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. Author manuscript; available in PMC: 2021 Apr 1.
Published in final edited form as: Biol Psychiatry. 2019 Sep 5;87(7):656–665. doi: 10.1016/j.biopsych.2019.08.020

Neuroprotective Peptide NAPVSIPQ Antagonizes Ethanol Inhibition of L1 Adhesion by Promoting the Dissociation of L1 and Ankyrin-G

Xiaowei Dou a, Jerry Y Lee a, Michael E Charness a,b,1
PMCID: PMC7056560  NIHMSID: NIHMS1539270  PMID: 31640849

Abstract

Background:

Ethanol causes developmental neurotoxicity partly by blocking adhesion mediated by the L1 neural cell adhesion molecule. This action of ethanol is antagonized by femtomolar concentrations of the neuropeptide NAPVSIPQ (NAP), an active fragment of activity dependent neuroprotective peptide (ADNP). How femtomolar concentrations of NAP antagonize millimolar concentrations of ethanol is unknown. L1 sensitivity to ethanol requires L1 association with ankyrin-G; therefore, we asked whether NAP promotes the dissociation of ankyrin-G and L1.

Methods:

L1-ankyrin-G association was studied using immunoprecipitation, Western blotting, and immunofluorescence in NIH/3T3 cells transfected with wild-type and mutated human L1 genes. Phosphorylation of the ankyrin-binding motif in the L1 cytoplasmic domain (L1-CD) was studied after NAP treatment of intact cells, rat brain homogenates, and purified protein fragments.

Results:

Femtomolar concentrations of NAP stimulated the phosphorylation of tyrosine-1229 (L1-Y1229) at the ankyrin binding motif of the L1-CD, leading to the dissociation of L1 from ankyrin-G and the spectrin-actin cytoskeleton. NAP increased the association of L1 and EphB2 and directly activated EphB2 phosphorylation of L1-Y1229. These actions of NAP were reproduced by P7A-NAP, a NAP variant that also blocks the teratogenic actions of ethanol, but not by I6A-NAP, which does not block ethanol teratogenesis as potently. Finally, knockdown of EphB2 prevented ethanol inhibition of L1 adhesion in NIH/3T3 cells.

Conclusions:

NAP potently antagonizes ethanol inhibition of L1 adhesion by stimulating EphB2 phosphorylation of L1-Y1229. EphB2 plays a critical role in synaptic development; its potent activation by NAP suggests that ADNP may mediate synaptic development partly by activating EphB2.

Keywords: L1, NAP, ethanol, EphB2, phosphorylation, Y1229

Introduction

Fetal alcohol spectrum disorders (FASD) are highly prevalent causes of developmental disability (1,2). Brain lesions of FASD resemble those of L1 syndrome, a disorder caused by mutations in the gene for the developmentally critical L1 neural cell adhesion molecule (3). This phenotypic similarity may be a consequence of the ability of ethanol to inhibit L1 adhesion at concentrations attained after just one drink (3). Ethanol interacts with an alcohol binding site in the extracellular domain of L1 (L1-ECD) at the interface between the Ig1 and Ig4 domains (4, 5), a location essential for stabilizing a horseshoe conformation of L1 that favors L1 homophilic binding (6). The alcohol binding site discriminates the size and shape of diverse alcohols, and several alcohols antagonize ethanol inhibition of L1 adhesion (7). One such alcohol, 1-octanol, also interacts with the alcohol binding site (4) and prevents ethanol-induced growth retardation in mouse embryos at micromolar concentrations (8).

A second class of ethanol antagonists is exemplified by the peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL). NAP and SAL are active fragments of the developmentally important activity dependent neuroprotective protein (ADNP) and activity-dependent neurotrophic factor (ADNF), respectively (9, 10). NAP and SAL both antagonize alcohol inhibition of L1 adhesion (11–13), and NAP protects against alcohol-induced growth retardation and delayed closure of the neural tube at femtomolar concentrations (12–14). ADNP and NAP may even protect against excessive alcohol consumption (15). Although NAP potently protects against a broad array of other CNS insults, NAP appears to block ethanol teratogenesis primarily by antagonizing ethanol inhibition of L1 adhesion, rather than through its broad neuroprotective actions (12). How femtomolar concentrations of NAP antagonize the actions of millimolar concentrations of ethanol is unknown. This stoichiometry strongly suggests that NAP acts through a catalytic mechanism, rather than by interacting directly with the alcohol binding site on the L1-ECD.

L1 sensitivity to ethanol requires the association of L1 with ankyrin-G and the spectrin-actin cytoskeleton (16, 17). Linkage of L1 to these cytoskeletal elements constrains the lateral mobility of L1 within the cell membrane (18), a change that is postulated to stabilize a conformation of the L1-ECD that favors the interaction of ethanol with the alcohol binding pocket (17). In contrast, dissociation of L1 from ankyrin-G and the spectrin-actin cytoskeleton increases the lateral mobility of L1, which may stabilize a conformation of the L1-ECD that excludes ethanol from the alcohol binding pocket. L1 association with ankyrin-G requires dephosphorylation of Y1229 within the L1 cytoplasmic domain (L1-CD) (19, 20). Therefore, we asked whether NAP antagonizes ethanol inhibition of L1 adhesion by activating the phosphorylation of L1-Y1229, thereby promoting the dissociation of L1 and ankyrin. Here we show that NAP, but not octanol, potently induces the dissociation of L1 and ankyrin-G by activating EphB2, a kinase that phosphorylates L1-Y1229 (21, 22). Moreover, knockdown of EphB2 abolishes NAP antagonism of ethanol inhibition of L1 adhesion.

