Ethanol Inhibits L1-mediated Neurite Outgrowth in Postnatal Rat Cerebellar Granule Cells*

Cynthia F. BearerDagger §, Alan R. SwickDagger , Mary Ann O'RiordanDagger , and Guanghui Cheng§parallel

From the Departments of Dagger  Pediatrics and § Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The neuropathology of the effects of ethanol on the developing central nervous system are similar to those of patients with mutations in L1, a neural cell adhesion molecule. This observation suggests that inhibition of L1 plays a role in the pathogenesis of alcohol-related neurodevelopmental disorders. Here we examine the effects of ethanol on L1 homophilic binding and on L1-mediated neurite outgrowth. Ethanol had no effect on cell adhesion or aggregation in a myeloma cell line expressing full-length human L1. In contrast, the rate of L1-mediated neurite outgrowth of rat postnatal day 6 cerebellar granule cells grown on a substratum of NgCAM, the chick homologue of L1, was inhibited by 48.6% in the presence of ethanol with a half-maximal concentration of 4.7 mM. The same effect was found with soluble L1-Fc, thus showing that the inhibitory effect is not dependent on cell adhesion. In contrast, neither laminin nor N-cadherin-mediated neurite outgrowth was inhibited by physiologic concentrations of ethanol. We conclude that one mechanism of ethanol's toxicity to the developing central nervous system may be the inhibition of L1-mediated neurite outgrowth.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ethanol is a known human teratogen of immense public health concern. The characteristic pattern of malformations now called fetal alcohol syndrome (FAS)1 was first described in 1968 (1). The criteria for diagnosis, established by the Fetal Alcohol Syndrome Study Group, are as follows: 1) pre- or postnatal growth retardation, 2) craniofacial dysmorphology including microphthalmia, and 3) neurologic abnormalities including mental retardation (2). Conservative estimates place the overall rate of FAS at 0.33/1000, with 1200 children/year born with FAS (3). Alcohol-related birth and neurodevelopmental defects are thought to be anywhere from 3-4 times as common as FAS (4). The Institute of Medicine has recently divided FAS and other effects into five separate categories, including a category for patients with only neurodevelopmental pathology, alcohol-related neurodevelopmental disorder (5). Although multiple organ systems are affected in FAS, the central nervous system appears to be particularly sensitive. The list of neuropathological anomalies found in FAS infants and children include neuronal-glial heterotopias, cerebellar dysplasia, agenesis of the corpus callosum, hydrocephalus, enlarged lateral ventricles, and microcephaly (2, 4, 6). Magnetic resonance imaging in 10 patients with FAS revealed central nervous system anomalies in all 10 (7). Six of these patients had midline defects including partial to complete agenesis of the corpus callosum, hypoplastic corpus callosum, cavum septum pellucidi, and cavum vergae. The other four had microcephaly.

Overlap of the neuropathological abnormalities observed in FAS with those of patients with L1 mutations has led to the hypothesis that ethanol acts via disruption of L1-mediated events (8). L1 is a member of the Ig superfamily of cell adhesion molecules (9). L1 was initially identified when antibodies to L1 disrupted migration of granule cells in vitro (9). Cell lines that express L1 support migration of cerebellar neurons (10). L1 binds to another molecule of L1 on an opposed surface in homophilic binding (11) and enables growth cones to extend rapidly along a bundle of pre-existing axons. Several second messenger systems appear to be involved in L1-mediated neurite outgrowth including the following: 1) serine phosphorylation (12-14),2 2) tyrosine phosphorylation (15), 3) nonreceptor tyrosine kinase activation (16), 4) fibroblast growth factor receptor activation (17-19), and 5) calcium influx (17, 18, 20-22). L1 can be purified from brain and used as a substratum for axon growth (23). Rat postnatal day 6 cerebellar neurons have been shown to extend neurites when cultured on either rat L1 or the chick homologue of L1, NgCAM.

