NAPVSIPQ (NAP), an active fragment of the glial-derived activitydependent neuroprotective protein, is protective at femtomolar concentrations against a wide array of neural insults and prevents ethanol-induced fetal wastage and growth retardation in mice. NAP also antagonizes ethanol inhibition of L1-mediated cell adhesion (ethanol antagonism). We performed an Ala scanning substitution of NAP to determine the role of ethanol antagonism and neuroprotection in NAP prevention of ethanol embryotoxicity. The Ser-Ile-Pro region of NAP was crucial for both ethanol antagonism and protection of cortical neurons from tetrodotoxin toxicity (neuroprotection). Ala replacement of either Ser-5 or Pro-7 (P7A-NAP) abolished NAP neuroprotection but minimally changed the efficacy of NAP ethanol antagonism. In contrast, Ala replacement of Ile-6 (I6A-NAP) caused a decrease in potency (>2 logarithmic orders) with only a small reduction (<10%) in the efficacy of NAP neuroprotection but markedly reduced the efficacy (50%) and the potency (5 logarithmic orders) of NAP ethanol antagonism. Ethanol significantly reduced the number of paired somites in mouse whole-embryo culture; this effect was prevented significantly by 100 pM NAP or by 100 pM P7A-NAP, but not by 100 pM I6A-NAP. The structure-activity relation for NAP prevention of ethanol embryotoxicity was similar to that for NAP ethanol antagonism and different from that for NAP neuroprotection. These findings support the hypothesis that NAP antagonism of ethanol inhibition of L1 adhesion plays a central role in NAP prevention of ethanol embryotoxicity and highlight the potential importance of ethanol effects on L1 in the pathophysiology of fetal alcohol syndrome. Fetal alcohol syndrome (FAS) is the most common preventable cause of mental retardation (1). Ethanol has multiple cellular targets in the nervous system (2); hence, it is not surprising that it damages the fetus through a variety of mechanisms: oxidative injury, induction of apoptosis, suppression of neurogenesis, disruption of cell-cell interactions, and alterations in the release and signaling of growth factors, morphogens, and chemical messengers (3-10). Several drugs that block specific molecular actions of ethanol have been shown to prevent or mitigate ethanol's teratogenesis in animal models (11,12), an unexpected finding, given the complex pathophysiology of FAS. Delineating the mechanism of action of these drugs would help to identify the most critical mechanisms that underlie ethanol's teratogenesis.Brain lesions in children with FAS resemble those of children with mutations in the gene for the L1 cell adhesion molecule, suggesting that ethanol might perturb fetal development in part by disrupting the actions of L1 (7). Interestingly, ethanol potently inhibits L1-mediated cell-cell adhesion (7, 13, 14) and L1-mediated neurite extension (15). A series of straight, cyclic, and branched alcohols shows unexpectedly strict structural requirements for alcohol inhibition of cell adhesion, consistent with a ligand-receptor i...

Ethanol inhibits cell-cell adhesion mediated by the L1 cell adhesion molecule. 1-Octanol potently antagonizes this cellular action of ethanol and also prevents ethanol-induced dysmorphology and cell death in mouse whole embryo culture. NAPVSIPQ (NAP) and SALLRSIPA (SAL) are active peptide fragments of two neuroprotective proteins: activity-dependent neuroprotective protein and activity-dependent neurotrophic factor. NAP and SAL are neuroprotective at femtomolar concentrations against a variety of neurotoxins and also prevent ethanol teratogenesis in mice. To explore the cellular basis for this action, we asked whether NAP and SAL antagonize ethanol inhibition of L1 adhesion. Aggregation assays were carried out in ethanol-sensitive, human L1-transfected NIH/3T3 cells in the absence and presence of NAP and SAL. Neither NAP nor SAL altered L1 adhesion or L1 expression; however, both peptides potently and completely antagonized the inhibition of L1 adhesion by 100 mM ethanol (EC 50 : NAP, 6 ϫ 10 Ϫ14 M; SAL, 4 ϫ 10 Ϫ11 M). NAP also antagonized ethanol inhibition of cell-cell adhesion in bone morphogenetic protein-7-treated NG108-15 cells. In L1-expressing NIH/3T3 cells, SAL antagonism was reversible and could be overcome by increasing concentrations of ethanol. In contrast, NAP antagonism was irreversible and could not be overcome by increasing agonist concentration. Two scrambled NAP peptides (ASPNQPIV and PNIQVASP) were not antagonists at concentrations as high as 10 Ϫ7 M. Thus, two structurally unrelated classes of compounds, alcohols and small polypeptides, share two common actions: antagonism of ethanol inhibition of L1-mediated cell adhesion and prevention of ethanol teratogenesis. These findings support the hypothesis that ethanol inhibition of L1 adhesion contributes to ethanol teratogenesis.

