Differential effects of ethanol antagonism and neuroprotection in peptide fragment NAPVSIPQ prevention of ethanol-induced developmental toxicity

Abstract

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...

An alcohol binding site on the neural cell adhesion molecule L1

“…However, 14 M 3-azioctanol completely antagonized ethanol inhibition of L1 adhesion. The effects of 3-azibutanol and 3-azioctanol are comparable to those reported for other alcohol antagonists and antagonists (14). These data establish that 3-azibutanol and 3-azioctanol are suitable photolabels to probe alcohol binding sites that modulate L1 adhesion.…”

“…Within the 1-alcohol series, methanol through 1-butanol show progressively increasing potency, whereas 1-pentanol and higher 1-alcohols are inactive, consistent with a size limitation for alcohol interactions with a hydrophobic site on L1 (4). Also, the agonist activity of 1-butanol, the most potent alcohol agonist, is eliminated by chemical modifications that constrain movement between carbon 3 and 4 or sterically hinder its hydroxyl group (13,14). Finally, 1-octanol and a number of alcohols and small peptides that lack agonist activity block ethanol inhibition of L1 adhesion (10,11,(13)(14)(15).…”

“…The concentrations of ethanol that affect L1 function are among the lowest reported for any ethanol target (4,8), and the ability of other alcohols to inhibit L1 adhesion is subject to surprisingly strict structural requirements (13,14). Within the 1-alcohol series, methanol through 1-butanol show progressively increasing potency, whereas 1-pentanol and higher 1-alcohols are inactive, consistent with a size limitation for alcohol interactions with a hydrophobic site on L1 (4).…”

“…Also, the agonist activity of 1-butanol, the most potent alcohol agonist, is eliminated by chemical modifications that constrain movement between carbon 3 and 4 or sterically hinder its hydroxyl group (13,14). Finally, 1-octanol and a number of alcohols and small peptides that lack agonist activity block ethanol inhibition of L1 adhesion (10,11,(13)(14)(15). In mouse embryos several of these ethanol antagonists also prevent ethanol-induced apoptosis, growth retardation, and delayed closure of the neural tube (9-12, 16, 17).…”

Linking acquired neurodevelopmental disorders to defects in cell adhesion

“…Within the latter group are proteins, such as neural cell adhesion molecule, L1, and deleted in colon cancer (DCC), that are prominently expressed in the nervous system (2). In this issue of PNAS, investigators from the laboratory of Dr. Michael Charness (3) report findings that link these molecules to an important, preventable mental retardation syndrome.…”