Voltage-dependent anion channel (VDAC) participates in amyloid beta-induced toxicity and interacts...
Transcript of Voltage-dependent anion channel (VDAC) participates in amyloid beta-induced toxicity and interacts...
Voltage-dependent anion channel (VDAC) participates in amyloidbeta-induced toxicity and interacts with plasma membrane estrogenreceptor a in septal and hippocampal neurons
RAQUEL MARIN1*, CRISTINA M. RAMIREZ1*, MIRIAM GONZALEZ1,
ELENA GONZALEZ-MUNOZ2, ANTONIO ZORZANO2, MARTA CAMPS2,
RAFAEL ALONSO1, & MARIO DIAZ3
1Laboratory of Cellular Neurobiology, Department of Physiology & Institute of Biomedical Technologies, University of La
Laguna, School of Medicine, Santa Cruz de Tenerife, Spain, 2Department of Biochemistry and Molecular Biology, Faculty of
Biology, University of Barcelona, and IRBB-PCB, Parc Cientific of Barcelona, Barcelona, Spain, and 3Laboratory of
Animal Physiology, Department of Animal Biology & Institute of Biomedical Technologies, Faculty of Biology, University of
La Laguna, Santa Cruz de Tenerife, Spain
(Received 15 May 2006; and in revised form 19 September 2006)
AbstractVoltage-dependent anion channel (VDAC) is a porin known by its role in metabolite transport across mitochondria andparticipation in apoptotic processes. Although traditionally accepted to be located within mitochondrial outer membrane,some data has also reported its presence at the plasma membrane level where it seems to participate in regulation of normalredox homeostasis and apoptosis. Here, exposure of septal SN56 and hippocampal HT22 cells to specific anti-VDACantibodies prior to amyloid beta (Ab) peptide was observed to prevent neurotoxicity. In these cell lines, we identified aVDAC form associated with the plasma membrane that seems to be particularly abundant in caveolae. The two membrane-related isoforms of estrogen receptor a (mERa) (80 and 67 kDa), known in SN56 cells to participate in estrogen-inducedneuroprotection against Ab injury, were also observed to be present in caveolae. Interestingly, we demonstrated for the firsttime that both VDAC and mERa interact at the plasma membrane of these neurons as well as in microsomal fractions of thecorresponding murine septal and hippocampal tissues. These proteins were also shown to associate with caveolin-1, therebycorroborating their presence in caveolar microdomains. Taken together, these results suggest that VDAC-mERa associationat the plasma membrane level may participate in the modulation of Ab-induced cell death.
Keywords: Voltage-dependent anion channels, amyloid beta toxicity, estrogen receptor, caveolae, SN56 cells, HT22 cells
Abbreviations: VDAC, voltage-dependent anion channel; ER, estrogen receptor; Amyloid beta, Ab; MAPK,
mitogen-activated protein kinase.
Introduction
Voltage-dependent anion channels (VDACs), also
named porins, are traditionally known to be located
at the outer mitochondrial membrane as compo-
nents of the multiprotein pore complex, where they
participate in the transport of ATP and other
metabolites (Colombini, 1994). VDAC has also
been proposed to be involved in apoptotic events
through direct interactions with apoptogenic factors
such as Bcl-2 family, thereby provoking the release of
cytochrome c from mitochondria and caspases
activation (Shimizu et al. 1999, Harris & Thompson
2000, Tsujimoto & Shimizu 2000). Moreover,
increasing evidence has claimed the location of
VDAC within the plasma membrane in a variety of
cellular types (Thinnes et al. 1989, Dermietzel et al.
1994, Reymann et al. 1995, Buettner et al. 2000,
Bahamonde & Valverde 2003), although its presence
in neural tissue remains to be explored. Even though
the role of this channel at the plasma membrane has
not been still elucidated, some recent data have
*These authors contributed equally to this work.
Correspondence: Dr Raquel Marin, Laboratory of Cellular Neurobiology, Department of Physiology, University of La Laguna, School of
Medicine, La Laguna, 38071 Santa Cruz de Tenerife, Spain. Fax: �/34 922 31 93 97. E-mail: [email protected]
Molecular Membrane Biology, March�April 2007; 24(2): 148�160
ISSN 0968-7688 print/ISSN 1464-5203 online # 2007 Informa UK Ltd
DOI: 10.1080/09687860601055559
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suggested a dual action, on one hand, to participate
in the maintenance of normal redox homeostasis
and, on the other hand, to modulate apoptotic
events (Elinder et al. 2005). The modulation of
VDAC at the plasma membrane level is still largely
unknown, and only a few data in a variety of cell
types have suggested that overall Maxi Cl� channels
may be modulated by different factors such as
unsaturated fatty acids (Riquelme & Parra 1999),
GTP-binding proteins (Schwiebert et al. 1990,
McGill et al. 1993), nucleotides (Schwiebert et al.
1992, Mitchell et al. 1997), phosphorylation (Paha-
pill & Schlichter 1992, Liberatori et al. 2004), and
direct interaction with hormones and anti-hormones
(Kajita et al. 1995, Li et al. 2000, Dıaz et al. 2001,
Valverde et al. 2002).
