Michael F. Wilkemeyer

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

NAPVSIPQ (NAP), an active fragment of the glial-derived activity-dependent 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.

Octanol antagonism of ethanol teratogenesis

Alcohol may cause birth defects in part by disrupting the developmentally critical L1 cell adhesion molecule. Because 1‐octanol antagonizes ethanol inhibition of L1‐mediated cell adhesion, we asked whether 1‐octanol also prevents ethanol teratogenicity. L1 was detected in control mouse embryos as early as gestational day 8 (GD8), an early stage of vulnerability to ethanol teratogenesis. Culture of GD8 embryos with ethanol increased apoptotic cell death and decreased numbers of somite pairs. 1‐Octanol markedly reduced both ethanol effects. The paradoxical inhibition of ethanol teratogenicity by a longer chain 1‐alcohol suggests a strategy for developing compounds that prevent alcohol‐related birth defects.

Peptide-mediated protection from ethanol-induced neural tube defects.

Ethanol inhibition of L1-mediated cell adhesion may contribute to the spectrum of neurological, behavioral and morphological abnormalities associated with prenatal ethanol exposure. We showed previously that the neuroprotective peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL) antagonize ethanol inhibition of L1 adhesion and prevent ethanol-induced growth retardation in mouse whole embryo culture. Here we ask whether NAP and SAL also prevent ethanol-induced major malformations of the nervous system. Gestational day 8.0 (3–5 somites) C57BL/6J mouse embryos were grown for 6 h in control medium, 100 mM ethanol and 10–10M peptides and then maintained for an additional 20 h in control medium. At the end of the culture period, only embryos having 18–19 somite pairs were examined and compared for the degree of neural tube closure. Ethanol exposure resulted in neural tube defects (NTDs) consistent with total dysraphia and anencephaly. Co-incubation with ethanol and L-NAP (all L-amino acids), D-NAP (all D-amino acids) or SAL significantly increased the percentage of embryos that had begun to close their neural folds at the level of the forebrain/midbrain junction or that had progressed beyond this stage of closure. P7A-NAP (NAPVSIAQ), which lacks neuroprotective activity, but retains activity as an antagonist of ethanol inhibition of L1 adhesion, was effective in preventing ethanol-induced NTDs. In contrast, I6A-NAP (NAPVSAPQ), which shows reduced efficacy as an ethanol antagonist but retains its neuroprotective efficacy, did not significantly diminish the induction of NTDs by ethanol. These findings demonstrate the ability of NAP and SAL to prevent ethanol-induced NTDs and support the hypothesis that ethanol teratogenesis is caused in part by ethanol inhibition of L1-mediated cell adhesion.

An alcohol binding site on the neural cell adhesion molecule L1

Prenatal ethanol exposure causes fetal alcohol spectrum disorders (FASD) in part by disrupting the neural cell adhesion molecule L1. L1 gene mutations cause neuropathological abnormalities similar to those of FASD. Ethanol and 1-butanol inhibit L1-mediated cell–cell adhesion (L1 adhesion), whereas 1-octanol antagonizes this action. To test the hypothesis that there are alcohol binding sites on L1, we used 3-azibutanol and 3-azioctanol, the photoactivatable analogs of 1-butanol and 1-octanol, to photolabel the purified Ig1–4 domain of human L1 (hL1 Ig1–4). 3-Azibutanol (11 mM), like ethanol, inhibited L1 adhesion in NIH/3T3 cells stably transfected with hL1, whereas subanesthetic concentrations of 3-azioctanol (14 μM) antagonized ethanol inhibition of L1 adhesion. 3-Azibutanol (100–1,000 μM) and 3-azioctanol (10–100 μM) photoincorporated into Tyr-418 on Ig4 and into two adjacent regions in the N terminus, Glu-33 and Glu-24 to Glu-27. A homology model of hL1 Ig1–4 (residues 33–422), based on the structure of the Ig1–4 domains of axonin-1, suggests that Glu-33 and Tyr-418 hydrogen-bond at the interface of Ig1 and Ig4 to stabilize a horseshoe conformation of L1 that favors homophilic binding. Furthermore, this alcohol binding pocket lies within 7 Å of Leu-120 and Gly-121, residues in which missense mutations cause neurological disorders similar to FASD. These data suggest that ethanol or selected mutations produce neuropathological abnormalities by disrupting the domain interface between Ig1 and Ig4. Characterization of alcohol agonist and antagonist binding sites on L1 will aid in understanding the molecular basis for FASD and might accelerate the development of ethanol antagonists.

Characterization of Ethanol-Sensitive and Insensitive Fibroblast Cell Lines Expressing Human L1

Abstract: Ethanol inhibits L1‐mediated cell‐cell adhesion in fibroblast cell lines stably transfected with human L1. Here we show that this action of ethanol is present in only a subset of transfected NIH/3T3 and L cell clonal cell lines. All L1‐expressing cell lines had higher levels of cell adhesion than cell lines transfected with empty vector. In all ethanol‐sensitive cell lines, L1‐mediated adhesion was inhibited by ethanol (IC50 5–10 mM), 2 mM butanol, but not 5 mM pentanol. In contrast, ethanol‐insensitive cell lines were not inhibited by up to 200 mM ethanol, 2 mM butanol, or 5 mM pentanol. Ethanol sensitivity or insensitivity was a stable property of each cell line and was not associated with differences in electrophoretic mobility, abundance, or cell surface localization of L1. Fab fragments prepared from anti‐L1 polyclonal antisera inhibited cell adhesion only in the ethanol‐sensitive cell lines. These data suggest that L1 may exist in an alcohol‐sensitive or an alcohol‐insensitive state that may be governed by host cell factors.

Mitogen-activated protein kinase modulates ethanol inhibition of cell adhesion mediated by the L1 neural cell adhesion molecule

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.

Fetal alcohol effects : Potential treatments from basic science

This article represents the proceedings of a symposium presented at the 2004 annual meeting of the International Society for Biomedical Research on Alcoholism, held in Mannheim, Germany. The presentations were as follows: 1) “Antioxidants Prevent Ethanol-Induced Cell Death in Developing Brain and in Cultured Neural Cells” by M. Pascual, M. C. Garcia-Minguillan, and Consuelo Guerri; 2) “Rational Development of Ethanol Antagonists” by Michael E. Charness and Michael F. Wilkemeyer; 3) “Choline Supplementation as a Treatment for Fetal Alcohol Effects” by Jennifer D. Thomas and Hector D. Dominguez; 4) “Cerebellar and Cortical Plasticity After Neonatal Alcohol Exposure: Model of Intervention” by Anna Y. Klintsova, Charles R. Goodlett, and William T. Greenough; and 5) “Circadian Rhythms in Prenatally Ethanol-Exposed Rats” by Hiromi Sakata-Haga.