TOXICOLOGICAL SCIENCES 115(1), 202–213 (2010)
doi:10.1093/toxsci/kfq040
Advance Access publication February 4, 2010
Endocytosis in Cultured Neurons Is Altered by Chronic Alcohol Exposure
Marı́a P. Marı́n,*,1 Guillermo Esteban-Pretel,*,1 Xavier Ponsoda,† Ana M. Romero,* Raúl Ballestı́n,* Carlos López,†
Luis Megı́as,‡ Joaquı́n Timoneda,§ Asunción Molowny,† Juan J. Canales,{ and Jaime Renau-Piqueras*,2
*Section of Biologı́a y Patologı́a Celular, Centro de Investigación, Hospital Universitario ‘‘La Fe,’’ E-46009 Valencia, Spain; †Departamento de Biologı́a
Celular, Universidad de Valencia, E-46100, Valencia, Spain; ‡Departamento de Anatomı́a y Embriologı́a Humanas, Universidad de Granada, E-18071,
Granada, Spain; §Departamento de Bioquı́mica y Biologı́a Molecular, Universidad de Valencia, 46100, Valencia, Spain; and {Biopsychology and Comparative
Neuroscience Group, Cavanilles Institute (ICBiBE), Universidad de Valencia, E-46980, Valencia, Spain
2
1
These authors contributed equally to this work.
To whom correspondence should be addressed at Section of Biologı́a y Patologı́a Celular, Centro de Investigación, Hospital Universitario ‘‘La Fe,’’ Avda.
Campanar 21, E-46009 Valencia, Spain. Fax: þ34-96-197-30-18. E-mail: [email protected].
Received October 23, 2009; accepted January 29, 2010
Endocytosis is required for many cellular pivotal processes,
including membrane recycling, nutrient uptake, and signal
transduction. This complex process is particularly relevant in
polarized cells, such as neurons. Previous studies have demonstrated that alcohol alters intracellular traffic, including endocytosis, in several cell types. However, information on the effect of
chronic alcohol exposure on this process in neurons is scarce. As
an approach, we investigated the effect of alcohol exposure on the
internalization of two widely used endocytic markers, albumin
and transferrin, in developing hippocampal neurons in primary
culture. The effect of this treatment on the levels of several
representative proteins involved in the endocytic process was
also analyzed. Some of these proteins are also involved in the
organization of the actin cytoskeleton. Pretreatment of cells with
inhibitors chlorpromazine or nystatin indicates that albumin is
internalized mainly by caveolin-dependent endocytosis. On the
other hand, alcohol decreases the endocytosis of both markers,
although no qualitative changes in the distribution of either of
these molecules were observed. Finally, the effect of ethanol on the
proteins analyzed was heterogeneous. Alcohol decreases the levels
of clathrin, AP-2, SNX9, Rab5, Rab11, EEA1, Cdc42, or RhoA but
increases the amount of Arf6. Moreover, alcohol does not affect
the levels of caveolin1, dynamin1, Rab7, and LAMP2. This toxic
effect of alcohol on endocytosis could affect some of the important
neuronal activities, which depend on this process, including cell
signaling. Our results in neurons also stress the notion that one of
the main targets of ethanol is intracellular transport.
Key Words: neurons; alcohol exposure; endocytosis; albumin;
transferrin.
Cell surface proteins, ligands, lipids, and other solutes can be
introduced into the cell through several endocytic routes that
start with the internalization of these structures and molecules
into primary endocytic vesicles (Peterson, 2008; Watson et al.,
2004), which are frequently coated with the protein clathrin
and have a variety of adaptors and accessory factors (Benmerah
and Lamaze, 2007). After uncoating, endocytic vesicles fuse
with each other or with preexisting endosomes to form early
endosomes. In addition, there are several routes whose cargo
can be taken from early endosomes (Schmidt and Haucke,
2007; Watson et al., 2004). The regulation of vesicular
transport is carried out by the Rab proteins, which are
small GTPases (Deinhardt et al., 2006b). Moreover, there are
other types of endocytosis, which do not depend on clathrin,
for instance, caveolin-dependent endocytosis (Sandvig et al.,
2008; Watson et al., 2004), clathrin- and caveolin-independent
pathways, and fluid-phase endocytosis (Bonanomi et al., 2008;
Doherty and McMahon, 2009; Mayor and Pagano, 2007).
Endosomal traffic appears to be of considerable importance
in polarized cells, such as neurons. These cells possess
elaborate membrane systems, which serve to spatially segregate incoming signals from outgoing signals via the formation
and maintenance of distinct membrane domains (Schmidt and
Haucke, 2007). One of the major neuronal functions of
clathrin-mediated endocytosis is synaptic vesicle recycling.
Clathrin-mediated endocytosis also regulates the activitydependent exo-endocytic trafficking of postsynaptic receptors
(Gundelfinger et al., 2003) and plays an active role during
polarized axon elongation and growth cone navigation (Zhou
et al., 2007). Moreover, an interplay between clathrin-coated
vesicles and cell signaling has been suggested, and functional
analyses of clathrin genes have provided insight into the
functions of clathrins and their interactions with other proteins
in cell signaling. In fact, clathrin-coated vesicle formation is
a principal uptake route in response to signaling events (Inoue
et al., 2007; Mills, 2007). In addition, the endosome function is
altered in a range of neurodegenerative disorders and hereditary
axonal peripheral neuropathies (Nixon, 2005). Despite very
little being known about the events that modulate caveolin in
neurons, this protein could play an important role in processes
The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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ALCOHOL AND ENDOCYTOSIS IN NEURONS
such as neuronal migration and hippocampal plasticity (Lentini
et al., 2008).