Methods and Materials

Reagents

The following antibodies were utilized: from Santa Cruz Biotech: goat polyclonal antibody (pAb) against the L1-CD (SC-1508, RRID: AB_631086) (17, 23, 24), measuring total L1; mAb against the L1-ECD (SC-53386, UJ127, RRID: AB_628937) (25), measuring total L1; rabbit pAb against C-terminus of ankyrin-G (SC-28561, RRID: AB_633909) (17, 26, 27); mAb against L1 (5G3) was produced by Maine Biotech Services (28); mAb against spectrin (SC-46696, RRID: AB_671135); goat pAb against actin (SC-1616 , RRID: AB630836); mAB against EphB2 (SC-130752, RRID: AB_2099957); rabbit pAb against Src (SC-18, RRID:AB_631324). Antibody mAB anti-Phosphotyrosine (ab10321, RRID: AB_297058) was from Abcam; HRP-conjugated secondary antibodies against mouse (115-035-062, RRID: AB_2338504), rabbit (711-035-152, RRID: AB_10015282), and Goat (705-035-003, RRID: AB_2340390) were from Jackson ImmunoResearch Laboratories. Recombinant Human EphB2 Protein (PV3625) and Kinase assay buffer (PV3189), goat anti-mouse IgG conjugated with Alexa Fluor-546 (A-11003, RRID: AB_2534071) and goat anti-rabbit IgG conjugated with Alexa Fluor 488 (R37120, RRID: AB_2556548) were from Thermo Fisher Scientific. mAB to β-tubulin was from Cell Signaling Technology (#86298, RRID:AB_2715541). NAP peptides were custom synthesized by New England Peptide Inc., and FIGQY peptides were synthesized by Genscript USA Inc.

Cell Culture

NIH/3T3 clonal cell line 2A2-L1s (ethanol-sensitive) stably expressing human L1 (hL1) were employed to evaluate the effects of experimental manipulations on wild-type L1 (17), and NIH/3T3 cells transiently transfected with L1 mutations in the ankyrin binding region were used to study L1-ankyrin interactions, as described (20). Cells were cultured in DMEM plus 10% bovine serum (BS) at 37°C with 10% CO2 atmosphere, as described (17).

Immunocytochemistry

NIH/3T3 cells were plated in 9 mm petri dishes in DMEM supplemented with 10% BS and transiently transfected with wild type L1 or L1-Y1229F. After 48 hours of culture, cells were treated with 10−12 M NAP for one hour and fixed in 4% paraformaldehyde for 30 minutes, blocked with PBS supplemented with 5% BS, and incubated overnight at 4°C with L1 mAb 5G3 (5) and rabbit pAb against ankyrin-G in PBS plus 5% BS. Cells were washed three times with PBS and incubated with goat anti-mouse IgG conjugated with Alexa Fluor-546 and goat antirabbit IgG conjugated with Alexa Fluor 488 in PBS/BS. Cells were washed again with PBS and fixed in paraformaldehyde. Images were captured using a Nikon Eclipse Ti microscope, and the co-localization efficiency of L1 and ankyrin-G were calculated using NIS-Elements Microscope Imaging Software (V4.30) (29).

Immunoprecipitation and Western Blot Analysis

Immunoprecipitation and Western blot analysis were carried out as described (17). NIH/3T3 cells were lysed in NP40 lysis buffer plus Halt Protease and Phosphatase Inhibitor Cocktail (#1862495, Thermo Scientific). For L1 immunoprecipitation, whole cell lysates were incubated with mAb 5G3 (Maine Biotech Services) at 4°C for 2 - 4 hours, and protein A-agarose beads were added to precipitate the antigen-antibody complex. Images of protein bands were acquired on an Amersham Imager 600, and densitometry was quantified using Image J (30) (32). All data were normalized to values in 2A2-L1s cells or L1-WT transfected cells and plotted as mean ± SEM. The specificity of the L1 and ankyrin antibodies for immunoprecipitation and Western blotting is well established in the literature (17, 23, 31) and is further demonstrated in Supp Figs 1 and 2.

siRNA Transfection of 2A2-L1s cells

Cells were transfected with EphB2-siRNA (Santa Cruz, SC-39950), using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions (16). Scrambled siRNA was used as a control (SC-37007, Santa Cruz, Inc). Forty-eight hours after transfection, cells were harvested for cell aggregation assays, Western blots, and co-immunoprecipitation analysis (16). Levels of protein expression in Western blots were normalized to values from untreated 2A2-L1s cells.

Preparation of Rat Brain Lysate

Rat brain lysates were prepared from whole brain homogenates of Sprague Dawley rat pups (Charles River Breeding, Worcester, MA) at postnatal day 10. Pups were euthanized in a CO2 chamber. The brain was removed, the cerebellum was dissected away, and the remaining tissue was frozen in liquid nitrogen. Thawed brain tissue was homogenized in NP40 lysis buffer plus protease and phosphatase inhibitors by repeated passage through a 20-gauge needle. After centrifugation, the supernatant was removed for co-immunoprecipitation experiments. The handling, care, and treatment of animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of VA Boston Healthcare System.