Initial studies using a cell line that expresses L1 following incubation with human osteogenic protein-1 revealed a significant reduction of aggregation in the presence of ethanol. Half-maximal inhibition occurred at 7 mM ethanol with a maximum of 55% inhibition of aggregation (8). Experiments utilizing a human fibroblast line transfected with full-length human L1 confirmed these findings (24). In contrast, ethanol does not inhibit aggregation either in Drosophila S2 cells that express neuroglian, the Drosophila homologue of L1, or are transfected to express human L1 (25). We have further investigated the effect of ethanol on L1-mediated binding and neurite outgrowth.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Origin and Maintenance of Cells-- J558L immunoglobulin-deficient mouse myeloma cells transfected with full-length human L1 were prepared as described previously (26). A cDNA encoding the full-length L1 amino acid sequence including the alternatively spliced RSLE motif in the cytoplasmic domain and a 3' splicing donor site was constructed from clones C2, 3.1, and 17 of a human fetal brain library (27) in pBluescript vector (Stratagene). The coding region was then sequenced. The expression vector used for this study has been described previously (28). Briefly, the L1 was excised from the pBluescript vector with EcoRI and HindIII and ligated into pJanusin, replacing the Janusin cDNA, 5' to an Ig poly(A) tract. This placed the cDNA under transcriptional control of an Ig Vk promoter and an Ig k enhancer. The plasmid contained a minigene conferring resistance to histidinol. J558L immunoglobulin-deficient myeloma cells were transfected with 10-40 µg of DNA/107 cells by electroporation. The cells were grown in 96-well dishes for 48 h in medium (RPMI, 10% fetal bovine serum) to allow for expression of the histidinol resistance gene before the addition of selective medium (containing 2.5 mM histidinol). L1-expressing cells were identified by immunofluorescence. Live cells were incubated with rabbit polyclonal antibodies against human L1 at 1:250 for 30 min on ice. The cells were then washed three times in Ca2+/Mg2+-free phosphate-buffered saline (CMF) and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies at 1:500 for 30 min on ice. The cells were again washed three times in buffer and examined by fluorescence microscopy. Cells from positive wells were cloned using fluorescence-activated cell sorting (Coulter Elite ESP). One clone (phL1A/pJ, 2a10-2C8) was used for all experiments.

Rat cerebellar granule cells were obtained from postnatal day 6 Sprague-Dawley rat pups (Zivic-Miller). Cerebellums were dissected and incubated in 1% trypsin-EDTA for 15 min on ice and then triturated with fire-polished Pasteur pipettes in the presence of 0.05% DNase. The cells were allowed to settle for 5 min, and the supernatant was removed and centrifuged at 200 × g for 5 min. The cell pellet was resuspended in tissue culture medium consisting of Dulbecco's modified essential medium with 10% fetal horse serum and 2.5 mg/100 ml gentamicin (DMEM complete) and counted. Viability was assessed with trypan blue. Cells were seeded onto tissue culture plates at either 1.5 × 105 cells/dish for NgCAM and L1 or 105 for laminin. These cells have been extensively characterized as >90% cerebellar granule cells (29-32).

Preparation of Substrates-- Nitrocellulose was obtained from Schleicher and Schuell. Laminin was obtained from Life Technologies, Inc., Collaborative Research. Rat L1 and chick NgCAM were purified using an affinity column conjugated to 74-5H7 (11) or 8D9 (33) antibodies, respectively. N-cadherin was purified using an affinity column with antibody NCD-2 (34). L1-Fc was prepared as follows. Polymerase chain reaction was used to amplify a fragment of clone 17 that contains the extracellular domain of L1 with primers from 2901 to 2918 and 3336 to 3319 to create a whole extracellular domain of human L1 cDNA. The latter primer also had a 3' splice donor site and EcoRI restriction site. The amplified fragment was digested with BsiWI and EcoRI and ligated into a BsiWI/EcoRI-digested pBluescript vector containing the full-length L1 cDNA. The vector was sequenced across the entire amplified region and insertion sites. The truncated L1 cDNA containing the whole extracellular domain of L1 was excised from the vector with HindIII and EcoRI and ligated into the pIG vector (Ingenius), which contains the Fc region of human immunoglobin isotype 1. The completed vector was electroporated into Escherichia coli MC1061 cells. Plasmid DNA was purified by alkaline lysis and checked by agarose gel electrophoresis. The plasmid was transiently expressed in COS7 cells. L1-Fc was purified from the tissue culture media by affinity chromatography using protein A-Affi-Gel (Bio-Rad).