Exposure to ethanol causes a variety of anomalies in the
developing brain, including neuronal-glia heterotopias, cerebellar displasia, agenesis of the corpus callosum, hydrocephalus, and microcephaly (Streissguth et al., 1994). These toxic
effects are indicative of aberrant migration, decreased proliferation, and neural cell death (Goodlett et al., 2005;
Streissguth et al., 1994). No single mechanism has been
sufficient to account for these varying effects, and it is likely
that multiple factors are involved, and a number of mechanisms
have been proposed (Goodlett et al., 2005; Guerri et al., 2001;
Martı́nez and Egea, 2007). One such mechanism suggests that
ethanol perturbs protein trafficking. Thus, ethanol exposure
substantially impairs endocytosis, exocytosis, and nucleocytoplamic traffic in rat astrocytes in primary cultures (Marı́n et al.,
2008; Megı́as et al., 2000; Tomás et al., 2002; Tomás et al.,
2004, 2005). These effects could be attributable to a primary
alteration in the dynamics and organization of the cytoskeleton
(Guasch et al., 2003; Martı́nez et al., 2007; Tomás et al., 2003,
2005), although we have demonstrated that this treatment also
affects the Golgi complex (Tomás et al., 2005).
Despite the importance of endocytosis in neuronal cells,
studies on the possible effects of chronic ethanol exposure
on this process in neurons are scarce. Therefore, as a first
approach, we analyzed the effect of chronic ethanol exposure
(30mM ethanol for 14 days) on the following general aspects
of endocytosis in rat hyppocampal neurons in primary culture
(14 days in vitro [DIV]): (1) the endocytosis of the marker
bovine serum albumin (BSA), which is transported to late
endosomes and lysosomes for degradation (Megı́as et al.,
2000; Murk et al., 2003) and (2) the internalization of
transferrin (Tf), which is widely used as a model to study the
recycling of endosomes through clathrin-dependent endocytosis (Deinhardt et al., 2006a; Schmidt and Haucke, 2007). We
also analyzed the effect of ethanol on the levels of the various
proteins involved in several steps of endocytosis, some of
which are also related to the organization of the actin
cytoskeleton (Doherty and McMahon, 2009; Mayor and
Pagano, 2007).
MATERIALS AND METHODS
203
rabbit polyclonal antibody was obtained form Abcam (Cambridge, UK). AntiCdc42 rabbit polyclonal antibody was purchased from Chemicon (Millipore
Iberica S.A.U., Spain), and anti-RhoA monoclonal antibody was acquired from
Cytoskeleton (Denver, CO). Anti-MAP2 mouse monoclonal antibody, antineurofilament (NF) 200 rabbit polyclonal antibody, and anti-glial fibrillary
acidic protein (GFAP) rabbit polyclonal antibody were obtained from SigmaAldrich (Spain). Anti-synaptosome–associated protein of 25 kDa (SNAP-25)
and anti-postsynaptic density (PSD)-95 mouse monoclonal antibodies were
purchased from Abcam.
Secondary antibodies. Alkaline phosphatase–conjugated anti-rabbit, antimouse, and anti-goat IgG were obtained from Sigma-Aldrich. Alexa-488
F(ab#)2 fragments were obtained from Molecular Probes (Invitrogen,
Invitrogen S.A., Spain).
Other reagents. BSA conjugated with 10-nm gold particles was acquired
from EY Laboratories, Inc. (San Mateo, CA). Fluorescein iso thio cyanate
(FITC) BSA and FITC-Tf were purchased from Molecular Probes. Nystatin and
chlorpromazine were purchased from Sigma-Aldrich. Tissue culture reagents
were acquired from Invitrogen. The chemicals used for electron microscopy
were acquired from Electron Microscopy Science (EMS, PA). All the
remaining chemicals were acquired from Sigma-Aldrich.
Primary Culture of Hippocampal Neurons and Alcohol Treatment
The primary cultures of hippocampal neurons were prepared from the rat
fetuses of female rats on day 16 of gestation as described with minor
modifications (Braza-Boils et al., 2006; Brewer et al., 1993). Cells were
cultured in Neurobasal medium containing B27 and Glutamax, which were
maintained in a humidified atmosphere of 5% CO2 and 95% air at 37C. The
medium was changed every 2 days, and cells were maintained until day 14 (14
DIV). To analyze the possible toxic effects of alcohol on endocytosis during
neuronal development, some cells were grown in the presence of ethanol,
which was added to the culture medium on the day of plating. The ethanol
concentration in the medium was checked daily and adjusted to a final
concentration of 30mM (138 mg/dl, ethanol evaporation after 24 h was
10–20%), similar to the blood levels reported in pregnant chronic drinkers.
This is considered a moderate alcohol consumption (Eckardt et al., 1998). The
concentration of alcohol in the culture medium was determined using a kit from
Sigma-Aldrich (NAD-ADH Reagent Multiple Test Vial N7160). The purity of
neuron cultures was assessed by immunofluorescence using anti-MAP2 and
anti-NF antibodies (Braza-Boils et al., 2006), and the evaluation of the
morphological features of cells was carried out using phase contrast and
transmission electron microscopy. Possible contamination by astroglial cells
was determined using an anti-GFAP antibody (Renau-Piqueras et al., 1989).
The anti-SNAP-25 and PSD-25 antibodies were used as presynaptic and
postsynaptic markers (Chin et al., 2000; Staple et al., 2000). Cell viability was
determined by the trypan blue exclusion test. In some cultures, the possible
cytotoxic effect of alcohol exposure on neurons was also evaluated by
measuring lactate dehydrogenase release in the medium from neurons using
a nonradioactive cytotoxicity assay kit (Sigma-Aldrich, Cat. Num. TOX7)
(Pascual et al., 2003). All the experiments using rats were approved by the
appropriate institutional review committee and performed in strict compliance
with the European Community Guide for the Care and Use of Laboratory
Animals.