Cell Aggregation Assay

2A2-L1s or 3C3-L1i, cells stably expressing hL1 or NIH/3T3 cells transiently transfected with L1 constructs were harvested for cell aggregation assays, as described (5, 16). Cell aggregation assays were performed in the absence and presence of 100 mM ethanol, a maximally effective concentration. Although the half maximal concentration for ethanol inhibition of L1 adhesion is ~ 5 mM (3), we used high concentrations of ethanol to study the antagonist effects of NAP. Ethanol inhibition of L1 adhesion was normalized to values obtained in 2A2-L1s cells or the values obtained in cells transfected with wild-type L1 (L1-WT).

Phosphorylation of FIGQY-Peptide

Phosphorylation of CNEDGSFIGQYSGKKE (FIGQY-peptide) by EphB2 kinase, whole cell lysate or rat brain was performed on peptides conjugated to agarose beads according to the manufacturer’s manual (Pierce NHS-activated dry agarose beasds, cat. No. 26196, Thermofisher). Purified EphB2 (0.4 μg) protein or whole cell lysates (50 μg protein) were added to 2 μg of peptide-coated agarose beads in the absence or presence of NAP for the desired time in 1x kinase buffer (Thermo Fisher, cat# PV3189) supplemented with 2 mM ATP at a final volumn of 660 μl. The reaction was stopped by adding 1 ml PBS plus 2 mM EDTA, and the particulate and supernatant fractions were separated by centrifugation at 3000 rpm for 5 min at 4°C. The pellet was washed three times with 1 ml PBS, and incubated for one hour with anti-phosphotyrosine antibody ab10321 (Abcam) in 500 μl PBS plus 5% BSA. After three washes, the pellet was denatured at 95°C in 1X reducing SDS-sample buffer for Western blot analysis. Blotted membranes were incubated with HRP-conjugated secondary antibodies against mouse (115-035-062, RRID: AB_2338504), and the level of phosphotyrosine antibody retained on agarose beads was used as an index of tyrosine phosphorylation of FIGQY-peptide.

Statistical Analysis

Data are expressed as mean ± SEM, except as noted. Statistical analysis was performed by using the ANOVA and two-tailed paired t-tests using Prism 5 (Graphpad Software). Statistical significance was defined as P < 0.05. Levels of statistical significance were designated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.

Results

NAP potently reduces the association of L1 with ankyrin-G and the spectrin-actin cytoskeleton

We evaluated the effects of NAP on L1 coupling to ankyrin-G using immunoprecipitation and Western blotting from NIH/3T3 cells transiently transfected with wild-type human L1 (L1-WT). NAP caused a dose-dependent reduction in the association of WT-L1 with ankyrin-G (Fig. 1A). Significant inhibition was observed at NAP concentrations of 100 fM with maximal effects at 100 nM. Treatment with 10−9 M NAP also significantly decreased the association of L1 with spectrin and actin (Fig 1B). Interestingly, 100 μM 1-octanol, a maximal antagonist concentration, had no significant effect on L1-ankyrin-G association (Fig 2A). These findings suggest that NAP and 1-octanol antagonize ethanol inhibition of L1 adhesion through different mechanisms.

Figure 1.

Figure 1.

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NAP effect on the interaction of L1 with ankyrin-G (AnkG) and the spectrin-actin cytoskeleton. (A) L1 was immunoprecipitated using mAb 5G3 from whole cell lysates of NIH/3T3 cells expressing human L1 (2A2-L1s) that had been treated with NAP at various concentrations; co-immunoprecipitated proteins were separated and blotted with antibodies to L1 or AnkG. Ankyrin-G was detected as a band of approximately 190 kD, but occasionally, a second band was observed at approximately 150 kD, likely representing a splice variant (60). L1 was detected as a doublet at 210 kD and 190 kD. Protein band densities were normalized to values for total L1, and values were expressed as mean ± SEM % of control from 6 - 14 independent experiments (F = 6.59, p < 0.0001). (B) L1 was immunoprecipitated using mAb 5G3 from whole cell lysates of 2A2-L1s cells treated with 10−9 M NAP, and co-immunoprecipitated proteins were separated and blotted with antibodies to L1, AnkG, spectrin, or actin. Protein band densities were normalized to values for total L1, and values were expressed as mean ± SEM % control from 5-13 independent experiments (*t=4.4, *p=0.01; *t=2.6; *p=0.03; ***t=5.9, ***p=0.0004).

Figure 2.

Figure 2.