Preparation of Tissue Culture Plates-- Tissue culture plates were prepared as described previously (23). A 5-cm2 strip of nitrocellulose was dissolved in 12 ml of methanol. Aliquots of this solution were spread over the surface of Corning 35-mm tissue culture dishes and allowed to dry under a laminar flow hood. Substrates were then applied to the dishes by spreading 4 µl of substrate solution across a 0.7-cm diameter spot on the nitrocellulose-coated dish. Rat L1, chick NgCAM, N-cadherin, and laminin were used with protein concentrations of 0.8, 0.3, 0.014, and 0.1 mg/ml, respectively. After 10 min, the droplets were removed by aspiration, and the area was washed twice with CMF. The substrates were then blocked with 1 ml of DMEM complete for 30 min in a 37 °C 10% CO2 incubator. The substrate plates were washed twice with DMEM complete and were incubated at 37 °C in 10% CO2 while cerebellar cells were being prepared. For plates with bound L1-Fc, protein A was used as a 1 mg/ml solution and applied to 35-mm tissue culture dishes (35, 34). After 10 min, the solution was aspirated, and the dish was washed twice with CMF. 8.5 µg of L1-Fc was spread across a 0.7-cm diameter spot on the dish. After 10 min, the droplets were removed by aspiration, and the area was washed twice with CMF. The plates were then blocked as described for laminin and NgCAM. For experiments with soluble L1-Fc, nitrocellulose-coated tissue culture dishes were covered with 250 µl of 0.01% poly L-lysine. After 10 min, dishes were washed twice and blocked as described.

Cell Adhesion Assay-- Both transfected and untransfected J558L cells were gently triturated to form a single cell suspension and kept at 4 °C to prevent reaggregation. Affinity-purified NgCAM (80 µg/ml) was immobilized on a nitrocellulose-coated 35-mm tissue culture dish. The tissue culture dishes were preincubated at room temperature for 30 min in 0, 10, and 100 mM ethanol. Ethanol was added to the cell suspensions to give final concentrations of 0, 10, and 100 mM ethanol, and 2 × 106 cells were added to the corresponding dish. The tissue culture plates were immediately wrapped in parafilm and placed in a 10% CO2 incubator for 2 h. The plates were gently washed twice with Hanks' balanced salt solution and fixed in 4% formaldehyde, 0.1 M potassium phosphate buffer, pH 7.4, with 0.2% glutaraldehyde. A Zeiss microscope equipped with an Image-1 image analysis system was used to quantitate adherent cells.

Cell Aggregation Assay-- A stock of both transfected and untransfected J558L cells were gently triturated to form a suspension of single cells. Ethanol at various concentrations was added to the cells, and aliquots were placed in Coulter counter vials. Following 30 min of rotation at 30 rpm at 37 °C, cells were fixed with 1.5% glutaraldehyde, and aggregation was assessed by Coulter counter. Control cultures were fixed prior to rotation. The percentage of aggregation was calculated as 100 × (1 - (number of single cells at 30 min/number of single cells at 0 min)).

Neurite Outgrowth Assay-- After plating on nitrocellulose-coated dishes to which L1, NgCAM, laminin, or N-cadherin had been adsorbed, cerebellar cultures were incubated for 2 h at 37 °C in 10% CO2 to allow for cell adhesion. Ethanol was then added to half of the dishes at the indicated concentrations. Both control and ethanol-containing tissue culture dishes were tightly wrapped in parafilm and placed in separate incubators. The water pan in the incubator of the ethanol-exposed cultures contained the indicated amount of ethanol. The control and ethanol-containing incubators were switched every other experiment. At the indicated times, cerebellar cultures were washed twice with phosphate-buffered saline and fixed with 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Neurons were examined using a Zeiss microscope equipped with an Image-1 image analysis system. Neurite length was measured as the distance between the center of the cell soma and the tip of its longest neurite. The neurite had to meet the following requirements: it must emerge from an isolated cell (not a clump of cells), it must not contact other cells or neurites, and it must be longer than the diameter of the cell body.