Reagents
Fluorescence Microscopy
Primary antibodies. Anti-Clathrin goat polyclonal antibody, anti-Rab5
rabbit polyclonal antibody, anti-Rab7 rabbit polyclonal antibody, anti-sorting
nexin protein 9 (SNX9) goat polyclonal antibody, and anti-ADP-ribosylation
factor 6 (Arf6) monoclonal antibody were obtained from Santa Cruz
Biotechnology, Inc. (Heidelberg, Germany). Anti-a-adaptin (AP-2) monoclonal
antibody, anti-early endosomal antigen 1 (EEA1) rabbit polyclonal antibody,
anti-lysosomal associated protein 2 (LAMP2) rabbit polyclonal antibody, and
anti-dynamin I rabbit polyclonal antibody were purchased from Affinity
BioReagents (Golden, CO). Anti-Rab11 monoclonal antibody was obtained
from BD Transduction Laboratories (Franklin Lakes, NJ). Anti-caveolin1
The ability of control and alcohol-exposed neurons to internalize BSA and
Tf was visualized using qualitative fluorescence microscopy as previously
described in astrocytes (Megı́as et al., 2000). Briefly, neurons were washed and
incubated for 60 min at 37C in culture medium without B27 containing FITCBSA (0.1 mg/ml) or FITC-Tf (0.075 mg/ml). Then, cells were washed in PBS
and fixed in 4% formaldehyde in PBS, pH 7.4, and were mounted and
examined under an Olympus microscope BX50WI equipped with a Hamamatsu
ORCA digital camera controlled with the Aquacosmos software. Immunofluorescence of SNAP-25 and PSD-95 was carried out as previously described
(Marı́n et al., 2008; Tomás et al., 2005).
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MARÍN ET AL.
Quantitative Analysis of BSA and Tf Endocytosis. Inhibitors Treatment
The quantitative analysis of BSA and Tf endocytosis was carried out
according to Vaslin et al. (2007) with minor modifications. To saturate the
entire endocytic/recycling pathway, the neurons cultured in the presence or
absence of ethanol were washed three times at 37C with PBS and were then
exposed to FITC-BSA (0.1 mg/ml) or FITC-Tf (0.05 mg/ml) from 15 min to
6 h in a culture medium without B27 and Glutamax.
In other experiments, ‘‘single-round’’ kinetics of Tf uptake and recycling
were analyzed by first incubating the control and alcohol-exposed neurons with
Tf for 30 min at 0C in a medium containing Tf, which were washed three
times in cold PBS and incubated in a medium without Tf for 0, 2, 5, 15, 30, and
60 min at 37C (Babia et al., 2001; Kim et al., 2006; Megı́as et al., 2000). At
low temperatures, Tf bound to its receptor but internalization is inhibited
because endocytosis only occurs when cells are above the phase transition
temperature of the plasma membrane (~23C). For this reason, the
internalization of Tf bound to the membrane during cold incubation was
induced by transferring cells at 37C.
Dishes were then placed on ice and washed three times with cold PBSMgCl2-CaCl2 and were rinsed twice for 5 min each with cold Neurobasal
medium. After rinsing, cells were detached from the plastic with a rubber
policeman in 200 ll of lysis buffer (50 mmol/l Tris-HCl, pH 7.4, and 5 ll/ml
mammalian protease inhibitor cocktail) and were then sonicated three times in
a Branson sonifier (W150) for 5 s and centrifuged for 10 min at 300 3 g. This
process was carried out at 0C. The fluorescence from the cell suspensions was
measured at excitation k 494 nm and at emission k 520 nm in a Molecular
Probes Gemini XPS system. The results, expressed as the fluorescence intensity
per milligram of protein, are the mean ± SD of at least four plates per point
from three different cultures.
For the inhibition studies, control neurons were preincubated with
Neurobasal medium containing antibiotics for 30 min at 37C in the absence
or presence of the following inhibitors: chlorpromazine (CPZ), an inhibitor of
clathrin-mediated endocytosis at several doses (25–100lM), and nystatin, an
inhibitor of caveolae-mediated endocytosis at doses comprised between
10–40lM (Ivanov, 2008; Trushina et al., 2006). The final dimethyl sulfoxide
concentration, used as solvent for nystatin, was 0.13%, which had no
significant effect on the survival of neurons (Lu and Mattson, 2001). Protein
determination was carried out using the method described by Lowry.
Western Blotting
Cells were washed with PBS and homogenized in extraction buffer (6mM
Tris buffer, 10mM EDTA, and 2% SDS, pH 7.0) with 5 ll/ml mammalian
protease inhibitor cocktail. Then, the cell lysates obtained were processed for
Western blotting as previously described (Tomás et al., 2005). Proteins were
separated on either 4–12% Bis-Tris polyacrylamide gels or 3–8% Tris-acetate
gels by loading an equal amount of protein per lane (20 lg) using the
discontinuous buffer system described by Laemmli (1970). Following
electrophoresis, proteins were transferred to nitrocellulose sheets and incubated
for 120 min with a primary antibody and then incubated for 60 min with an
alkaline phosphatase–conjugated secondary antibody. After color development,
filters were washed and scanned. Gel quantification was conducted using the
Scion Image program (Beta 4.0.2; Scion Corporation, Frederick, MA).
Linearity ranges were established by blotting different amounts of each
sample. The results are shown as the mean values of at least four to six different
experiments per group.
Transmission Electron Microscopy
In order to both evaluate the morphology of the neurons in primary culture
and analyze the presence and characteristics of the different types of the
endocytic elements present in them, cells were processed by transmission
electron microscopy. Briefly, plastic culture dishes containing growing neurons
were randomly selected (five dishes per treatment, three different cultures),
washed three times in PBS, fixed in 1.5% glutaraldehyde þ 1.0% formaldehyde
in 0.1mM cacodylate buffer, pH 7.4, for 60 min at 4C, and embedded in Epon
812 (10 blocks per culture dish) (Mayordomo et al., 1992). In some cases, cells
were embedded ‘‘in flat’’ and the fragments of the Epon dish containing cells
were glued onto Epon blocks. Ultrathin sections were double counterstained
and examined at 80 kV in a Philips CM 100 Electron Microscope.
