2006 Progress Report: Neurotoxicant Effects on Cell Cycle Regulation of Neurogenesis

EPA Grant Number: R829391C001
Subproject: 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 , University of Medicine and Dentistry of New Jersey
EPA Project Officer: Louie, Nica
Project Period: November 1, 2001 through October 31, 2006
Project Period Covered by this Report: November 1, 2005 through October 31, 2006
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001) RFA Text |  Recipients Lists
Research Category: Children's Health , Health Effects , Health


The 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 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 continue to characterize changes in specific neuronal populations and define underlying cell cycle mechanisms, showing region-specific effects. We also, however, have expanded our studies to a recently identified animal model of autism susceptibility, specifically, the Engrailed 2 (En-2) mutant mouse, as collaborators James Millonig and Linda Brzustowicz have demonstrated strong genetic association with human autism.

Methylmercury (MeHg) Elicits Acute and Long-Term Effects on Newborn Rat Hippocampal Neurogenesis Selectively

To examine acute neurotoxicant effects on DNA synthesis, we injected postnatal day 7 (PND 7) rats with MeHg and then [3H]thymidine 6 hours later, measuring incorporation 2 hours later. At 8 hours, DNA synthesis was reduced 16 percent at 0.1, 50 percent at 3, and 80 percent at 30 µg/gbw in hippocampus, indicating effects on DNA synthesis. Two weeks later, rats injected with MeHg at PND 7 exhibited reduced hippocampal cell number, indicated by a 17 percent decrease in total DNA at 3 µg/g and a trend to reduction at a 10-fold lower dose. In contrast, MeHg did not affect DNA synthesis or cell number in cerebellum, though blood flow and Hg content were similar in both regions. Indeed, MeHg concentrations at 8 hours were between 1.5-3.1 ppb in saline injected animals, whereas following 3 µg/gbw injections of MeHg, concentrations were 714 (+ 68) in hippocampus and 920 (+ 122) in cerebellum (mean + SEM). Because DNA synthesis in isolated cerebellar granule precursors was inhibited 25 percent by MeHg, reflecting a sensitivity to the metal comparable to other cell types, we conclude that the in vivo cerebellar environment offers a neuroprotective effect, which may possibly reflect effects of local glia, a direction for future studies.

MeHg Decreases Cortical Precursor Survival in Culture and Blocks the G1 to S Phase Transition Via Selective Reduction in Cyclin E Protein Levels.

Using embryonic day 14.5 (E 14.5) 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 had 90 percent inhibition at 2.0 µM. There were parallel reductions in DNA synthesis at this time consistent with death of mitotic precursors. At 6 hours, however, DNA synthesis was reduced 49 percent at 3 and 98 percent at 10µM MeHg, with no change in cell number, suggesting that reduced DNA synthesis was a result of a G1/S block. In support of this proposal, we found that MeHg elicited a 75 percent reduction in cyclin E, the stimulatory subunit of the CDK2 kinase complex. In contrast, there was no wholesale reduction in all cell cycle proteins, as CDK2 levels were unchanged. Furthermore, MeHg did not increase either p27 or p57, CDK inhibitors commonly stimulated by endogenous antimitogens, such as pituitary adenylate cyclase-activating peptide (PACAP). These studies identify the cell cycle machinery as new MeHg targets, specifically cyclin E. The decreases in DNA synthesis at 6 hoursin hippocampus in vivo and cortical precursor culturessuggest MeHg inhibits neurogenesis by interfering with cell cycle progression. Furthermore, the studies indicate that MeHg rapidly and directly alters brain development through modulating regional neurogenesis. 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. Ongoing studies in vivo are examining levels of cell cycle markers, including bromodeoxyuridine (BrdU) nuclear labeling, cyclin E, p27/p57, and those for cell death, including terminal transferase-mediated dUTP nick-end labeling (TUNEL) and activated caspase 3.