Measurement of Ethanol-- 100 µl of media following the addition of ethanol and 1 ml of media at the time of fixation were taken to measure the ethanol concentration. Samples were placed in microcentrifuge tubes and stored at 4 °C until ethanol concentration was assayed. In control experiments, the size of the sample did not influence evaporative loss of ethanol. However, storage at -20 °C enhanced the rate of evaporation. Ethanol concentrations was measured in duplicate samples with an ethanol assay kit (Boehringer Mannheim) according to the manufacturer's instructions.

Protein Determination-- Protein concentrations were determined by Pierce Coomassie Blue Plus protein assay.

Statistical Analysis-- Univariate statistics were used to describe the cell adhesion, cell aggregation, and ethanol concentration data. The main outcome variable, mean neurite length, was determined for each condition from each cell preparation. Since the variable is the mean of greater than 30 measurements, it will be considered a normal distribution, and parametric tests of significance are used. Previous experiments have shown that there is very little variability within one cell preparation, while variability does exist between cell preparations. Therefore, the mean neurite length of all neurites measured under one condition was calculated per cell preparation. Descriptive statistics determined the mean ± S.E. of the mean neurite lengths from multiple cell preparations. Two group comparisons were made using the appropriate t test; comparisons involving more than two groups were made using one-way ANOVA with Duncan's New Range Test for multiple comparisons. For dose-response analyses, results were evaluated from each cell preparation separately to validate the best fit curve observed and confirm similarity of response across cell preparations. Best fit is defined as the simplest appropriate model that has general applicability. The concentrations of ethanol giving half the maximum inhibition (IC50) were predicted from these models.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The effect of ethanol on J558L cells transfected with full-length human L1 was first tested on the ability of the cells to adhere to an L1-coated surface that can support L1-mediated adhesion and neurite outgrowth for a variety of neurons (23). Only L1-transfected cells adhere to the substrate. The addition of either 10 or 100 mM ethanol did not alter the attachment of the cells to the L1 substrate (Fig. 1).


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Fig. 1.   Adhesion of L1-expressing myeloma cells to L1 substratum in the presence of ethanol. Both transfected (L1-expressing) and untransfected (no L1 expression) J558L myeloma cells were triturated to form a single cell suspension, mixed with ethanol, and then added to tissue culture dishes prepared with NgCAM for 2 h. The dishes were then washed twice with buffer and fixed with glutaraldehyde. Adherent cells were counted with a microscope equipped with an Image-1 computer-assisted image analysis system. No untransfected cells adhered to the plates. Results shown are for transfected cells from three separate experiments (mean ± S.E.). There was no significant difference in adhesion of transfected cells in the presence of ethanol (ANOVA, p = 0.93).

To further explore the effect of ethanol on L1 homophilic binding, the cells were then assayed for their ability to self-aggregate (26). In previous experiments, aggregation is completed within 30 min in this transfected cell line (26). Fig. 2 shows that, in agreement with Fig. 1, less than 10% of untransfected J558L cells self-aggregate at 30 min, whereas 50-60% of transfected cells aggregate. Ethanol in concentrations up to 100 mM had no effect on either transfected or untransfected J558L self-aggregation.


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Fig. 2.   Self-aggregation of L1-expressing myeloma cells in the presence of ethanol. Both transfected (L1-expressing, light gray bars) and untransfected (no L1 expression, dark gray bars) J558L myeloma cells were triturated to form a single cell suspension, mixed with ethanol, and then either fixed with glutaraldehyde or rotated for 30 min and then fixed. The number of single cells was determined by a Coulter counter. Percentage of aggregation was calculated as 100 × (1 - (single cells at 30 min/single cells at 0 min)). Results shown are from two cell preparations (mean ± S.E.). Transfection with L1 significantly increased cell aggregation, but there was no significant effect of ethanol at any concentration (two-way ANOVA: group effect, p < 0.001; dose effect, p = 0.75; group × dose, p = 0.9).

