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2005 Progress Report: Neurotoxicant Effects on Cell Cycle Regulation of Neurogenesis
EPA Grant Number: R829391C001Subproject: this is subproject number 001 , established and managed by the Center Director under grant R829391
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: CECEHDPR - University of Medicine and Dentistry of New Jersey Center for Childhood Neurotoxicology and Assessment
Center Director: Lambert, George H.
Title: Neurotoxicant Effects on Cell Cycle Regulation of Neurogenesis
Investigators: DiCicco-Bloom, Emanuel
Institution: University of Medicine and Dentistry of New Jersey
Current Institution: University of Medicine and Dentistry of New Jersey
EPA Project Officer: Fields, Nigel
Project Period: November 1, 2001 through October 31, 2006
Project Period Covered by this Report: November 1, 2004 through October 31, 2005
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001)
Research Category: Children's Health , Health Effects
Description:
Objective:The overall objective of this research project is to investigate the hypothesis that neurotoxic metals and teratogens disrupt neurogenesis in developing forebrain and hindbrain systems in vitro and in vivo, acting to inhibit proliferation by altering mitogenic growth factor receptors and cell cycle and signaling pathways.
Progress Summary:We have made significant progress this past year. Indeed, we have found evidence supporting our hypothesis that neurotoxic metals and teratogens disrupt neurogenesis in developing forebrain and hindbrain systems, altering precursor mitosis, survival and differentiation. We now are beginning to characterize changes in specific neuronal populations and define underlying cell cycle mechanisms.
Methylmercury (MeHg) Decreases Cortical Precursor Survival in Culture, and Blocks the G1 to S Phase Transition Via Reduced Cyclin E Protein Levels
Using Embryonic Day 14.5 (E14.5) cerebral cortical precursors in 24-hour cultures, we found that MeHg (0.01-3.0 µM) reduced cell survival approximately 50 percent at 1.0-1.5 µM and 90 percent inhibition at 2.0 µM. There were parallel reductions in DNA synthesis at this time, consistent with death of mitotic precursors. When we examined cells at 6 hours before cell death occurred, however, 3 µM MeHg elicited a 50 percent reduction in DNA synthesis. Significantly, the reduced DNA synthesis reflected less cells entering mitotic S phase, as there were similar decreases in the bromodeoxyuridine (BrdU) nuclear labeling index, a marker for cells synthesizing DNA. Moreover, we found a 75 percent reduction in the promitogenic regulator, cyclin E, which likely accounts for cells failing to enter S phase. The reduction in cyclin E was highly specific, as other cell cycle machinery proteins, such as CDK2 and p57, were unchanged. These studies identify a new locus for MeHg action in reducing the generation of forebrain neurons—effects on cell cycle components. In aggregate, our studies suggest that MeHg can alter brain neurogenesis by two mechanisms, including reducing the number of precursor cells undergoing cell division and inducing apoptosis of newly born neurons.
Lead Acetate (Pb) Enhances Cortical Neuron Survival and Process Outgrowth
Although previous literature would suggest that Pb should elicit negative effects during brain development and on cells in culture, our studies suggest more complex effects. Specifically, we found that Pb elicited trophic effects in cultures of primary cortical neurons: at doses relevant to that observed in human blood, Pb increased the initiation as well as the length of process outgrowth and markedly increased cell survival. Effects on process outgrowth included a 4-fold increase in process initiation at 8 hours as well as processes that were 2- to 4-fold longer by 1 and 2 days. Furthermore, by 2 days, a dose of 1 and 10 µg/mL Pb increased cell number by 2- to 4-fold and elicited 10-fold effects by 4 days. Extensive study indicated that Pb prevented the death of cortical neurons in vitro, as the precursors in control media underwent progressive apoptosis, and Pb did not alter DNA synthesis at these doses. These studies suggest that both the number and process outgrowth of cortical neurons could be altered by low levels of Pb exposure. If such effects occur in vivo, environmental Pb may affect human cognition by altering the composition and connectivity of brain circuitry. Excess brain neurons and processes have been suggested in autism pathology and neuroimaging. Furthermore, funded by an Environmental and Occupational Health Science Institute (EOHSI) pilot project grant, we have defined changes in gene expression, using an Affymetrix-based analysis in collaboration with the W.M. Keck Center for Collaborative Neuroscience, induced by Pb at 36 hours, a time that precedes alterations in cell survival. These candidates’ responsive genes now will serve as targets to examine effects in developing animals, after we confirm them by real-time PCR. The genes include recently defined survival promoting pathways related to B-cell CLL/lymphoma 2 (Bcl-2), to neurotrophin-3 (NT3), and several neuropeptides known to elicit increased cell survival in vitro, which we may begin to explore by selective blockade or over-expression strategies.
Valproic Acid Increases Cortical Precursor Cell Proliferation In Vitro and In Vivo
Using E14.4 cortical precursors, we have found that valproic acid (VPA) can elicit increased DNA synthesis at 0.3 to 1.5 mM concentration, with cell death induction thereafter. Significantly, the increased DNA synthesis is caused by more cells entering S phase, as indicated by increased BrdU labeling, as well as more cells remaining in the precursor state, identified by nestin expression. In contrast, less cells express the cytoskeletal molecule, beta III tubulin, and fewer cells elaborate neurites. These observations suggest that VPA maintains cells in a proliferative state. In turn, we have begun to define in vivo models to examine whether the drug has similar effects during prenatal cortical development. Indeed, VPA treatment of pregnant rats elicits region-specific stimulation of the cerebral cortex. This model suggests that at certain doses, VPA promotes cortical overgrowth, leading to mismatched neuronal populations during development. Significantly, recent neuroimaging studies indicate abnormal control of brain growth in autism. Furthermore, we have begun defining signaling pathways and cell cycle mechanisms underlying these changes in developing cortex, based on our ongoing studies in parallel culture models. Our observations suggest that VPA exposure indeed elicits forebrain overgrowth. Current studies are defining the exact changes in cortical cell composition and in collaboration, the behavioral consequences.
Future Activities:The investigators did not report any future activities at this time.
Journal Articles:No journal articles submitted with this report: View all 8 publications for this subproject
Supplemental Keywords:children’s health, disease and cumulative effects, ecological risk assessment, environmental chemistry, health risk assessment, risk assessments, susceptibility/sensitive population/genetic susceptibility, toxicology, genetic susceptibility, assessment of exposure, assessment technology, autism, behavioral assessment, behavioral deficits, childhood learning, children, developmental disorders, developmental effects, environmental health hazard, environmental toxicant, exposure assessment, gene-environment interaction, neurodevelopmental, neurological development, neuropathological damage, neurotoxic, neurotoxicity, outreach and education, public health,
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Scientific Discipline, Health, RFA, Health Risk Assessment, Children's Health, Biochemistry, neurodevelopmental toxicity, children's environmental health, environmental toxicant, developmental neurotoxicity, biological response, metals, growth & development
Progress and Final Reports:
2004 Progress Report
Original Abstract
2006 Progress Report
Main Center Abstract and Reports:
R829391 CECEHDPR - University of Medicine and Dentistry of New Jersey Center for Childhood Neurotoxicology and Assessment
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829391C001 Neurotoxicant Effects on Cell Cycle Regulation of Neurogenesis
R829391C002 Adhesion and Repulsion Molecules in Developmental Neurotoxic Injury
R829391C003 Disruption of Ontogenic Development of Cognitive and Sensory Motor Skills
R829391C004 Exposure Assessment and Intervention Project (EAIP)
R829391C005 Clinical Sciences Project