Endocytosis of BSA in the control and ethanol-exposed neurons was
assessed by incubating cells with culture medium containing BSA conjugated
with 10-nm gold particles (1:5 dilution, A520 ~5.0) (Megı́as et al., 2000) at
37C for 60 min to label the entire endocytic pathway. After this time, cells
were washed with cold PBS and processed for transmission electron
microscopy as described above. Sections were only counterstained with uranyl
acetate to avoid the possibility of lead precipitates.
Biochemical Determination of Total Cholesterol
Since most endocytic pathways are sensitive to cholesterol perturbation
(Mayor and Pagano, 2007; Takahashi and Kobayashi, 2008), to evaluate the
effect of alcohol exposure on the levels of cholesterol in culture neurons, cells
were detached from the plastic in radio-immunoprecipitation assay buffer
containing protease inhibitors. Determination of cholesterol was then carried
out using a kit from Spinreact (Spain). The results are expressed as micrograms
of cholesterol per milligram of protein.
Statistical Analysis
The results are expressed as mean ± SD. For statistical computation and
estimation of significance, we used the online software GraphPad (GraphPad
Software, www.graphpad.com). Statistical significance was accepted when we
obtained a p value of < 0.05 using the Student’s t-test.
RESULTS
Main Characteristics of Neurons in Primary Culture
The purity of the neuron cultures was assessed by
immunofluorescence using anti-GFAP as an astrocyte marker
and the anti-MAP2 and/or anti-NF antibodies to identify the
neurons. Our cell cultures contained 97–99% of neurons,
which agrees with previous studies, indicating that glial growth
in the Neurobasal/B27 medium is reduced to less than 0.5%,
which results in a nearly pure population of neurons (BrazaBoils et al., 2006; Brewer et al., 1993, 2008; Price and Brewer,
2001). Moreover, excellent long-term survival was achieved in
the control and alcohol-exposed cells after 2 weeks in culture
with more than 95% viability as determined by the dye
exclusion method using trypan blue or the TOX7 in vitro
toxicology assay kit.
The morphology of neurons in culture was evaluated by
phase contrast microscopy (Fig. 1A) and also by the presence
of the presynaptic and postsynaptic markers SNAP-25 and
PSD-95, respectively, whose distribution was qualitatively
evaluated by immunofluorescence (Figs. 1B and 1C). The
possible effect of ethanol on the levels of these synaptic
proteins was determined by Western blot. The results are
shown in Figure 1D. Both proteins were expressed in
the control and ethanol-exposed cells (data not shown). While
exposure to alcohol did not affect the morphology of the
neurons (data not shown), such treatment reduced the amount
of PSD-95 (Fig. 1D).
Figure 2 shows several details of the ultrastructure of
the control neurons, particularly from some of the structures participating in intracellular traffic. Cells presented a
ALCOHOL AND ENDOCYTOSIS IN NEURONS
205
of these endocytic elements in the alcohol-exposed cells was
similar to that of the control neurons (data not shown). Finally,
cytoskeleton elements were observed in many cells and
synaptic contacts were also frequently found (Fig. 2E).
Endocytosis of BSA and Tf
FIG. 1. The morphology of control neurons in primary culture observed by
phase contrast microscopy (A) and after immunostaining for the demonstration
of the presynaptic and postsynaptic markers SNAP-25 (B) and PSD-95 (C) is
shown. (D) The effects of alcohol exposure on the levels of SNAP-25 and
PSD-95 were analyzed by immunoblotting. The data used in the statistical
analyses are the mean ± SD of four to six independent experiments. Asterisks
indicate significant differences (p < 0.05). Student’s t-test; bars represent 500 nm.
well-preserved morphology and displayed a developed Golgi
complex (Figs. 2A and 2B), abundant lysosomes, and multivesicular bodies (Fig. 2B). Figures 2C and 2D show several
endocytic clathrin-coated vesicles and clathrin-coated pits,
which were mainly found in the basal membrane. Interestingly,
we found no structures resembling caveolae. The morphology
The endocytosis of markers FITC-BSA and FITC-Tf was
followed by fluorescence microscopy (Fig. 3) and showed
a labeling pattern that consisted in bright spots, similar to what
different authors have previously described (see, e.g., Blanpied
et al., 2002). These spots were distributed along the neurite
outgrowths and neuronal soma. This pattern was more evident
in those neurons whose soma appeared to be more flattened.
The qualitative comparison between the control and ethanolexposed neurons did not reveal apparent differences in the
distribution pattern after both stainings. BSA endocytosis was
also analyzed using gold-conjugated BSA and electron
microscopy. In the ultrathin sections (Fig. 4), gold particles
appeared to be located on the flattened portions of the plasma
membrane (Figs. 4A and 4B) and in several types of endocytic
elements, including endosomes, lysosomes, and multivesicular
bodies (Figs. 4C and 4D).
Endocytosis of BSA and Tf. Quantitative Analysis and Effect
of Inhibitors
Figure 5 summarizes the effect of ethanol exposure on the
endocytosis of BSA where alcohol did not affect the time
course of the FITC-BSA incorporation but reduced the FITCBSA incorporation at all the time points (Fig. 5A). This effect
FIG. 2. Transmission electron microscopy images illustrating some of the main ultrastructural features of the hippocampal control neurons in primary culture.
(A) Detail of the cytoplasm of a neuron showing a well-developed Golgi complex. (B) This image illustrates the presence of lysosomal elements, mainly
multivesicular bodies (L), a Golgi complex, and several coated vesicles (arrows), some of which bud from the Golgi cisternae. (C) Arrows indicate two coated pits
located in the basal membrane of a neuron. (D) A clathrin-coated vesicle near the plasma membrane is indicated (arrow). (E) This micrograph shows several
synaptic contacts and cytoskeletal elements; bars represent 50 lm.
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MARÍN ET AL.