Neurite growth of rat cerebellar granule cells was measured over 12 h of culture to measure the effect of ethanol on the functions of L1. The ethanol concentration of the tissue culture dishes was monitored over time. Table I shows the typical ethanol concentration of cultures maintained for 12 h. There was no significant change in the ethanol concentration over the duration of the experiment. When different concentrations of ethanol were required to determine the dose-response relationship of ethanol on neurite outgrowth, preliminary experiments were conducted to determine the concentration of ethanol in the water pan that maintained all ethanol concentrations. The water pan concentration of 25 mM ethanol was found to be optimal for maintaining ethanol concentrations in the tissue culture dishes over the range of ethanol used in this experiment. Table II shows the ethanol concentrations of the tissue culture dishes at 0 and 12 h with 25 mM ethanol in the incubator water pan. Concentrations of ethanol were significantly increased in the media after 12 h for several of the higher concentrations of ethanol. However, for most conditions these increases were not large. In addition, at the lower concentrations of ethanol, the concentration did not change with time.

                              
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Table I
Ethanol concentrations of tissue cultures over time of neurite outgrowth assay

                              
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Table II
Changes in ethanol concentrations of tissue cultures after 12 h of neurite outgrowth assay

Using these assay conditions, the neurite lengths of cerebellar cells grown on chick NgCAM were measured. NgCAM was used initially due to its availability. Fig. 3 shows the effect of 17 mM ethanol on the range of neurite lengths of granule cells grown on NgCAM measured at 2, 4, 8, and 12 h. The striking difference in neurite lengths between the control and ethanol-exposed cells becomes apparent at 4 h of culture. The mean neurite length was determined and plotted as a function of time. The results of three separate granule cell preparations are shown in Fig. 4. The mean neurite lengths are significantly shorter for the ethanol-treated cells than the controls at 8 and 12 h.


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Fig. 3.   Range of neurite lengths on NgCAM in the presence of 17 mM ethanol. Rat postnatal day 6 cerebellar cells were plated on tissue culture dishes prepared with NgCAM. After 2 h, ethanol was added to the tissue culture medium (open symbols). Control plates had no ethanol (closed symbols). At the indicated times, cells were washed and fixed with 1% glutaraldehyde, and neurite length was determined by the Image-1 computer-assisted image analysis system. Results shown are duplicate tissue culture plates for control and ethanol-treated cells from a single granule cell preparation.


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Fig. 4.   Mean neurite length of cells grown on NgCAM as a function of time in the presence of ethanol. Mean neurite length was determined from data obtained as shown in Fig. 3 and plotted as a function of time. Points represent three separate cell preparations (mean ± S.E.). Mean neurite length is significantly different between control (open squares) and ethanol (closed circles) at 8 and 12 h (Student's two-tailed t test, p < 0.01 and p < 0.02).

To determine the concentration dependence and the substrate specificity of this effect, cerebellar cells were plated as described using both NgCAM and laminin as substrates. Fig. 5 shows the results of ethanol exposure on NgCAM-mediated neurite outgrowth from one such experiment. As can be seen in Fig. 6A, ethanol had no effect on the mean neurite length of cells grown on laminin at the concentrations used by nonlinear regression analysis. However, mean neurite length of cells grown on NgCAM was shorter by approximately 50% at concentrations of ethanol greater than 40 mM, with a half-maximal effect at 4.7 ± 0.4 mM (Table III). This concentration is similar to the concentration of ethanol giving half-maximal inhibition of cell aggregation (5-7 mM) as reported by Charness et al. (8) and Ramanathan et al. (24).


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Fig. 5.   Neurite length at 12 h as a function of ethanol concentration. Neurite lengths are shown of rat postnatal day 6 cerebellar cells plated on NgCAM with different concentrations of ethanol. Cells were fixed at 12 h after the addition of ethanol. An example is given of measurements from a single cell preparation.