-L1) and mutated L1, we found that cysteine substitution of both residues (E33C/Y418C-L1) significantly increased L1 adhesion above levels observed for WT-L1 or the single cysteine substitutions E33C-L1 or Y418C-L1. The reducing agent ␤-mercaptoethanol (␤ME) reversibly decreased the adhesion of E33C/ Y418C-L1, but had no effect on WT-L1, E33C-L1, or Y418C-L1. Thus, disulfide bond formation occurs between Cys-33 and Cys-418, confirming both the close proximity of these residues and the importance of Ig1-Ig4 interactions in L1 adhesion. Maximal ethanol inhibition of cell adhesion was significantly lower in cells expressing E33C/Y418C-L1 than in those expressing WT-L1, E33C-L1, or Y418C-L1. Moreover, the effects of ␤ME and ethanol on E33C/Y418C-L1 adhesion were non-additive. The cutoff for alcohol inhibition of WT-L1 adhesion was between 1-butanol and 1-pentanol. Increasing the size of the alcohol binding pocket by mutating Glu-33 to Ala-33, increased the alcohol cutoff from 1-butanol to 1-decanol. These findings support the hypothesis that alcohol binding within a pocket bordered by Glu-33 and Tyr-418 inhibits L1 adhesion by disrupting the Ig1-Ig4 interaction.Alcohol exposure during pregnancy is the leading cause of preventable mental retardation in the Western world (1, 2).Depending on timing, dose, and duration of exposure, alcohol causes a range of facial and brain dysmorphology, growth retardation, and cognitive, neurological, and behavioral abnormalities, referred to as fetal alcohol spectrum disorders (FASD) 2 (3). Alcohol is a weak, pleiotropic drug that disrupts fetal development through a variety of mechanisms (4, 5). One potentially important target molecule for alcohol is the L1 neural cell adhesion molecule (CAM), a developmentally critical protein (6 -8).Children with mutations in the gene for L1 have brain lesions that resemble those of children with FASD, including hydrocephalus, agenesis, or hypoplasia of the corpus callosum, and cerebellar dysplasia (7, 9, 10). Concentrations of ethanol attained after one drink inhibit the adhesion of L1 expressed in fibroblasts, neural cell lines, and cerebellar granule neurons (CGN) (6, 7). Furthermore, ethanol inhibits L1-mediated neurite outgrowth in CGNs with similar potency to its inhibition of L1 adhesion (11, 12). Finally, drugs that block ethanol inhibition of L1 adhesion also prevent ethanol teratogenesis in mouse embryos (13)(14)(15)(16)(17)(18).L1 is an immunoglobulin transmembrane glycoprotein (8). The extracellular domain (ECD) includes six Ig domains and five fibronectin III repeats. The first four Ig domains (L1 Ig1-4 ) comprise the minimal elements required for L1 adhesion (19,20). The crystal structure of neurofascin, a member of the L1 family of CAMs, and homology modeling with related CAMs suggest that L1 Ig1-4 folds into a horseshoe structure, with Ig1 in apposition to Ig4 and Ig2 in apposition to (Fig. 6A). Electron microscopy has captured both a horseshoe and an extended conformation of L1 , and functional studies suggest that the hors...

There is a genetic contribution to fetal alcohol spectrum disorders (FASD), but the identification of candidate genes has been elusive. Ethanol may cause FASD in part by decreasing the adhesion of the developmentally critical L1 cell adhesion molecule through interactions with an alcohol binding pocket on the extracellular domain. Pharmacologic inhibition or genetic knockdown of ERK2 did not alter L1 adhesion, but markedly decreased ethanol inhibition of L1 adhesion in NIH/3T3 cells and NG108-15 cells. Likewise, leucine replacement of S1248, an ERK2 substrate on the L1 cytoplasmic domain, did not decrease L1 adhesion, but abolished ethanol inhibition of L1 adhesion. Stable transfection of NIH/3T3 cells with human L1 resulted in clonal cell lines in which L1 adhesion was consistently sensitive or insensitive to ethanol for more than a decade. ERK2 activity and S1248 phosphorylation were greater in ethanol-sensitive NIH/3T3 clonal cell lines than in their ethanol-insensitive counterparts. Ethanol-insensitive cells became ethanol sensitive after increasing ERK2 activity by transfection with a constitutively active MAP kinase kinase 1. Finally, embryos from two substrains of C57BL mice that differ in susceptibility to ethanol teratogenesis showed corresponding differences in MAPK activity. Our data suggest that ERK2 phosphorylation of S1248 modulates ethanol inhibition of L1 adhesion by inside-out signaling and that differential regulation of ERK2 signaling might contribute to genetic susceptibility to FASD. Moreover, identification of a specific locus that regulates ethanol sensitivity, but not L1 function, might facilitate the rational design of drugs that block ethanol neurotoxicity. P renatal alcohol exposure causes fetal alcohol spectrum disorders (FASD) in up to 2-5% of school-age children and is the leading preventable cause of mental retardation in the Western world (1, 2). The prevalence and presentation of FASD are influenced by the quantity, frequency, and timing of drinking and are modified by a variety of environmental, nutritional, epigenetic, and genetic factors (3-7). The observation that there is greater concordance for fetal alcohol syndrome (FAS) in monozygotic twins than in dizygotic twins suggests that there are susceptibility genes for FASD (8); however, their identification remains elusive. The identification of molecular pathways that regulate sensitivity to ethanol teratogenesis would be helpful in the search for FASD susceptibility genes.One potentially important target of ethanol in the pathogenesis of FASD is the developmentally critical immunoglobulin neural cell adhesion molecule, L1. The homophilic binding of L1 molecules on adjacent cells mediates neuronal migration, axon guidance, and axon fasciculation (9)-developmental events that are disrupted in FASD (10-12). Mutations in the human L1 gene cause brain lesions and neurological abnormalities. Some of these mutations also disrupt L1 homophilic binding (13-16). We noted that brain lesions in children with FASD resemble those of child...