Ethanol Inhibits L1-mediated Neurite Outgrowth in Postnatal
Rat Cerebellar Granule Cells*
Cynthia F.
Bearer
§¶,
Alan R.
Swick
,
Mary Ann
O'Riordan
, and
Guanghui
Cheng§
From the Departments of
Pediatrics and
§ Neurosciences, Case Western Reserve University School of
Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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.
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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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 |
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).
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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).
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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.
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).
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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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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).
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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.
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DISCUSSION |
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.
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.
 |
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