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Fig. 6.   A, mean neurite length of cells grown on NgCAM and laminin as a function of ethanol concentration. Mean neurite length was calculated from data obtained as shown in Fig. 5 for cells grown on either NgCAM (open circles) or laminin (closed squares) and plotted as a function of ethanol concentration. Results shown are from three separate cell preparations (mean ± S.E.). Data points were fit to the nonlinear curve with the best fit. For laminin, within these concentrations of ethanol, the curve was a straight line with no slope. For NgCAM, the best fit was obtained with the equation, y = b0 + b12-x/b2. B, mean neurite length of cells grown on laminin as a function of ethanol concentration. Postnatal day 6 rat cerebellar cells were plated on tissue culture dishes prepared with laminin. Following a 2-h incubation to allow cells to adhere, ethanol was added to the media in the concentrations shown. After a further 12-h incubation, cells were fixed with glutaraldehyde, and neurite length was measured. Symbols represent five separate preparations of cells. Using nonlinear regression analysis, data points were best described by the logistic dose response equation, y = a + b/(1 + (x/c)d).

                              
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Table III
Effect of ethanol on neurite outgrowth mediated by different substrates

Since ethanol is a known metabolic poison, it should inhibit neurite outgrowth nonspecifically at high concentrations. To ensure that the neurite outgrowth assay was capable of detecting this nonspecific effect, neurite outgrowth on laminin was measured in the presence of high concentrations of ethanol. Fig. 6B shows that the mean neurite length of granule cells grown on laminin for 12 h is shorter at high concentrations of ethanol. From nonlinear regression analysis, the concentration for half-maximal effect is approximately 400 mM, 100 times that needed for half the maximal effect on L1-mediated neurite outgrowth (Table III). This result is in contrast to that of Matsuzawa et al. (36), where the mean neurite length of hippocampal neurons plated on laminin and grown in 100 mM ethanol was 71% that of control. This difference may be due to different sensitivities to ethanol of hippocampal neurons compared with cerebellar granule cells. However, even this concentration of 100 mM is 25 times that required for half-maximal inhibition of L1-mediated neurite outgrowth. The difference in ethanol concentration required for half-maximal inhibition of neurite outgrowth mediated by L1 and laminin suggests that different mechanisms underlie these inhibitory effects and that the mechanism of inhibition of L1-mediated neurite outgrowth is not due to a simple nonspecific mechanism such as ATP depletion.

To ensure that this effect was not due to an interspecies sensitivity to ethanol between rat cerebellar cells and chick NgCAM, L1 was purified from fetal rats, and the ability of ethanol to inhibit L1-mediated neurite outgrowth of rat cerebellar granule cells was tested. Table III summarizes the results. Mean neurite length of rat cerebellar neurons on L1 was shorter in the presence of ethanol by 41.9%, with a half-maximal effect at a concentration of 3.8 ± 0.7 mM. The effect of ethanol on neurite length was identical for NgCAM and L1, showing that the observed effect of ethanol was not due to an interspecies effect.

A chimeric protein, L1-Fc, which contains the extracellular domain of human L1 fused to the constant domain of immunoglobulin was used to determine if ethanol inhibits L1-mediated neurite outgrowth in the absence of L1-mediated cell attachment. As can be seen in Table III, ethanol inhibits neurite outgrowth of cerebellar cells mediated both by soluble L1-Fc and L1-Fc presented as a substratum. The extent of inhibition is approximately 40%, with a half-maximal effect at 3-5 mM ethanol, similar to that found with Ng-CAM and rat L1.

To determine whether the remaining neurite outgrowth in the presence of high concentrations of ethanol is due to L1 or to some other process, anti-L1 Fabs known to block L1 binding (26) were added to the media of cerebellar granule neurons plated on poly-L-lysine. After 2 h, L1-Fc (5 µg/ml) and 25 mM ethanol were added. Neurite length was measured after a further 12-h incubation. If L1-mediated neurite outgrowth is completely inhibited by ethanol and the remaining outgrowth is due to other factors, antibody to L1 will have no effect on the remaining range of neurite lengths. In contrast, if L1-mediated neurite outgrowth is only partially inhibited by ethanol and the remaining outgrowth is still L1-mediated, then antibody to L1 will abolish this growth. The results of this experiment are shown in Fig. 7. The addition of anti-L1 Fabs to the cultures resulted in complete inhibition of L1-stimulated neurite outgrowth, and completely abolished the remaining L1-stimulated neurite outgrowth in the presence of 25 mM ethanol. Therefore, ethanol added 2 h after the addition of L1-Fc only partially inhibits L1-mediated neurite outgrowth and does not stimulate neurite outgrowth.


