ALCOHOL INHIBITION OF NEURONAL CELL ADHESION

胎儿酒精综合征(FAS)是已知的主要原因之一 在美国，智力低下的人占所有智障人数的5% 先天畸形。Fas中的一些脑部病变与 伴随着神经元的增殖、迁移、束状化和 突触形成--发育事件严重依赖于 细胞黏附分子(CAM)的图案化表达和功能。我们 已经表明，人类重组OP-1(HOP-1)是 转化生长因子β超家族，强诱导细胞 在增殖的NG108-15细胞中聚集和聚集，部分是通过 诱导神经细胞黏附分子N-CAM和L1。我们的预赛 数据表明，乙醇抑制HOP-1的形态发生(超过2-4天) 和细胞-细胞黏附(超过30分钟)，以剂量依赖的方式，贯穿始终 在社交饮酒过程中达到的浓度范围。我们的长- Range的目标是了解乙醇如何抑制细胞间神经元 粘连导致胎儿酒精综合征和其他并发症 酗酒。NG108-15细胞将与硫代细胞黏附分子共同培养 确定乙醇是否抑制HOP-1的反义寡核苷酸 破坏特定凸轮的诱导或功能的形态发生。 HOP-1的形态发生将通过光学显微镜、计算机化进行量化 图像分析，并通过微板阅读器读取的光透过率。 为了确定乙醇是否抑制了亲水性N-CAM结合，我们将 研究可溶性N-CAM与N-CAM包衣胶乳微珠、脂质体或 HOP-1作用于NG108-15细胞。将进行类似的实验以 L1。我们将研究N-CAM和L1包覆乳胶珠的聚集和 脂质体相互作用并向经HOP-1处理的NG108-15细胞学习 乙醇是否通过直接抑制N-CAM抑制细胞与细胞间的黏附 或L1结合，N-CAM-S或L1‘S脂环境改变，或 N-CAM或L1耦合效应器。我们将使用转基因的小鼠成纤维细胞 用L1或N-CAM直接研究乙醇是否抑制粘连 这些细胞黏附分子的性质。在研究同型 转基因细胞的黏附及转基因细胞的异型黏附 细胞和神经元，我们将了解乙醇是否抑制L1和N- CaM介导的细胞黏附依赖于细胞类型，其中 分子被表达出来。我们将确定宽容是否会发展到 乙醇对HOP-1形态发生和细胞间黏附的抑制作用。至 针对这些现象的共性，我们将研究乙醇 抑制HOP-1的形态发生和细胞与细胞的黏附 神经元和神经胶质细胞系，一种人类皮质细胞系，以及原代 大鼠神经系统神经元和星形胶质细胞的培养。建议数 研究将有助于确定怀孕的分子机制 接触乙醇会破坏神经系统发育。在识别 在这样的机制下，我们或许能够了解为什么有些女性 比其他人更容易受到乙醇致畸作用的影响。这一知识可能会 导致了一种筛查风险最高的女性的方法。

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.