Moderate Lead Exposure Elicits Neurotrophic Effects in Cerebral Cortical Precursor Cells in Culture

Lead (Pb) persists as an environmental toxicant despite aggressive environmental and occupational regulation. Neurotoxicological effects of acute Pb poisoning range from subtle cognitive deficits, to clumsiness and ataxia, to coma and seizures. In adult neurotoxicity, reductions of blood Pb levels often are associated with reversal of clinical signs. In children, however, the effects are more likely to endure, with even low levels of chronic Pb exposure correlating with decreasing IQ. These persistent effects likely result from neurodevelopmental insults, such as altered cell survival or maturation, although the mechanisms have not been defined fully. In the present study we define the effects of moderate-level Pb exposure on mammalian neurogenesis using a well-characterized cortical precursor model. Gestational day 14.5 rat cerebral cortical precursor cells were cultured in defined media, and cell number, precursor proliferation, apoptosis, and neuritic process outgrowth were assessed following exposure to a range of Pb acetate concentrations. Surprisingly, whereas a concentration of 30 µg/mL Pb acetate was acutely toxic to neurons, concentrations between 1 and 10 µg/mL Pb acetate (3 µM and 30 µM Pb, respectively) increased cell number: 10 times as many cells exposed to 10 µg/mL Pb were present on day 4 as compared to control. The increase in cell number was not a result of increased proliferation, however, as DNA synthesis did not increase. Rather, Pb exposure maintained the survival of cortical precursors, as the progressive apoptosis occurring under control conditions was reduced markedly by the metal. Additionally, neuritic process initiation and outgrowth increased in a concentration-dependent manner, with processes four times as abundant on day 1 and twice as long on day 2. These results suggest that brief exposure to Pb during neurogenesis directly affects cell survival and process development, potentially altering cortical arrangement. Consequently, alterations in neural circuitry may underlie some of the neurological effects of Pb exposure during brain development.

En-2, an Autism-Associated Gene, Regulates Neurogenesis During Brain Development

Although the signs of autistic spectrum disorder (ASD), including abnormal language and social skills and repetitive and restricted motor behaviors, indicate forebrain dysfunction, there is growing evidence of hindbrain pathology, including newly identified linkage and association to En-2, a gene patterning development of hindbrain, especially the cerebellum. Indeed, the most consistent pathological abnormalities of ASD are deficiencies of cerebellar Purkinje and granule neurons, suggesting relevance to disease susceptibility. We hypothesize that normal cerebellar development depends on proper timing, localization, and regulation of En-2 expression, the disruption of which contributes to ASD cerebellar pathology. Whereas the En-2 mutant mouse exhibits pathological phenotypes similar to ASD, including small posterior vermis, as observed in human neuroimaging, and deficits in Purkinje and granule neurons, as defined on neuropathology, the cellular mechanisms of action of En-2 are wholly unexplored. We currently are performing a variety of in vivo and in vitro studies to define En-2 expression and function during brain development. Although En-2 appears early in mouse hindbrain development, at E9.5, throughout the mid/hindbrain, and then as stripes in tissues of E17.5 cerebellum, we are focusing on its postnatal role in newborn cerebellum, because it appears solely in the postmitotic granule neurons, suggesting it may play a role in cell cycle exit and/or neuronal differentiation. These studies will lay the groundwork for later tests of effects on neurotoxicants and teratogens.

Given this association, we have begun to define human EN-2 functions during neurogenesis. A mouse En-2 cDNA was cloned into an expression vector bicistronic with enhanced green fluorescent protein (GFP) and transfected into rat E14.5 cortical precursors. The vector alone and En-2 cDNA cloned in the reverse orientation were used as negative controls. En-2 protein was detected by immunostaining only in En-2-transfected GFP+ cells. Morphological analysis of GFP+ cells 24-hour posttransfection indicates that a higher percentage of En-2+ cells are nonneuronal than GFP+/En-2- cells (p = 0.014). Furthermore, En-2+ cells that resembled neurons exhibited shorter processes with fewer branches. These data suggest En-2 represses neuronal differentiation when misexpressed in cortical precursors, suggesting dysregulation of human EN-2 may underlie abnormal neurodevelopment identified in autism. Future experiments will investigate En-2 neurogenetic effects in cerebellar granule precursors.