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Fig. 7.   Effect of anti-L1 Fab on remaining neurite outgrowth in the presence of ethanol. Rat postnatal day 6 cerebellar cells were isolated and plated on poly-L-lysine-coated tissue culture dishes. Following a 2-h incubation, the following additions were made: L1-Fc, L1-Fc plus 25 mM ethanol, L1-Fc plus anti-L1 Fab, or L1-Fc with both 25 mM ethanol and anti-L1 Fab. Cells were incubated for an additional 12 h and then fixed, and neurite length was measured. Results from three separate cell preparations are shown (mean ± S.E.). There was no significant difference between mean neurite length from control (no addition), L1-Fc + Fab, and L1-Fc + Fab + ethanol. L1-Fc significantly increased mean neurite length compared with control. The addition of ethanol with L1-Fc significantly reduced the mean neurite length compared with L1-Fc alone, but the mean neurite length remained significantly greater than the other three (overall ANOVA, p < 0.00002; post hoc multiple comparisons using Duncan's new multiple range test).

It has been proposed that L1 promotes neurite outgrowth via an interaction with the fibroblast growth factor receptor and that this pathway is shared by the neural cell adhesion molecule and N-cadherin (37). To determine whether ethanol acts on this common pathway, the effect of ethanol on N-cadherin-mediated neurite outgrowth was determined (Fig. 8). Ethanol at both 10 and 100 mM had no effect on mean neurite length at 12 h of cerebellar granule cells plated on N-cadherin. If one assumes that the fibroblast growth factor receptor (FGFr) is critical in L1-mediated neurite outgrowth, these data suggest that ethanol acts directly on L1 prior to the FGFr signal cascade or interferes with some other aspect of L1-mediated neurite outgrowth not shared with N-cadherin. For example, ethanol may interfere with L1 interactions with the ankyrin cytoskeleton (38) or with L1 internalization (39).


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Fig. 8.   Effect of ethanol on N-cadherin-mediated neurite outgrowth. Rat postnatal day 6 cerebellar cells were isolated and plated on N-cadherin-coated tissue culture dishes. Following a 30-min incubation, 10 or 100 mM ethanol was added to the plates. The plates were incubated for 12 h at 37 °C in a CO2 incubator. Cells were fixed with glutaraldehyde, and neurite length was measured. Mean neurite length was calculated and normalized to mean neurite length in the absence of ethanol. There was no significant effect of ethanol at either concentration.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study is the first report of ethanol inhibiting neurite outgrowth mediated by a specific cell adhesion molecule. Ethanol has been reported to have multiple effects on neurite outgrowth. In some experimental systems, it enhances growth, whereas in others it inhibits (36, 40-52). In these systems, the effects of ethanol are only seen starting at concentrations of ethanol of 25 mM, which are approaching nonphysiologic levels. Since neurite outgrowth is a complex process mediated by many different cell adhesion molecules and growth factors, simple cell culture systems permit the isolation and careful characterization of specific factors influenced by ethanol. Using such a system, this study found a specific inhibitory effect of ethanol at concentrations of 3-5 mM on L1-mediated neurite outgrowth of cerebellar granule cells.

There are four likely mechanisms by which ethanol could perturb L1-mediated neurite outgrowth. The first is that L1 homophilic binding is disrupted by ethanol. Although initial studies reported such an effect (8, 24), subsequent studies have not supported this finding (25, 52). Our own results reported here do not show an effect of ethanol on L1-mediated cell adhesion or aggregation.