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L1 association with AnkG and ethanol inhibition of L1 adhesion in cells expressing wild-type L1 (L1-WT) or L1 in which Y1229 was mutated to phenylalanine (L1-Y1229F). (A) NIH/3T3 cells were transfected with L1-WT or L1-1229F in the absence and presence of 10−12 M NAP or 100 μM octanol. L1 was immunoprecipitated from whole cell lysates using mAb 5G3, and coimmunoprecipitated proteins were separated and blotted with antibodies to L1 and AnkG. Densities of AnkG bands were normalized to values for L1 in corresponding experiments and then expressed as a percentage of values in untreated cells expressing L1-WT. Shown are mean ± SEM % association of L1 and AnkG from 4 - 13 independent experiments (F =2.94, p< 0.05); *** t=7.18, p < 0.001. (B) Co-localization of L1 (red) and AnkG (green) immunostaining in L1-WT and L1-Y1229F-expressing cells treated for 1 hour in the absence and presence of 10−12 M NAP. Mander’s overlap coefficient indicated significant differences in the co-localization of L1 and AnkG under various experimental conditions (F = 3.2, p<0.05); ** t=5.26, p <0.01, n = 14-15. (C) and (D) The L1-Y1229F mutation abolished NAP (10−9 M) and okadaic acid (100 μM) antagonism of ethanol inhibition of L1 adhesion. NIH/3T3 cells were transiently transfected with L1-WT and L1-Y1229F. Cells were treated with 10−9 M NAP or 100 μM okadaic acid for one hour, and cells were harvested for cell aggregation assays performed in the absence and presence of 100 mM ethanol. Ethanol inhibition of L1 adhesion in L1-Y1229F expressing cells was expressed as a percentage of that obtained in L1-WT expressing cells (47.7 ± 4.4%). Shown are mean ± SEM relative levels of ethanol inhibition in (C) (F = 16.77; p < 0.0001); *** t=5.938, p*** = 0.0000, n = 12-32; (D) (F = 3.90; p < 0.05); ** t = 6.93 , p** =0.002, n = 5.

The L1-Y1229F mutation blocks the actions of NAP on L1

Ankyrin-G binds to L1 at the consensus sequence FIGQY1229 in the L1-CD, and this binding requires dephosphorylation of L1-Y1229 (18, 21). If NAP acts by inducing the dissociation of L1 and ankyrin-G, then stabilizing the association of L1 and ankyrin-G by preventing the phosphorylation of L1-Y1229 might eliminate NAP’s antagonist activity. Mutation of Y1229 to phenylalanine (Y1229F) prevents phosphorylation at the 1229 site and stabilizes ankyrin-G association with L1 (17, 20, 33, 34). We used this mutation to test whether NAP retains its antagonist properties when the residue at the 1229 position can no longer be phosphorylated. Immunoprecipitation studies showed that NAP had no effect on the association of ankyrin-G with L1-Y1229F, in contrast to its actions on L1-WT (Fig 2A). The effects of NAP on the co-localization of L1 and ankyrin-G were also visualized using immunofluorescence staining of L1 and ankyrin G. As expected, NAP significantly reduced the co-localization of L1 and ankyrin-G in cells transfected with L1-WT but had no effect on their co-localization in cells transfected with L1-Y1229F (Fig 2B). Of note, both NAP and the phosphatase inhibitor okadaic acid blocked ethanol inhibition of L1 adhesion in cells transfected with L1-WT but not in cells transfected with L1-Y1229F (Fig 2C,D). These data imply that NAP promotes the dissociation of L1 and ankyrin-G and antagonizes ethanol inhibition of L1 adhesion by increasing the phosphorylation of L1-Y1229.

Lysates from NAP-treated NIH/3T3 cells or rat brain increase tyrosine phosphorylation of FIQGY-peptide

We next sought evidence that NAP increases the phosphorylation of L1-Y1229. We were unable to raise an antibody that selectively recognizes phospho- or dephospho-L1-Y1229 (21). Therefore, we used an anti-phosphotyrosine (pY) antibody to determine the phosphorylation state of a synthetic 16-amino acid peptide fragment of the L1-CD comprising a single tyrosine residue located within the FIGQY ankyrin binding site (FIGQY-peptide) (35). 2A2-L1s is an ethanol-sensitive NIH/3T3 clonal cell line that stably expresses human L1 (16, 17, 34, 36). We exposed 2A2-L1s cells to 10−9 M NAP for various periods of time and then incubated whole cell lysates from NAP-treated 2A2-L1s cells with FIGQY-peptide conjugated to agarose beads. Levels of phosphorylated FIGQY-peptide were determined by Western blot analysis. NAP increased tyrosine phosphorylation of FIGQY-peptide more than two-fold, with peak effects occurring after 10 minutes of exposure (Fig 3A). A 10-minute NAP exposure caused a dose dependent increase in FIGQY-peptide phosphorylation, with significant effects observed at 10−16 M and half maximal effects at approximately 3x10−15 M (Fig 3B). NAP also caused a greater than 2-fold increase in FIGQY-peptide phosphorylation in lysates from rat cerebral cortex (Fig 3C). These findings support the hypothesis that NAP acts by increasing the phosphorylation of L1-Y1229.

Figure 3.

Figure 3.

