizures or behavioral abnormalities. The diagnosis is further supported by MRI imaging or histological examination of the hippocampus. More recently, DAT has been classified along a continuum ranging from acute to chronic, with animals with the former recovering over time and the latter progressing to status epilepticus as a result of cerebral anoxia and chronic hippocampal damage.3 Currently markers of DAT are limited and often provide inconclusive results. Discovery and validation of protein markers of DAT could greatly facilitate diagnosis and guide therapy. For this reason we evaluated whether protein differences exist in the cerebral spinal fluid (CSF) that could be utilized as markers of DAT. CSF samples from 11 sea lions (3 without DAT, 2 with acute-DAT and 6 with chronic-DAT) were acquired from The Marine Mammal Center, and proteins were digested with trypsin for analysis by tandem mass spectrometry. Data were searched using Mascot against a mammalian database, with identifications supported by Scaffold. We identified 150 experiment-wide proteins with a protein false discovery rate of < 0.1%. Relative protein abundance was estimated by spectral counting across all samples, and differences were detected using a Wilcoxon rank-sum test. Five proteins were significantly different (p < 0.05) between non-DAT and DAT animals - four of which were higher in animals with DAT. Although not different by proteomic measures (p = 0.121), the 180 kDa isoform of Reelin was found to be lower in DAT animals by immunoblotting (p < 0.001). Overall, Dickkopf-3, Gelsolin, Neural cell adhesion molecule 1, and Oligodendrocyte-myelin glycoprotein were elevated in sea lions with DAT, while Reelin and Ig lambda-6 chain C region were decreased in sea lions with DAT. Interestingly, Dickkopf-3, Gelsolin, and Reelin have been implicated in the progression of Alzheimer's disease, temporal lobe epilepsy, and schizophrenia in humans. These data suggest that sea lions with DAT exhibit a CSF protein phenotype that overlaps with several neurodegenerative diseases. Interestingly, transcripts belonging to pathways associated with Alzheimer's disease are prominent in blood cells from sea lions with DAT.4 Future studies to qualify these proteins as markers of DAT are necessary and ongoing.

Acknowledgements

Funding was provided by the Office of Naval Research: N00014-08-1-0341. The authors would like to thank the veterinarians and technicians at The Marine Mammal Center. Also, thanks are extended to the MUSC Nephrology Proteomics Laboratory for technical assistance.

References

1.  Bejarano AC, Gulland FM, Goldstein T, et al. Demographics and spatio-temporal signature of the biotoxin domoic acid in California sea lion (Zalophus californianus) stranding records. Mar Mamm Sci. 2008;24:899–912.

2.  Pulido OM. Domoic acid toxicologic pathology: a review. Mar Drugs. 2008;6:180–219.

3.  Goldstein T, Mazet JAK, Zabka TS, et al. Novel symptomatology and changing epidemiology of domoic acid toxicosis in California sea lions (Zalophus californianus): an increasing risk to marine mammal health. Proc R Soc B. 2008;275:267–276.

4.  Mancia A, Ryan JC, Chapman RW, Wu Q, Warr GW, Gulland FM, Van Dolah FM. Health status, infection and disease in California sea lions (Zalophus californianus) studied using a canine microarray platform and machine-learning approaches. Dev Comp Immunol. 2012;36:629–637.

  

Speaker Information
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Benjamin A. Neely
Department of Medicine, Division of Nephrology
Medical University of South Carolina
Charleston, SC, USA


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