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2004 Progress Report: Adhesion and Repulsion Molecules in Developmental Neurotoxic Injury
EPA Grant Number: R829391C002Subproject: this is subproject number 002 , 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: Adhesion and Repulsion Molecules in Developmental Neurotoxic Injury
Investigators: Reuhl, Kenneth R.
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, 2003 through October 31, 2004
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001)
Research Category: Health Effects , Children's Health
Description:
Objective:Normal brain development depends on the appropriate temporal and spatial expression of neural adhesion and repulsion molecules, several families of membrane proteins that provide instructive and permissive guidance for neuronal and neuritic movement. We postulate that neurotoxic metals perturb brain development/morphogenesis by disrupting the regulated expression and function of critical morphoregulatory adhesion and repulsion molecules. We further postulate that defects in these critical molecules give rise to morphological, biochemical, and behavioral effects of relevance to the study of autism spectrum disorder.
The objective of this research project is to address four specific questions: (1) Does exposure to neurotoxic metals alter the expression of adhesion and repulsion molecules during critical stages of brain development and thereby compromise morphogenesis? (2) Do selective transcriptional, translational, or post-translational processes mediate metal-induced changes in adhesion and repulsion molecules? (3) What are the behavioral consequences of toxicant-disturbed adhesion and repulsion molecules? (4) Can the deleterious effects of toxic metals on morphoregulatory molecules be modified or ameliorated by intervention strategies?
The studies utilize complementary biochemical and behavioral assessment to characterize the effects of toxic metals on neural cell adhesion molecules (NCAM), L-1, and the Eph family of tyrosine kinase repulsion molecules.
Progress Summary:Studies during Year 3 of the project have focused on both in vivo and in vitro assessment of methylmercury (MeHg) effects on the Eph repulsion molecules. The Eph receptors and their ligand (the ephrins) are involved in several critical steps in brain formation and particularly in the topographic structuring of specific neural fields. The Eph/ephrin molecules help specify the pattern of connectivity of neuronal populations and serve to prevent inappropriate synaptic contacts. Data from our studies strongly support an effect of the metal on the transcriptional control of Eph.
In Vivo Studies
Pregnant C57 mice were administered 2.5 mg/kg MeHg by gavage on alternate days beginning on day 14 of gestation and continuing to sacrifice on postnatal day 1, 5, 10, or 15. Brains were removed; dissected into cerebellum, hippocampus, and rest of brain; and examined for Eph/ephrin expression using a ribonuclease (RNase) protection assayfor mRNA, which allows for the simultaneous assessment of 12 Eph receptors and 8 ephrin ligands. Western blotting was used to quantitate protein expression. Results of RNAse protection assays revealed changes in Eph and ephrin RNA that differed according to the age of the pups and the region of brain examined. Significant increases of Eph A8, Eph B6, and ephrin-B2 were observed on postnatal day 5, and increases of ephrin-A5 in hippocampus were observed on day 10. In cerebellum, Eph A4 was decreased on day 15.
Several other Ephs showed a consistent trend for increased expression. Although these did not reach statistical significance, the consistency of the trend suggests that the results are biologically real.
The mouse studies have been supported by behavioral analysis. Mice treated with MeHg were examined for early reflexive behaviors and later-developing spatial navigation skills. MeHg slowed the acquisition of reflexive behaviors and impaired spatial memory; these defects correlated with reductions in polysialated NCAM (PSA-NCAM). Eph A5 and Eph A4 increased in response to learning tasks, but these increases were attenuated in MeHg-treated animals.
In Vitro Studies
To characterize the expression of Ephs and ephrins during neural lineage commitment and differentiation, we exploited the P19 embryonal carcinoma cell model. These pluripotent cells differentiate into neuroectoderm on treatment with retinoic acid and express neural-specific molecular markers in the same temporal sequence as neurons in vivo. Undifferentiated P19 cells express only Eph A2. Retinoic acid treatment, however, results in expression of B-class Ephs by day 2 of differentiation. A-class Eph receptors do not appear until day 6 of differentiation. Similarly, ephrins are not expressed in undifferentiated P19 cells, but both A- and B-class ephrin ligands are present by day 2 of differentiation. The fact that A-class ligands are expressed in the absence of A-class receptors implies that the ligands have another function in lineage commitment other than class-specific binding. It has been speculated that the A-class receptors play a predominant role during topographical mapping and the B-receptors are more involved in neural movement and neurite fasciculation.
Treatment of P19s with MeHg resulted in changes in Eph and ephrin expression in a concentration-related fashion. Effects were particularly evident in day 6 cultures, where 0.5 and 1.5 µM concentrations caused a general increase in most receptor and ligand levels, whereas 3.0 µM caused increased levels of Ephs A2, A4, A8, B2, B2, and B6. Other receptors and ligands were either unaltered or decreased. Paradoxically, Western blot analysis showed an increase in protein expression at 0.5 and 1.5 µM but a decrease at 3.0 µM, suggesting increased transcription but impaired translation of Eph/ephrin mRNA.
Significance
Both in vivo and in vitro data indicate that MeHg effects the expression of specific members of the Eph/ephrin family of tyrosine kinase repulsion molecules. Significantly, to toxicant results in increased expression of many Eph and/or ephrins, rather than generalized decreases in expression as would be predicted if the effect as nonspecific. The functional consequence of overexpression of the repulsion molecules would be failure of the neurons to appropriately target their synaptic partners, leading to errors in topographic mapping and pathway development. Defects of this type have been proposed to underlie some of the functional deficits observed in individuals with autism. The behavioral studies have provided a strong linkage between changes in behavioral performance and the expression of adhesion and repulsion molecules. MeHg-treated animals showed delayed acquisition of reflexive behaviors and spatial navigation skills that correlated with reduced PSA-NCAM expression. In untreated mice, acquisition of learning corresponded to increased expression of Ephs A4 and A5. Mice treated with MeHg during development and trained on a water maze task showed a reduced increase in Eph A5 and exhibited persistent behavioral effects as adults.
Future Activities:Work during the next year will focus on establishing structural and functional correlations between the changes in Eph/ephrin expression and MeHg exposure. We have begun to characterize the cellular expression of the specific molecules using in situ hybridization techniques. These results will be correlated with the structure of specific neural pathways (e.g., hippocamposeptal) using DiI labeling. The exact site of the dysregulation of Eph/ephrin RNA expression will be sought by examining the molecular machinery of RNA transcription. Working in collaboration with Dr. Wagner, we will examine further the relationship between the expression of morphoregulatory molecules and behavior, focusing on the effects on early-onset behaviors. We are particularly interested in expression of these molecules at the synapse, where their dysregulation would have immediate and persistent effects on the function of the brain.
Journal Articles on this Report: 1 Displayed | Download in RIS Format
| Other subproject views: | All 23 publications | 9 publications in selected types | All 9 journal articles |
| Other center views: | All 101 publications | 52 publications in selected types | All 51 journal articles |
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Prozialeck WC, Grunwald GB, Dey PM, Reuhl KR, Parrish AR. Cadherins and NCAM as potential targets in metal toxicity. Toxicology and Applied Pharmacology 2002;182(3):255-265. |
R829391 (2004) R829391 (2005) R829391 (2006) R829391C002 (2004) |
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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, brain development, neurodevelopmental toxicity, autism, children's environmental health, environmental toxicant, developmental neurotoxicity, biological response, metals, growth & development
Progress and Final Reports:
Original Abstract
2005 Progress Report
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