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NAP effect on tyrosine phosphorylation (pY) of the 16-amino acid FIQGY-peptide fragment derived from the L1-CD, containing a single tyrosine. (A) 2A2-L1s cells were incubated in the absence and presence of 10−9 M NAP, and extracted cell lysates were incubated with FIGQY-peptide conjugated to agarose beads at room temperature for the indicated time (Methods). FIGQpY-peptide was measured using mouse anti-pY mAb (PY20). The magnitude of pY in FIGQY-peptide was quantified by densitometric analysis of anti-pY antibody-agarose bead bands separated by SDS-PAGE. Densities of FIGQpY-peptide bands were normalized to values obtained at time 0. Shown are mean ± SEM % FIGQpY-peptide compared to values at time 0 from 5 independent experiments (F =9.84, p< 0.0001). Peak phosphorylation occurred at 10 minutes following NAP treatment of intact cells. (B) Dose response curve for NAP phosphorylation of FIQGY-peptide by cell lysate from NAP-treated 2A2-L1s cells. Reactions were carried out for 10 min at room temperature in the presence of the indicated concentrations of NAP. pY levels were normalized to values in cells that were not treated with NAP (lane 0). Shown is a representative gel from 12 experiments. Densitometry was obtained from 7 – 12 independent experiments (F = 2.48, p < 0.05). (C) Lysate of cerebral cortex from post-natal day 10 rat pups was incubated in the absence and presence of 10−12 M NAP for 10 minutes. NAP treatment of rat brain lysates significantly increased phosphorylation of FIGQY-peptide (n=7; **t=2.888, ** p < 0.01). (D) 2A2-L1s cells were incubated for 15 minutes at room temperature in the absence and presence of 10−9 M NAP and 20 uM PP2, and cell lysates were then incubated with FIGQY-peptide to determine the effect of drug treatments on pY; *t=3.45, *p=0.010, n=8; **t=3.55, **p=0.0085, n=8.

NAP Stimulates the Association of EphB2 and L1

The kinase EphB2 phosphorylates the FIGQY consensus sequence of L1 (21, 22). We next asked whether NAP stimulates the association of EphB2 and L1 in NIH/3T3 cells transiently transfected with L1-WT. Treatment of these cells with 10−9 M NAP significantly increased the association of L1 with EphB2, as determined by immunoprecipitation (Fig 4A). Hence, NAP treatment of intact cells brings EphB2 and L1 into proximity, thereby increasing the probability of EphB2 phosphorylation of L1-Y1229.

Figure 4.

Figure 4.

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NAP activation of EphB2 phosphorylation of L1. (A) Effect of NAP on association of L1 with EphB2. L1 was immunoprecipitated with mAb 5G3 from 2A2-L1s cells in the absence and presence of 10−9 M NAP, and coimmunoprecipitated proteins were separated and blotted with antibodies to L1 and EphB2. Densities of EphB2 bands were normalized to those for L1, and values for NAP treatment were expressed as a percentage of control values. Shown is the mean ± SEM % increase in L1 association with EphB2 following NAP treatment derived from 8-9 independent experiments; *t=3.17,*p <0.05, n = 8. (B) Dose-dependent stimulation by NAP of tyrosine phosphorylation of FIGQY-peptide by recombinant EphB2 (F = 2.45, p<0.05). pY levels following NAP treatment were normalized to control values (0 NAP). Shown is the mean ± SEM % increase in pY levels following treatment with the indicated concentrations of NAP derived from 6-9 independent experiments (C) Stimulation of EphB2 phosphorylation of L1 by 10−9 M NAP and P7A-NAP (P7A), but not by 10−9 M I6A-NAP (I6A), SAL, or octanol (Oct). Shown is a representative gel and densitometric analysis from 7 independent experiments. pY levels for each drug treatment were normalized to values obtained in the absence of drugs (Control) (F = 2.84, p<0.05); **t = 4.01, **p = 0.0070; *t= 3.01, *p=0.0235, n=7.

NAP and its active homologues stimulate EphB2 phosphorylation of FIGQY-peptide

To determine whether NAP directly activates EphB2, we incubated purified EphB2 with FIGQY-peptide in the absence and presence of various concentrations of NAP. NAP caused a dose-dependent increase in EphB2 phosphorylation of FIGQY-peptide, with half maximal effects occurring between 10 and 100 fM (Fig 4B). NAP did not increase phosphorylation of FIGQY-peptide in the absence of EphB2.

Dai et al. (21) showed that EphB2 phosphorylation of L1-Y1229 in HEK293 cells is reduced by PP2, a Src family kinase (SFK) inhibitor, suggesting that Src may be a downstream mediator of EphB2 phosphorylation of L1. In contrast, in our 2A2-L1s cells, 20 μM PP2 had no effect on NAP stimulation of FIGQY-peptide phosphorylation, both in whole-cell lysates and with purified EphB2 (Fig 3D). Furthermore, NAP did not increase Src association with L1 in NIH/3T3 cells (69.8 ± 12.4 % of control; n=6; p = 0.059)(Supp Fig 3). These experiments suggest that in our model system, NAP activates EphB2, and EphB2 directly phosphorylates L1.

A structure-activity analysis of NAP revealed that alanine replacement of proline (NAP-P7A) does not decrease NAP antagonism of ethanol inhibition of L1 adhesion or NAP inhibition of ethanol teratogenesis (12). In contrast, alanine replacement of isoleucine (NAP-I6A) greatly reduces both actions of NAP. Consistent with this structure-activity relationship, NAP-P7A was as effective as NAP in stimulating EphB2 phosphorylation of the FIGQY-peptide, whereas NAP-I6A had no significant effect (Fig 4C). Neither SAL nor 1-octanol significantly activated EphB2 phosphorylation of FIGQY-peptide.

