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，精神发育迟滞占所有人的5%， 先天性异常 FAS中的一些脑部病变与 患有神经元增殖、迁移、肌束震颤和 突触的形成--发育过程很大程度上依赖于 细胞粘附分子（CAM）的模式化表达和功能。 我们 已经表明，人重组OP-1（hOP-1）， 转化生长因子β超家族，强烈诱导细胞 在增殖的NG 108 -15细胞中聚集和成团，部分是由于 诱导神经细胞粘附分子N-CAM和L1。 我们的初步 数据表明乙醇抑制hOP-1形态发生（2-4天） 和细胞-细胞粘附（超过30分钟）的剂量依赖性方式，在整个 在社交饮酒期间达到的浓度范围。 我们长久以来- 范围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 酒精中毒 NG 108 -15细胞将与硫代磷酸酯CAM一起培养 反义寡核苷酸，以确定乙醇是否抑制hOP-1 通过破坏特异性CAM的诱导或功能来实现形态发生。 hOP-1形态发生将通过光学显微镜、计算机化 图像分析，并通过酶标仪读取透光率。 为了确定乙醇是否抑制嗜同性N-CAM结合，我们将 研究可溶性N-CAM与N-CAM包被的乳胶珠、脂质体或 hOP-1处理NG 108 -15细胞。 将进行类似的实验， L1。 我们将研究N-CAM和L1包被的乳胶珠的聚集， 脂质体相互作用以及hOP-1处理的NG 108 -15细胞，以了解 乙醇是否通过直接抑制N-CAM来抑制细胞-细胞粘附 或L1结合，N-CAM-s或L1脂质环境的改变，或 N-CAM或L1偶联效应物。 我们将使用小鼠成纤维细胞转染 用L1或N-CAM直接研究乙醇是否抑制粘附 这些细胞粘附分子的特性。 在研究同性恋 转染细胞的粘附和转染细胞的异型粘附 细胞和神经元，我们将了解是否乙醇抑制L1和N- CAM介导的细胞粘附依赖于细胞类型，其中这些细胞粘附在细胞中。 分子表达。 我们将确定耐受性是否会发展到 乙醇抑制hOP-1形态发生和细胞-细胞粘附。 到 为了解决这些现象的普遍性，我们将研究乙醇 抑制hOP-1形态发生和细胞-细胞粘附 神经元和神经胶质细胞系，人皮质细胞系，和原代 大鼠神经系统神经元和星形胶质细胞的培养。 拟议 研究将有助于确定妊娠期高血压疾病的分子机制， 接触乙醇会破坏神经系统的发育。 查明 这样一个机制，我们也许能够了解为什么有些妇女 比其他人更容易受到乙醇致畸性的影响。 这些知识可以 从而找到一种筛查高危妇女的方法。

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.