FIG. 3. (A) and (B) illustrate the FITC-BSA uptake in a control neuron (A)
and an alcohol-exposed neuron (B). The marker appears in bright spots
distributed along the neurite outgrowths and neuronal soma. (C) and (D) show
the images corresponding to the endocytosis of FITC-Tf in the control (C) and
treated (D) neurons. The trace accumulated in the intracellular clusters of
endosomes. No significant labeling on the cell surface was observed. No
qualitative differences between the staining pattern distribution of the different
markers in the controls and the alcohol-exposed neurons were found; bars
represent 10 lm.
was significant after a 15-min incubation, and it continued for
6 h. After this period, the amount of internalized BSA was 20%
higher in the controls than in the treated neurons. As previously
described (Mayor and Pagano, 2007), whereas Tf uptake
occurs via clathrin-coated vesicles, BSA may either be
internalized by various mechanisms in different cell types or
FIG. 4. Electron microscopy of neurons incubated with gold-BSA. In (A)
and (B), 10-nm gold particles appeared to be associated with the flattened
portions of the neuronal plasma membrane. In (C) and (D), the presence of gold
particles in the matrix of the endosomal elements is illustrated; bars represent
100 nm.
switch pathways in a single cell type under different
conditions. Therefore, to better characterize BSA uptake in
our cultures, we examined the effect of several concentrations
of CPZ and nystatin on this process in the control cells. As
Figure 5B depicts, CPZ has little effect on FITC-BSA uptake.
In contrast, the pretreatment of the control neurons with
nystatin inhibited BSA uptake by ~50% (Fig. 5C).
On the other hand, when cells were incubated for different
periods with the continuous presence of FITC-Tf in the
culture medium to saturate the entire endocytic/recycling
pathway, no significant differences between the control and
alcohol-exposed neurons were observed, which suggests
a lack of effect of alcohol on Tf endocytosis (data not
shown). To further elucidate this result, other single round of
the Tf uptake experiment series were done in which cells were
incubated at 0C in a medium containing FITC-Tf, washed
thoroughly in cold medium, and were then incubated for
several periods in a medium without Tf at 37C to induce
endocytosis. The results of these experiments are summarized
in Figure 6 and indicate that Tf endocytosed rapidly in the
control cells and that the internalized Tf was efficiently
recycled back to the plasma membrane. Tf uptake was
reduced in alcohol-exposed neurons after 2 min when
compared with control cells. After 15 min, however, the
amount of Tf inside the cells was greater in alcohol-exposed
cells than that in the controls.
Western Blot Analysis
The effect of ethanol on endocytosis in cultured neurons was
also evaluated by analyzing whether alcohol alters the amount
of the several representative proteins involved in both clathrindependent endocytosis and clathrin-independent endocytosis
pathways. The results are summarized in Table 1 and in
Figures 7 and 8. We show that the effect of alcohol on these
proteins was heterogeneous. Thus, alcohol exposure neither
affects the levels of caveolin1 nor the amount of dynamin1. In
contrast, chronic alcohol exposure clearly alters the levels of
the several proteins involved in endocytosis. Thus, we
observed a significant alcohol-induced reduction in the amount
of clathrin, AP-2, Cdc42, RhoA, and SNX9 and of RabGTPases, Rab5, and Rab11. In contrast, alcohol exposure induced
no significant variations in the levels of Rab7. Moreover, the
analysis of markers LAMP2 (data not shown) and EEA1
indicate that alcohol decreased only the level of the latter. An
important exception was observed when Arf6 was analyzed.
This protein showed an important increment in alcoholexposed neurons compared with the control neurons.
Total Cholesterol Determination
Extracts from the alcohol-exposed neurons showed a significant increase (~34%) in cholesterol content when compared
with the controls (Fig. 9). Thus, the amount of cholesterol
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ALCOHOL AND ENDOCYTOSIS IN NEURONS
FIG. 6. Effect of alcohol exposure on Tf endocytosis and recycling.
Single-round kinetics of Tf uptake and recycling were determined incubating
neurons at 0C in a medium containing FITC-Tf. After washout in cold
medium, cells were incubated for several periods in a medium without Tf at
37C to induce endocytosis. Each point represents the mean ± SD of four plates
from three independent experiments. Asterisks indicate significant differences
(p < 0.05). Student’s t-test.
DISCUSSION
FIG. 5. (A) Time dependence of FITC-BSA uptake by control and alcoholexposed (EtOH) neurons. The marker was taken up by the neurons at 37C and
the uptake increased for up to 6 h. The FITC-BSA uptake decreased in those
neurons exposed to EtOH. (B) and (C) show the effect of inhibitors nystatin and
chlorpromazine (CPZ in the text) on FITC-BSA uptake by the control neurons.
As shown, whereas CPZ has little effect on FITC-BSA uptake, the pretreatment
of cells with nystatin inhibited BSA uptake by ~50%. Each point represents the
mean ± SD of four plates from three independent experiments. Asterisks
indicate significant differences (p < 0.05). Student’s t-test.
content in the control cells was 34.66 ± 2.69 lg/mg protein,
whereas this value was 46.34 ± 3.74 lg/mg protein in the
alcohol-treated neurons.
The principal aim of the present work was to analyze
whether chronic ethanol exposure affects endocytosis by the
neurons of Tf and BSA, two widely used markers in the
analysis of this process in different cellular types. Our results
clearly demonstrate that alcohol alters the endocytosis of both
markers in cultured rat hippocampal neurons. However, to
better characterize the mechanisms involved in these effects,
we furthermore analyzed the effect of ethanol on some of the
proteins that participate in the routes involved in the
endocytosis of these markers. Whereas it is accepted that Tf
is internalized through the clathrin pathway, different mechanisms have been described for BSA endocytosis (Mayor and
Pagano, 2007; Trushina et al., 2006). From these, caveolae are
apparently the predominant route by which albumin is
internalized by a cell. Moreover, it has been recently shown
that the albumin uptake in primary striatal neurons occurs
through a clathrin-independent and caveolar-related pathways
(Trushina et al., 2006). However, very little is known about the
events that modulate albumin uptake in neurons. Interestingly,
several studies indicate that neurons in culture do not contain
morphologically detectable caveolae (Trushina et al., 2006),
and we were unable to visualize the caveolae in our cultures
using electron microscopy. Nevertheless, the expression of
caveolin1 in the neurons in culture has been demonstrated not
only in previous studies but also in the present study,
suggesting that caveolins could be implicated in physiological
roles irrespectively of caveolae, including caveolin1-related
endocytosis (Trushina et al., 2006).