Knockdown of EphB2 blocks NAP antagonism of ethanol inhibition of L1 adhesion

If NAP antagonizes ethanol inhibition of L1 adhesion by stimulating EphB2 phosphorylation of L1-Y1229, then knockdown of EphB2 should reduce NAP antagonism. We transfected 2A2-L1s cells with an EphB2 siRNA, resulting in a 37±9% reduction in EphB2 protein expression (n=9; p=0.0047) (Fig 5). This reduction in EphB2 expression abolished NAP stimulation of FIGQY-peptide phosphorylation and eliminated NAP antagonism of ethanol inhibition of L1 adhesion. A scrambled siRNA had no effect on EphB2 expression or the actions of NAP.

Figure 5.

Figure 5.

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Effect of EphB2 knockdown on NAP antagonism of ethanol inhibition of L1 adhesion. (A) 2A2-L1s cells were treated with an EphB2 siRNA or a scrambled siRNA. L1 and actin were used as loading controls, and densities of protein bands were normalized to those obtained in untreated cells (Control). Shown is a representative gel and mean ± SEM % changes in EphB2 expression derived from 9 independent experiments (F=9.33, p< 0.001); * t =1.07, p <0.0.0044, n=10. (B) EphB2 siRNA specifically reduced while scrambled siRNA had no effect on the phosphorylation of FIGQY-peptide from lysates of 2A2-L1s cells treated with 10−12 M NAP. Values for pY in NAP-treated siRNA-treated cells were normalized to values in control cells that were not treated with NAP (F = 2.77, p < 0.05); *t = 2.47, p =0.04, n=8; t = 2.54, p=0.039, n =8. (C) EphB2-siRNA specifically reduced while scrambled siRNA had no effect on NAP antagonism of ethanol inhibition of L1 adhesion. Values for ethanol inhibition of L1 adhesion in the presence of NAP were normalized to values obtained in the absence of NAP (38.6 ± 6.9%) (F = 7.17, p < 0.0001); t= 9.30, ***p =0.0000, n = 9; t = 7.41, ***p = 0.0001, n =9).

Discussion

These experiments identify a possible mechanism by which femtomolar concentrations of NAP antagonize the effects of millimolar concentrations of ethanol (Fig 6). NAP potently activates EphB2, leading to the association of EphB2 with L1 and the direct phosphorylation by EphB2 of Y1229 on the L1-CD. Phosphorylation of L1-Y1229 promotes the uncoupling of L1 from ankyrin-G and the spectrin-actin cytoskeleton. The resulting increase in lateral mobility of L1 in the cell membrane may then induce a conformational change in the L1-ECD that restricts access of ethanol to the alcohol binding pocket in the L1-ECD. In effect, an intracellular catalytic action of NAP produces an extracellular change that renders L1 insensitive to ethanol.

Figure 6.

Figure 6.

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NAP antagonizes ethanol inhibition of L1 adhesion by activating EphB2 phosphorylation of L1-Y1229, leading to the dissociation of L1 from ankyrin-G and the spectrin-actin cytoskeleton. L1 is sensitive to ethanol only when it is associated with ankyrin-G.

What is the evidence for this mechanism? Previous work demonstrated that L1 association with ankyrin-G was necessary for L1 sensitivity to ethanol (17). Phosphorylation of four separate residues on the L1-CD was required for ethanol inhibition of L1 adhesion (16, 17). Each of these phosphorylation events led to an increase in L1 association with ankyrin-G and the spectrin-actin cytoskeleton. Dephosphorylation of each of these residues had no effect on L1 adhesion but reduced or abolished inhibition of L1 adhesion by ethanol, but not by methanol. The effects of dephosphorylation of these residues could be overcome by stabilizing the association of L1 and ankyrin-G through the mutation of L1-Y1229 to phenylalanine, a residue that cannot be phosphorylated. Furthermore, ethanol inhibition of L1 adhesion could be abolished by knockdown of ankyrin-G. Collectively, these findings suggest that the dissociation of L1 from ankyrin-G leads to a small conformational change in the alcohol binding site in the L1-ECD that excludes ethanol (17).

Ankyrin-G binds to L1 at the highly conserved consensus sequence FIGQY1229 only when Y1229 is dephosphorylated (18, 37). Because ankyrin binding to L1 is necessary for ethanol inhibition of L1 adhesion, drugs that stimulate the phosphorylation or inhibit the dephosphorylation of Y1229 will antagonize ethanol inhibition of L1 adhesion. We found that okadaic acid, a phosphatase inhibitor, blocked ethanol inhibition of L1-WT adhesion but had no effect on ethanol inhibition of L1-1229F adhesion. These findings imply that okadaic acid antagonizes ethanol inhibition of L1 adhesion by inhibiting dephosphorylation of L1-1229. Our data do not indicate whether NAP also acts as a phosphatase inhibitor; however, we found strong evidence that NAP antagonizes ethanol inhibition of L1 adhesion by stimulating the phosphorylation of Y1229.

NAP stimulated the dissociation of L1 and ankyrin-G at femtomolar concentrations. The potency of NAP for inducing the dissociation of ankyrin-G and L1 was comparable to that observed for NAP antagonism of ethanol inhibition of L1 adhesion (11–13), consistent with a common mechanism of action. Like okadaic acid, NAP did not reduce ethanol inhibition of L1-Y1229F adhesion or promote the dissociation of L1-Y1229F and ankyrin-G. These findings support the hypothesis that NAP antagonizes ethanol inhibition of L1 adhesion by increasing the phosphorylation of L1-Y1229 and promoting the dissociation of L1 and ankyrin.