A second possibility is that ethanol alters the cell surface expression of L1. Although the lack of effect of ethanol on cell adhesion or aggregation measured in our laboratory would suggest that the total amount of L1 on the cell surface is not altered, there may be effects on the dynamics of L1 cell surface expression. L1 must be dynamically regulated to allow for adhesion and nonadhesion to occur, allowing the growth cone to move forward over L1 (53). If L1 is too adhesive, it may retard neurite outgrowth. There are few published studies on the dynamic aspects of the cell surface expression of L1. L1 on the cell surface may be identified by its susceptibility to trypsin proteolysis. Trypsin cleaves L1 at a single extracellular site, generating peptide fragments of 140 and 80 kDa from the parent 200-kDa form, all three of which are membrane-bound (54). Immunoprecipitation of metabolically labeled L1 following trypsinization of live cells showed retention of some label in the 200-kDa band. Complete loss of label in the 200-kDa band was accomplished following trypsinization of permeabilized cells, demonstrating that some L1 is intracellular in location (54). Neither the kinetics nor the location of the internalized L1 were described. Recent experiments show that L1 is targeted to the axon and growth cone (55) and that L1 is endocytosed via a clathrin-mediated pathway, and colocalizes with the transferrin receptor. L1 endocytosis is most active in the rear of the growth cone, implying a role in axon growth (39). These experiments suggest that L1 may be internalized to allow for forward movement of the growth cone. Further studies are needed to explore possible ethanol disruption of this process.

Disruption of the association between the cytoskeleton and L1 is a third possible mechanism for the ethanol effect. L1 has been shown to associate with ankyrin B (15, 38). Mice lacking the ankyrinB gene exhibit a phenotype similar but more severe than L1 knockout mice (56), which share phenotypic similarity with mice models of fetal alcohol syndrome. Thus, ethanol may be disrupting the L1-ankyrinB interaction.

The fourth possible mechanism of the inhibitory effect of ethanol on L1-mediated neurite outgrowth is disruption of L1 signal transduction. Ethanol has been shown to affect a number of signal cascades and second messenger systems (for a review, see Ref. 57). Homophilic binding of L1 is followed by a cascade of well defined signaling events. These events include 1) serine phosphorylation of L1 on serines 1152, 1181, and 1204 on the cytoplasmic domain (12, 13)2; 2) phosphorylation of L1 on a highly conserved tyrosine on the cytoplasmic domain (15); 3) activation of pp60csrc (58), ERK2, and Raf-12; 4) interaction with the FGFr with activation of its tyrosine kinase (37); and 5) calcium influx (17, 18, 20-22). Ethanol may act at one or several locations within these signaling cascades. Our results showing no inhibition of N-cadherin-mediated neurite growth at 100 mM ethanol are consistent with the hypothesis that L1 is not acting downstream of the FGFr receptor. Further studies investigating the effect of ethanol on the distribution of L1, the phosphorylation state of L1, and its downstream signaling events are needed.

Our results highlight the potential importance of ethanol's effects on cell adhesion molecules during brain development. Several underlying mechanisms may account for these effects. Future research should address the mechanism underlying the inhibitory effect of ethanol on L1-mediated neurite outgrowth.

    ACKNOWLEDGEMENTS

We thank Dr. Vance Lemmon for providing NgCAM, rat L1, anti-L1 Fabs, and the L1-Fc construct; Dr. Sue Burden-Gulley for providing N-cadherin; Kevin Buck for excellent technical assistance; and both Dr. Vance Lemmon and Dr. Al Malouf for helpful comments.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel Supported by National Institutes of Health Grants EY5285 and NS34252 (to Vance Lemmon).

Supported by a Faculty Fund Grant. To whom correspondence should be addressed: Dept. of Pediatrics, Rainbow Babies and Children's Hospital, 11100 Euclid Ave., Suite 3100, Cleveland, OH 44106. Tel.: 216-844-5249; Fax: 216-844-3380; E-mail: cfb3{at}po.cwru.edu.

2 A. W. Schaefer, H. Kamiguchi, E. V. Wong, C. M. Beach, G. Landreth, and V. Lemmon, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: FAS, fetal alcohol syndrome; CMF, Ca2+/Mg2+-free phosphate-buffered saline; FGFr, fibroblast growth factor receptor; ANOVA, analysis of variance.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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