To characterize the mechanism of BSA internalization, we
tested the effects of inhibitors CPZ and nystatin. CPZ had little
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MARÍN ET AL.
TABLE 1
Effects of Alcohol Exposure (30mM, 14DIV) on the Levels of Several Proteins Involved in Endocytosis in Neurons in Primary Culture
Protein
Caveolinb
Clathrinc,d
AP-2e
SNX9f
Main function and references (see below)
Variation (%)
Endocytic
mechanism
affecteda
0.0 (¼)
31.2 (Y)
16.0 (Y)
50.4 (Y)
—
CDE
CDE
CDE
Dynamin 1g
Arf6h
Integral membrane proteins that are essential structural components of caveolae
Protein that forms the basketwork around a coated vesicle
One of the multimeric adaptor proteins present in clathrin-associated complexes
This sorting nexin plays an important role in multiple membrane remodeling events, including clathrin-dependent
endocytosis, clathrin-independent, actin-dependent fluid-phase endocytosis, and dorsal ruffle formation
A GTPase expressed exclusively in neurons that participate in the cellular process of clathrin-mediated endocytosis
Involved in plasma membrane/endosomes trafficking and cortical actin reorganization
0.0 (¼)
þ 25.6 ([)
Rab5i
The small GTPase Rab5 plays an important role in budding, motility, and fusion in endocytosis
24.1 (Y)
Rab7i
Rab11i
Rab7 is located in the late endosomes and participates in the transport from early to late endosomes
Involved in the transport from early endosomes to the trans-Golgi network
0.0 (¼)
25.5 (Y)
EEA1j
Cdc42k
EEA1 has been shown to be specifically associated with the cytoplasmic face of the early endosome membrane
A small GTPase belonging to the Rho-subfamily, which regulates signaling pathways that control several cellular
functions, including endocytosis. Cdc42 is also involved in actin polymerization
A Rho GTPase that plays a key role in the regulation of actin cytoskeleton dynamics
25.0 (Y)
35.4 (Y)
—
CDE
Arf6-CIE
CDE
Cav-CIE
Cdc42-CIE
—
CDE
Arf6-CIE
Cdc42-CIE
Cdc42-CIE
34.3 (Y)
RhoA-CIE
RhoAl
Note. CDE, clathrin-dependent endocytosis; CIE, clathrin-independent endocytosis.
a
See ‘‘Discussion’’ section and Mayor and Pagano (2007).
b
Sandvig et al. (2008).
c
Peterson (2008).
d
Watson et al. (2004).
e
Benmerah and Lamaze (2007).
f
Lundmark and Carlsson (2009).
g
Smillie and Cousin (2005).
h
Jaworski (2007).
i
Ng and Tang (2008).
j
Barysch et al. (2009).
k
Kho (2006).
l
Wheeler and Rydley (2004).
effect on BSA internalization. In addition, electron microscopy
using gold-conjugated BSA reveals that gold particles were
located on the flattened portions of the plasma membrane, but
never on the clathrin-coated pits, thus indicating that BSA
uptake in the neurons was a clathrin-independent process. In
contrast, the incubation of neurons with nystatin inhibited BSA
uptake by ~50%. This suggests two things that the caveolindependent route was not efficiently inhibited by nystatin and
that there are other BSA clathrin- and caveolin-independent
mechanisms of endocytosis in neurons, which are sensitive to
neither CPZ nor nystatin. Thus, in addition to caveolinmediated endocytosis, other alternative clathrin- and caveolinindependent pathways have been described, including the
Cdc42-regulated, RhoA-regulated, and Arf6-dependent mechanisms, as well as fluid-phase endocytosis (Bonanomi et al.,
2008; Doherty and McMahon, 2009; Mayor and Pagano,
2007). The existence of different endocytic routes in neurons is
supported by the fact that the endocytosis rates vary from
several milliseconds, as with membrane retrieval at the
presynaptic terminal, to tens of minutes for the G protein–
coupled receptor internalization after agonist stimulation
(Kennedy and Ehlers, 2006). Of these routes, caveolar
endocytosis and the RhoA-regulated mechanism require
dynamin (Donaldson et al., 2009; Mayor and Pagano, 2007;
Sandvig et al., 2008). Therefore, the alcohol-induced reduction
of BSA endocytosis described herein could be the result of the
action of ethanol on one or several of the proteins that integrate
the machinery associated with these pathways. Unfortunately,
there are no well-established protocols to inhibit these routes
(Mayor and Pagano, 2007). So, although we found no changes
in the content of two important components in the caveolinmediated endocytosis, caveolin1, and dynamin1, other proteins
such as Rab5 showed significant variations after alcohol
exposure. We also found alcohol-induced variations in the
levels of Cdc42, Rab5, and EEA1 inside the Cdc42-regulated
pathway and also in the amount of Arf6 and Rab11, two
proteins associated with the Arf6-regulated pathway machinery. Alcohol also lowered the RhoA levels, suggesting that this
route could also be affected by alcohol exposure. Previous
studies have reported conflicting results of the effect of ethanol
ALCOHOL AND ENDOCYTOSIS IN NEURONS
209
FIG. 9. This graph shows the effect of chronic alcohol exposure on the
total cholesterol levels in cultured neurons. The data used in the statistical
analyses are the mean ± SD of four to six independent experiments. Asterisks
indicate significant differences (p < 0.05). Student’s t-test.