We were not able to raise antibodies that would have allowed us to measure phosphorylation of Y1229 as part of the full L1 molecule within intact cells. Rather, our assay depended on the detection of tyrosine phosphorylation of a peptide fragment of L1 that contained Y1229 as the sole tyrosine, an approach that has proved effective in other proteins (35). Past work showing phosphorylation of L1 by EphB2 in intact cells increases the likelihood that our in vitro phosphorylation assay reflects in vivo events. Our data identify EphB2 as one kinase that NAP activates to phosphorylate Y1229. The ability to block the antagonist action of NAP by partial knockdown of EphB2 in intact cells further implies that NAP activation of EphB2 and EphB2 phosphorylation of L1-Y1229 constitute the primary mechanism responsible for NAP antagonism of ethanol inhibition of L1 adhesion.

NAP treatment of mouse embryos prevents ethanol-induced fetal demise, growth retardation, premature closure of the neural tube, and cognitive deficits in adult mice (12, 14, 38, 39). NAP may decrease ethanol embryotoxicity through diverse mechanisms, including reducing ethanol-induced increases in pro-inflammatory cytokines (38, 40), decreases in vasoactive intestinal peptide (41) and brain derived neurotrophic factor (42), neuroprotection (43), and antagonism of ethanol inhibition of L1 adhesion (12). Systematic replacement of individual amino acids of NAP with alanine produced a series of NAP homologues that differed markedly in their antagonism of ethanol inhibition of L1 adhesion and their inhibition of tetrodotoxin neurotoxicity (neuroprotection) (12). Alanine replacement of proline 7 (P7A-NAP) abolished NAP neuroprotection but did not reduce antagonism of either ethanol inhibition of L1 adhesion or embryotoxicity. In contrast, alanine replacement of isoleuceine-6 (I6A-NAP) preserved neuroprotection but greatly reduced antagonism of both ethanol inhibition of L1 adhesion and embryotoxicity. These findings suggested that NAP antagonism of ethanol inhibition of L1 adhesion was more important than neuroprotection in preventing ethanol embryotoxicity in mice (12). Our current experiments demonstrate that P7A-NAP is also more effective than I6A-NAP in stimulating the phosphorylation of L1-Y1229. These findings suggest that NAP stimulation of L1-Y1229 phosphorylation plays a pivotal role in preventing ethanol embryotoxicity in mice. In contrast, the neuroprotective peptide SALLRSIPA did not activate L1-Y1229 phosphorylation and may therefore prevent ethanol embryotoxicity (44) through a different mechanism.

NAP directly stimulated the activation of EphB2, leading to phosphorylation of L1-Y1229. In our experiments, EphB2 appeared to phosphorylate L1-Y1229 directly. In contrast, EphB2 phosphorylation of L1-Y1229 in HEK293 cells requires activation of Src (21). We could not block EphB2 activation of L1-Y1229 phosphorylation with the SFK inhibitor PP2, and we did not find that NAP promoted the association of Src with L1 in NIH/3T3 cells. Therefore, this activation does not appear to be necessary for NAP activation of EphB2 or EphB2 phosphorylation of L1-Y1229, at least in our model system.

Octanol also antagonizes ethanol inhibition of L1 adhesion (7); however, it appears to do so by a different mechanism than NAP. NAP stimulated the dissociation of L1 and ankyrin-G and stimulated EphB2 phosphorylation of FIGQY-peptide, whereas 1-octanol showed neither of those effects. Previous work demonstrated that 1-octanol interacts directly with the alcohol binding pocket in the L1-ECD (4). The effective concentrations of 1-octanol and ethanol are more similar than those for NAP and ethanol, and, based on differences in membrane-buffer partition coefficient, the membrane concentration of 10 μM 1-octanol and 100 μM ethanol are likely very similar (7). Hence, 1-octanol may act as a competitive antagonist, whereas NAP may act non-competitively through a catalytic mechanism.

The Eph family of signaling molecules are receptor tyrosine kinases that interact with ephrins on adjacent cellular processes to promote bidirectional signaling (45, 46). EphB2 and its ephrin ligands guide pathfinding of neuronal growth cones (46, 47), and EphB2 plays a critical role in synapse formation and synaptic stabilization (46). The kinetics of EphB2 activation in cortical dendritic filopodia mediates the sampling and selection of axonal processes for synapse formation (48).

The interaction of EphB2 and L1 contribute to forward signaling in hippocampal development (47) and in retinocollicular synaptic mapping (21). Disruption of the EphB2 signaling pathway leads to aberrant retinocollicular mapping. Furthermore, deletions of the genes for the EphB2, EphB3, and L1 lead to defective pathfinding of commissural axons, resulting in agenesis of the anterior commissure or the corpus callosum (49–51). Dysgenesis of the corpus callosum also occurs in FASD (52), perhaps reflecting the linkage between ethanol inhibition of L1 adhesion and ethanol embryotoxicity (3).

