The fetal alcohol syndrome (FAS) is one of the leading known cause of mental retardation in the United States and accounts for up to 5% of all congenital anomalies. Some of the brain lesions in FAS are consistent with a disorder of neuronal proliferation, migration, fasciculation, and synapse formation -- developmental events that depend heavily on the patterned expression and function of cell adhesion molecules (CAMs). We have shown that human recombinant OP-1 (hOP-1), a member of the transforming growth factor beta superfamily, strongly induces cell clustering and clumping in proliferating NG108-15 cells, in part by inducing the neural cell adhesion molecules N-CAM and L1. Our preliminary data indicate that ethanol inhibits hOP-1 morphogenesis (over 2-4 days) and cell-cell adhesion (over 30 min) in dose dependent fashion, throughout the range of concentrations achieved during social drinking. Our long- range goal is to learn how ethanol inhibition of intercellular neuronal adhesion contributes to the fetal alcohol syndrome and other complications of alcoholism. NG108-15 cells will be cultured with phosphorothioate CAM antisense oligonucleotides to determine whether ethanol inhibits hOP-1 morphogenesis by disrupting the induction or function of specific CAMs. hOP-1 morphogenesis will be quantitated by light microscopy, computerized image analysis, and by light transmittance read by a microplate reader. To determine whether ethanol inhibits homophilic N-CAM binding, we will study soluble N-CAM binding to N-CAM-coated latex beads, liposomes, or hOP-1 treated NG108-15 cells. Similar experiments will be conducted for L1. We will study the aggregation of N-CAM and L1 coated latex beads and liposomes to each other and to hOP-1 treated NG108-15 cells to learn whether ethanol inhibits cell-cell adhesion by direct inhibition of N-CAM or L1 binding, alterations in N-CAM-s or L1's lipid milieu, or changes in N-CAM- or L1-coupled effectors. We will use mouse fibroblasts transfected with L1 or N-CAM to study directly whether ethanol inhibits the adhesive properties of these cell adhesion molecules. In studying the homotypic adhesion of transfected cells and the heterotypic adhesion of transfected cells and neurons we will learn whether ethanol inhibition of L1 and N- CAM-mediated cell adhesion is dependent on the cell type in which these molecules are expressed. We will determine whether tolerance develops to ethanol inhibition of hOP-1 morphogenesis and cell-cell adhesion. To address the generality of these phenomena, we will study ethanol inhibition of hOP-1 morphogenesis and cell-cell adhesion in several neuronal and glial cell lines, a human cortical cell line, and primary cultures of neurons and astrocytes from rat nervous system. The proposed studies will help define the molecular mechanisms by which gestational exposure to ethanol disrupts nervous system development. In identifying such a mechanism, we may be able to learn why some women are more vulnerable than others to ethanol's teratogenicity. This knowledge could lead to a means for screening women at greatest risk.
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