About GSBS   |  FAQ  |  Job Opportunities  |  Search UMDNJ
     




Effects of methylmercury on the developing hippocampus: a molecular, cellular, and behavioral analysis

by
Katie Sokolowski
B.A., Rutgers University - 2004


Thesis Advisor: Emanuel DiCicco-Bloom, M.D.
Graduate Program in Toxicology

CABM, Room 010
Piscataway, NJ

Friday, November 5, 2010
12:00 noon


Abstract

Through diet, humans are exposed to methylmercury (MeHg). By virtue of its molecular mimicry, MeHg readily crosses the blood-brain and blood-placental barriers. Studies from poisonings in Japan and Iraq have shown the particular sensitivity of the developing brain. In adults, MeHg-induced lesions were primarily in the cerebellar granule cells and the calcarine cortex, while exposures during development showed a more diffuse pattern of neuropathology. Even at lower levels of exposure, epidemiological studies have raised concern for the developing brain with respect to behavioral outcomes such as learning and memory. Recent studies have drawn attention to one part of the brain involved in learning and memory, the hippocampus, as a sensitive target for MeHg. We sought to better understand mechanisms of teratogenic effects of MeHg on developing hippocampal neurons, later cell number and behavior.

The present results demonstrated that after a single sc injection of 0.6 µg/g MeHg in the P7 Sprague-Dawley male rat, Hg concentrations peaked at 679ppb at 24h and fell to control levels by P21. Using a ten-fold higher dose (5 µg/g), Hg concentrations were above controls by 2h, peaked at 2,129ppb by 24h, and fell to control levels by P35. Levels of Hg in our models are significantly higher than controls during postnatal neurogenesis of the dentate gyrus and approximate Hg levels found in brain tissues from humans consuming fish and from animal models. Mechanistic analysis revealed increased levels of proteins associated with both the mitochondrial-dependent and –independent pathways: 38% increases in Bax protein levels as early as 2h after exposure to 5 µg/g MeHg, increases in cytosolic/mitochondrial cytochrome C at 4h, increases in caspase-9 (40% at 12h and 33% at 24h) and a trend to increased caspase-8 at 24h. However, inhibition of caspase-9 prevented caspase-3 and caspase-8 activation suggesting that MeHg-induced caspase-3 activation is primarily, but not exclusively, through the mitochondrial-dependent cascade. Single cell analysis of the endpoint protein, caspase-3, revealed hippocampal cell death in vivo at ten-fold lower levels of MeHg exposure (0.6 µg/g), previously shown to be ineffective using whole hippocampal homogenate assays.

Single cell analysis 24h after exposure to both 5 µg/g and 0.6 µg/g MeHg demonstrated the sensitivity of mitotic cells to MeHg. BrdU labeled cells were decreased by 30% in the hilus 24h after 0.6 µg/g MeHg, suggesting mitotic cells are sensitive. This exposure induced a 2-3-fold increase in cleaved caspase-3+ cells as well as pyknotic nuclei, indicating enhanced apoptosis. Furthermore, the overwhelming majority of MeHg-induced caspase-3+ cells colocalized the stem cell markers nestin and Sox2, suggesting that proliferative precursors are especially vulnerable in vivo. Decreases in neural stem cells from caspase-3-induced apoptosis were correlated with cellular and behavioral deficits weeks after the initial insult. After a single P7 injection of 0.6 µg/g MeHg, cell number decreased to 22% of control in hilus (p<0.05) and a 27% decrease in the granule cell layer (p<0.01) at P21. At P35 subtle memory impairments were detected using the Morris water maze (2-day memory escape latencies: con: 18+3 sec, MeHg: 28+4 sec, p<0.05).
Together, results of this dissertation demonstrate that MeHg works to activate caspase-3 primarily through the mitochondrial-dependent cascade in hippocampal neural stem cells, leading to decreased cell number at P21 and hippocampal-dependent behavioral deficits during adolescence.


Return to Dissertation list

 

Newark Campus - Piscataway Campus - Stratford Campus
About GSBS - FAQ - Job Opportunities - Search UMDNJ