FIG. 7. The effects of alcohol exposure on the amount of clathrin-coat
associated proteins (clathrin, AP-2, SNX9, and Arf6) as well as on the levels of the
several proteins involved in caveolin-dependent and caveolin-independent endocytoses (caveolin1, RhoA, Cdc42, and dynamin 1) were analyzed by immunoblotting.
The data used in the statistical analyses are the mean ± SD of four to six independent
experiments. Asterisks indicate significant differences (p < 0.05). Student’s t-test.
on the activity and/or the levels of RhoA and Cdc42 (Guasch
et al., 2003; Martı́nez et al., 2007; Schaffert et al., 2006). In
summary, our results suggest that, in addition to a partial
inhibition (~50%) of caveolin-dependent endocytosis, alcohol
exposure also affects clathrin- and caveolin-independent pathways. Nevertheless, more work would be necessary to clarify
this point.
The actin cytoskeleton plays a pivotal role in many clathrindependent and clathrin-independent endocytic pathways
FIG. 8. Quantitative analysis of the immunoblots corresponding to the
levels of the several RabGTPases involved in endocytosis (Rab5, Rab7, and
Rab11). The level of the marker EEA1 was also examined. The data used in the
statistical analyses are the mean ± SD of four to six independent experiments.
Asterisks indicate significant differences (p < 0.05). Student’s t-test.
(Mayor and Pagano, 2007; Smythe and Ayscough, 2006),
and several of our previous studies and those of other groups
demonstrate that alcohol exposure affects actin remodeling
(Guasch et al., 2003; Sordella and Van Aelst, 2006; Tomás
et al., 2003). It is interesting to note, therefore, that several of
the proteins affected by ethanol in the present study, such as
Cdc42, Arf6, SNX9, or RhoA, are involved in the organization
and dynamics of the actin cytoskeleton since this suggests that,
as other contexts have previously proposed (Martı́nez et al.,
2007; Sordella and Van Aelst, 2006), the inhibition of
endocytosis of BSA in the ethanol-exposed neurons described
in our study could also be the result of an alcohol-induced
alteration of some regulators of actin dynamics.
Cholesterol plays a major role not only in modulating the
membrane bilayer structure but also in signal transduction and
membrane trafficking (Maxfield and Mondal, 2006). Therefore,
another factor that might be involved in the effect of ethanol on
the clathrin-independent endocytosis could be the cholesterol
level in the plasma membrane (Mayor and Pagano, 2007). This
study and previous studies have demonstrated that alcohol
exposure alters the cholesterol levels in different cell types,
including neurons that can alter the fluidity of the plasma
membranes (Parmahamsa et al., 2004; Renau-Piqueras et al.,
1987). In addition, cholesterol has been proposed to also
regulate the intracellular distribution of Rab proteins, thus
affecting membrane traffic (Takahashi and Kobayashi, 2008).
However, whereas cholesterol depletion has been reported to
affect clathrin-independent endocytosis (Mayor and Pagano,
2007), there is no information of the inverse situation, i.e., the
effect of increased cholesterol levels on this process.
We not only analyzed the effect of ethanol exposure on the
endocytosis of Tf, the most widely used model for studies into
‘‘general’’ clathrin-dependent internalization, but we also
analyzed it as a specific marker of the recycling pathway
(Maxfiel and McGraw, 2004). Surprisingly, we found no
differences between controls and alcohol-exposed cells when
210
MARÍN ET AL.
neurons were incubated with Tf under similar conditions to
those of BSA; i.e., at 37C in a medium containing FITC-Tf
over several periods of time. This first suggests that the
endocytosis of Tf is not affected by alcohol and, second, that
the possible effects of ethanol on the different steps of the
process under the conditions of a continuous presence of FITCTf have been balanced out (or compensated) with time. To
clarify this result, we carried out single round of Tf endocytic/
recycling experiments. It is well known that after Tf binds to its
receptor, it is subjected to endocytosis. Then, it is rapidly
delivered to the sorting endosome and is subsequently
transported to the recycling compartment from which it returns
to the plasma membrane. These experiments appear to confirm
the second possibility, i.e., they indicate that alcohol exposure
affects both the internalization of the cargo and its return to
the plasma membrane. Thus, whereas the initial rate of Tf
uptake in the alcohol-exposed neurons decreased in relation to
the controls, the amount of fluorescence present in the treated
cells after 15 min was greater in these cells than that in the
control neurons. This delay in Tf recycling back to the plasma
membrane in the alcohol-exposed neurons could be due to the
initial delay in Tf internalization. This effect suggests that
chronic alcohol exposure affects the fine-tuning of this process
in neurons. As suggested for BSA endocytosis, the effect of
ethanol on the Tf endocytosis could be the result of the sum of
the effects of ethanol exposure on several of the structural
proteins involved in the clathrin-mediated endocytosis or on
some of the factors modulating this process, such as the
integrity of the actin cytoskeleton. Thus, apart from decreasing
the amount of clathrin in the ethanol-exposed neurons, we also
found alcohol-induced significant variations in the adaptor
protein AP-2, which could have important effects on some
neuronal functions. Some studies have indicated that AP-2 is
one of several endocytic adaptors required for the uptake of
certain cargo proteins, including the Tf receptor (Motley et al.,
2003). It has also been reported that AP-2 is indispensable for
early embryonic development (Mitsunari et al., 2005).
Moreover, phosphatidylinositol (4,5)-bisphosphate (PIP2) has
been reported to regulate both the clathrin-mediated endocytosis of receptors throughout its binding to AP-2 and the
recycling of internalized receptors back to the plasma
membrane (Abe et al., 2008; Di Paolo and De Camilli, 2006;
Kim et al., 2006; Rohde et al., 2002) In fact, it has been shown
that the decrease of intracellular PIP2 levels lead to a loss of
endocytic clathrin-coated pits and induces severe defects in the
internalization and recycling of Tf in COS-7 cells (Kim et al.,
2006; Zoncu et al., 2007). Thus, the analysis of single-round
kinetics of Tf uptake and recycling in these PIP2-depleted cells
shows an initial delay in Tf internalization, followed by a delay
in Tf recycling back to the plasma (Kim et al., 2006).