NAP is an active motif of activity dependent neuroprotective protein (ADNP), a molecule that is critical for normal development (53, 54). ADNP gene mutations are among the most common genetic abnormalities in autism spectrum disorder (55), which arises in part due to abnormal synaptic formation. ADNP regulates the activity of numerous genes by chromatin remodeling (56). Among its many actions, NAP is believed to strengthen synaptic connections by binding to microtubule end-binding proteins (EB3) and promoting axonal transport (9). The potent activation of EphB2 by NAP suggests that ADNP may mediate normal synaptic development in part by stimulating EphB2 activity. EphrinB1 is a microtubule-associated protein (57), and it is conceivable that the cognate Eph ligands interact with ephrins on a microtubule scaffold. Indeed, there is evidence that EphB2 may help shape dendritic morphogenesis through its interaction with glutamate receptor interacting protein (GRIP1) and kinesin-1, a microtubule motor protein (58). Hence, NAP might activate EphB2 through its interaction with microtubules or other microtubule-associated proteins (59). Further work is necessary to support this hypothesis.

Supplementary Material

2

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Resource Type Specific Reagent or Resource Source or Reference Identifiers Additional Information
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Antibody goat polyclonal antibody (pAb) against the L1-CD Santa Cruz Biotech SC-1508, RRID: AB_631086
mAb against the L1-ECD Santa Cruz Biotech SC-53386 RRID: AB_628937
total L1; rabbit pAb against C-terminus of ankyrin-G Santa Cruz Biotech SC-28561, RRID: AB_633909
mAb against spectrin Santa Cruz Biotech SC-46696, RRID: AB_671135
goat pAb against actin Santa Cruz Biotech SC-1616 , RRID: AB630836
mAB against EphB2 Santa Cruz Biotech SC-130752, RRID: AB_2099957
goat pAb to actin Santa Cruz Biotech SC-1616, RRID AB630836
rabbit pAb against Src Santa Cruz Biotech SC-18, RRID:AB_631324
Antibody mAB anti-Phosphotyrosine Abcam ab10321, RRID: AB_297058
HRP-conjugated secondary antibodies against mouse Jackson ImmunoResearch Laboratories 115-035-062, RRID: AB_2338504
rHRP-conjugated secondary antibodies against rabbit Jackson ImmunoResearch Laboratories 711-035-152, RRID: AB_10015282
rHRP-conjugated secondary antibodies against Goat Jackson ImmunoResearch Laboratories 705-035-003, RRID: AB_2340390
mAb 5G3 Maine Biotech Services ascites production service
Bacterial or Viral Strain goat anti-mouse IgG conjugated with Alexa Fluor-546 Thermo Fisher Scientific A-11003, RRID: AB_2534071
goat anti-rabbit IgG conjugated with Alexa Fluor 488 Thermo Fisher Scientific R37120, RRID: AB_2556548
mAB to b-tubulin Cell Signaling Technology #86298, RRID:AB_2715541
Biological Sample Recombinant Human EphB2 Protein Thermo Fisher Scientific PV3625
Kinase assay buffer Thermo Fisher Scientific PV3189
Biological Sample Sprague Dawley rat pups Charles River Breeding, Worcester, MA
Cell Line NIH/3T3 American Type Culture Collection (ATCC) CRL-1658
Chemical Compound or Drug
Commercial Assay Or Kit Halt Protease and Phosphatase Inhibitor Cocktail Thermo Scientific Cat #: 1862495
Lipofectamine 2000 Invitrogen Cat#: 11668027
NHS-activated dry agarose beasds Thermofisher Cat #: 26196,
Deposited Data; Public Database
Genetic Reagent
Organism/Strain
Peptide, Recombinant Protein NAP New England Peptide Inc
FIGQY peptides Genscript USA Inc.
Recombinant DNA plasmid, pcDNA3+ wild-type human L1 (hL1) cDNA gene Dr. Patricia Maness, The University of Nother Carolina at Chapell Hill LK, Thelen K, Maness PF. J Neurosci. 2001 Mar 1;21(5):1490-500. Cytoplasmic domain mutations of the L1 cell adhesion molecule reduce L1-ankyrin interactions.
Sequence-Based Reagent EphB2-siRNA Santa Cruz Biotech, Inc SC-39950
Scrambled siRNA Santa Cruz Biotech, Inc SC-37007
ankyrin-G siRNA Santa Cruz Biotech, Inc SC-43268
Software; Algorithm Image J Schneider, C. A.; Rasband, W. S. & Eliceiri, K. W. (2012), “NIH Image to ImageJ: 25 years of image analysis”,Nature methods9(7): 671-675, PMID 22930834. Schindelin, J.; Rueden, C. T. & Hiner, M. C. et al. (2015), “The ImageJ ecosystem: An open platform for biomedical image analysis”, Molecular Reproduction and Development, PMID 26153368.
Prism 5 GraphPad Software, La Jolla California USA, www.graphpad.com
Transfected Construct 2A2-L1s American Type Culture Collection (ATCC) subcloned NIH/3T3 cell line expressing human L1
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Acknowledgements

This work was supported by NIAAA Grant R01AA012974 (M.E.C.); U24AA014811 as a component of the Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD) (M.E.C.); The Medical Research Service and VA Merit Review 5I01BX002374 (M.E.C.); DoD grant W81XWH-12-2-0048 Subaward 8742sc (M.E.C). Part of this work was done in conjunction with the Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD), which is funded by grants from the National Institute on Alcohol Abuse and Alcoholism (NIAAA). Additional information about CIFASD can be found at www.cifasd.org.

Footnotes

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Disclosures

All authors report no biomedical financial interests or potential conflicts of interest.

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