Extracellular Growth Factors Interact With En-2, an Autism-Associated Gene, to Control Cerebellar Development

Interactions of genes with the environment, including extracellular signaling molecules, control neurodevelopment. Disrupting these interactions alters neurogenesis, potentially producing developmental disorders, such as autism. In mice, disrupted En-2 expression reduces cerebellar Purkinje and granule cell numbers, similar to human autism neuropathology. To define En-2 cerebellar functions, we examined DNA synthesis in whole cerebellum and granule neuron precursors (GNPs) in vivo, as well as GNPs in culture in response to growth signals, using C57/BL6 (WT) and En-2 knockout (KO) mouse pups. GNPs were isolated by genotype, cultured for 24 hours in defined media, and assessed for 3H-deoxythymidine (3H-dT) or BrdU incorporation. Proliferation in vivo was reduced in whole KO cerebellum and GNPs, suggesting disordered growth regulation. In culture, no genotype-dependent differences in control GNP mitosis were present. Insulin-like growth factor 1 (IGF1), however, elicited a two-fold increase in DNA synthesis in WT, and a six-fold increase in KO, paralleled by BrdU labeling, suggesting En-2 expression negatively regulates IGF1 mitogenic signaling. Consistent with this model, KO GNPs exhibited decreased process outgrowth (WT=42%; KO=25%; % neurite-bearing cells). There were no genotype-dependent differences in cell survival. No other growth factors engaging tyrosine kinase receptors (basic fibroblast growth factor, brain-derived neurotrophic factor, epidermal growth factor) nor nonkinase receptors (Shh, PACAP) elicited genotype-dependent effects, suggesting cell cycle regulation is comparable among genotypes. Together, these data identify previously unrecognized interactions between IGF1 and En-2 expression, suggesting altered En-2 activity may contribute to abnormal cerebellar growth via aberrant IGF1 signaling.

Future Activities:

The investigators did not report any future activities at this time.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 8 publications 2 publications in selected types All 2 journal articles
Other center views: All 86 publications 50 publications in selected types All 49 journal articles
Type Citation Sub Project Document Sources
Journal Article Benayed R, Gharani N, Rossman I, Mancuso V, Lazar G, Kamdar S, Bruse SE, Tischfield S, Smith BJ, Zimmerman RA, DiCicco-Bloom E, Brzustowicz LM, Millonig JH. Support for the homeobox transcription factor gene ENGRAILED 2 as an autism spectrum disorder susceptibility locus. American Journal of Human Genetics 2005;77(5):851-868. R829391 (2004)
R829391 (2005)
R829391 (2006)
R829391C001 (2006)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: ScienceDirect-Full Text HTML
  • Other: ScienceDirect-Full Text PDF
  • Journal Article Davidovics Z, DiCicco-Bloom E. Moderate lead exposure elicits neurotrophic effects in cerebral cortical precursor cells in culture. Journal of Neuroscience Research 2005;80(6):817-825. R829391 (2004)
    R829391 (2005)
    R829391 (2006)
    R829391C001 (2006)
  • Abstract from PubMed
  • Abstract: Wiley-Abstract
  • 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,, RFA, Health, Scientific Discipline, Health Risk Assessment, Biochemistry, Children's Health, developmental neurotoxicity, biological response, neurodevelopmental toxicity, children's environmental health, environmental health hazard, environmental toxicant, growth & development

    Progress and Final Reports:

    Original Abstract
  • 2002
  • 2003
  • 2004 Progress Report
  • 2005 Progress Report
  • Final

  • 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