Interestingly, these results are similar to those we observed in
the ethanol-exposed neurons and, furthermore, we have
previously reported that ethanol decreases the content of PIP2
in astrocytes, which accompanied by changes in the actin
organization (Martı́nez et al., 2007). Therefore, the effect of
ethanol on the endocytosis of Tf in neurons could also be
mediated by a decrease in the PIP2 levels. Since PIP2
participates in both cell signaling and multiple trafficking
and/or sorting events during endocytosis (Abe et al., 2008; Di
Paolo and De Camilli, 2006; Haucke, 2005; Kim et al., 2006),
the effects of ethanol on this compound should be studied in
more detail.
Alcohol also increases the levels of Arf6, which plays
a major role in clathrin-mediated endocytosis by controlling the
assembly of the AP-2/clathrin coat (Jaworski, 2007). As
commentred, Arf6 is implicated in cortical actin reorganization
and the regulation of both clathrin-dependent and clathrinindependent endocytosis (Jaworski, 2007). SNX9, which is
also affected by alcohol exposure, is another protein involved
in the formation of endocytic-coated vesicles. This protein also
helps to release the coated vesicle through the recruitment and
activation of the actin regulator N-WASP (Lundmark and
Carlsson, 2009). Moreover, actin plays an important role in the
regulation of endocytosis and clathrin coats at specialized
endocytic zones of spines and dendrites and also at late stages
of trafficking, such as returning presynaptic endocytic vesicles
back to the synaptic vesicle cluster (Blanpied et al., 2002; Jung
and Haucke, 2007; Kennedy and Ehlers, 2006). Therefore, an
alcohol-induced alteration in actin organization could also
affect these processes. In this sense, an effect of chronic alcohol
exposure on actin and PSD-95 at the PSD has been reported
(Mulholland and Chandler, 2007; Samudio-Ruiz et al., 2010),
and this study has also found a significant alteration in the
amount of PSD-95 in the alcohol-exposed neurons.
Thus, these results presented herein suggest that alcohol could
induce alterations in several processes in which clathrindependent endocytosis plays a key role, including cell signaling
and numerous cell functions, such as nutrient uptake, regulation
of the number of signaling receptors on the cell surface, and the
recycling of synaptic vesicles at nerve terminals (Inoue et al.,
2007). Moreover, alcohol could also affect processes such as
polarized axon elongation, growth cone navigation, and neuronal
migration, which depend, in part, on clathrin-dependent endocytosis (Levecque et al., 2009; Zhou et al., 2007).
In addition to the effect of alcohol on the proteins that take
part in the formation of the coat in the clathin-coated vesicles,
we also analyzed the toxic effect of this drug on the other
important factors involved in both clathrin-dependent and
clathrin-independent endocytosis. We selected three proteins
belonging to the Rab family of small GTPases, which serve as
cellular regulators of vesicular transport, particularly the
endosomal Rabs, Rab5, Rab7, and Rab11 (Deinhardt et al.,
2006b; Kho, 2006; Mayor and Pagano, 2007; Ng and Tang,
2008). It is interesting to note that many signaling receptors
traverse the endocytic pathway in a regulated manner and that
the endosomal system is intimately involved in cell signaling
(Bronfman et al., 2007; von Zastrow and Sorkin, 2007; Zoncu
et al., 2009). An example of this link between endocytosis and
ALCOHOL AND ENDOCYTOSIS IN NEURONS
cell signaling in neurons is neurotrophins, which are
synthesized in postsynaptic cells and activate presynaptic
nerve growth factor receptors, and the signal is transported
from distal axons to the soma (Sorkin and von Zastrow, 2009).
Therefore, alcohol-induced alterations to proteins from the
endosomal system could result in alterations in cell signaling.
Indeed, it has been demonstrated that ethanol affects intracellular signal pathways in neurons (De la Monte et al.,
2001; Lee et al., 2007). As mentioned, endosomal Rab5 and
Rab11 were affected by alcohol exposure. Nonetheless, we
found that alcohol also affects the levels of EEA1, which is
specifically associated with the cytoplasmic face of the early
endosome membrane (Barysch et al., 2009).
In conclusion, the present study shows that the exposure of
neurons in primary culture to chronic ethanol (0 to 14DIV,
30mM) affects both clathrin-dependent and clathrin-independent
endocytoses by probably acting not only on the several proteins
that are directly involved in the several steps of these processes
but also on the proteins that also participate in the organization
and dynamics of the actin cytoskeleton as well as on cholesterol
cellular levels. In fact, studies on the effect of the ethanol on
actin organization and dynamics in neurons are in progress.
A toxic effect of alcohol on endocytosis could affect some of
the important neuronal activities, which depend on the endocytic
process, including synaptic vesicle recycling, regulation of
the number of signaling receptors, trafficking of postsynaptic
receptors, polarized axon elongation, growth cone navigation,
neuronal migration, and hippocampal plasticity. The results that
we have obtained in neurons support the idea that one of the
main targets of ethanol is intracellular transport.
FUNDING
Ministerio de Ciencia e Innovación (SAF2008-00305);
Addictive Disorders Network (Red de Trastornos Adictivos,
Redes Temáticas de Investigación Cooperativa en Salud,
Instituto de Salud Carlos III, RD06/0001/0009 and RD06/
0001/0032), Plan Nacional sobre Drogas (PND2008-057);
Fundación de la Comunidad Valenciana para el Estudio,
Prevención y Asistencia a las Drogodependencias (FEPAD).
Fondo de Investigaciones Sanitarias fellowships to M.P.M and
G.E.-P. FEPAD fellowships to G.E.-P. and R.B.
ACKNOWLEDGMENTS
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