Amyloid beta peptide (Ab) is a 39�43 amino acid
peptide known to accumulate in senile plaques
which represents one of the pathological hallmarks
of Alzheimer’s disease (AD) (Yankner et al. 1989).
Ab-induced cytotoxicity has been shown in different
cultured cells to be preceded by mitochondrial
dysfunction and signalling events characteristic of
apoptosis (Takuma et al. 2005). Of particular
clinical interest is the fact that numerous in vivo
and in vitro paradigms of AD pathology have
demonstrated that estrogen can prevent neuronal
death from Ab toxicity through still unclear mechan-
isms (Garcia-Segura et al. 2001, Wise et al. 2001;
Wise 2002). We have previously demonstrated in
septal-derived SN56 cells that, apart from classical
ERa (Marin et al. 2003a), a membrane-related ERa-
like (mER) is also involved in cell survival against Abtoxicity (Marin et al. 2003b). Other examples of
mER mediation in estrogen neuroprotective effects
have also been reported in response to different
toxicities, including glutamate, serum deprivation,
and different Ab fragments (Toran-Allerand 2004).
However, important questions remain with respect
to the plasma membrane integration of these alter-
native receptors within this hydrophobic structure,
and their overall functional regulation at this parti-
cular domain.
Here, we have explored whether VDAC may be
participating in the modulation of Ab neurotoxicity
in septal- and hippocampal-derived cell lines.
Furthermore, the possibility that VDAC may be
located at the plasma membrane prompted us to
explore the putative interactions of this porin with
ERs at the plasma membrane of these cultured
neurons as well as in microsomal fractions from
their corresponding brain areas, known to be da-
maged in neurodegenerative disorders.
Materials and methods
Materials
SN56 cells and HT22 cells were provided, respec-
tively, by Dr Bruce Wainer, Wesley Woods Health
Center, Atlanta, GA, USA, and Dr Schubert, The
Salk Institute for Biological Studies, La Jolla, CA,
USA. Nalco 1060 colloidal silica was obtained from
Nalco Chemical Co. (Chicago, IL, USA). The
primary polyclonal anti-caveolin-1 (N-20) antibody
and anti-ERa (MC-20) antibody were from Santa
Cruz Biotechnology (Inc. Sta. Cruz, California,
USA). The primary monoclonal anti-VDAC direc-
ted to the amino terminal region of human porin,
anti-flotillin and anti-Hsp90 antibodies were pur-
chased from, respectively, Calbiochem (La Jolla,
California, USA), BD Transduction laboratories
(Madrid, Spain), and Stressgen biotechnologies
(Victoria, Canada). Polyclonal anti-VDAC antibody
directed to carboxi terminal region and polyclonal
uncoupling protein 1 (UCP-1) antibody were from
Affinity Bioreagents (Madrid). Amyloid beta 1�40
and 25�35 fragments were purchased from Calbio-
chem. Complete protease inhibitor cocktail and cell
proliferation reagent WST-1 were obtained from
Roche Diagnost (Manheim, Germany). The poly-
clonal antibody directed to Na��K�-ATPase a1
subunit was from Upstate (Madrid, Spain). Dyna-
beads sheep anti-rabbit and anti-mouse IgG were
from Dynal (Oslo, Norway). The cyanine 2 and
cyanine 3 dye-conjugated streptavidin were from
Jackson Laboratories (Baltimore, PA, USA). Mito-
tracker Red 580 was from Invitrogen (Paisley, UK).
Imaging Densitometer was obtained from Bio-Rad
laboratories (Hertfordshire, England), and laser
scanning confocal imaging system (FluoView
1000) was from Olympus Optical Espana (Barce-
lona, Spain).
Methods
Ab toxicity quantification and competition assays with
anti-VDAC antibodies. SN56 and HT22 cells were
grown under conditions previously described (Marin
et al. 2001). For culture treatments, cells were
subcultured in 96-well plates at a density of 104/ml
for 24 h. Then, cells were exposed to VDAC specific
antibodies directed to either amino- or carboxi-
terminal at 10 nM for 2 h. After exposure to the
antibodies, toxicity was induced with 5 mM Ab1�40
in the case of SN56 cells or with 5 mM Ab25�35 in
the case of HT22 cells, both diluted in 0.05%
DMSO for 24 h, and cells were finally proceeded
to survival quantification. Cell viability was assessed
by a colorimetric assay based on the cleavage of
VDAC-ER association at nerve plasma membranes 149
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tetrazolium salt WST-1 in viable cells, following the
manufacturer’s instructions.
As a control of Ab1�40 and Ab25�35 toxicity, some
cultures were exposed to either peptide alone. Data
were referred to cell death relative to cultures treated
with Ab vehicle and antibody vehicles.
Isolation of cell plasma membrane and mouse brain
microsomal fractions. Highly pure plasma membranes
of both SN56 and HT22 cultures were isolated using
the cationic colloidal silica technique (Chaney &
Jacobson 1983). Septal and hippocampal microso-
mal fractions were obtained from mice aged 60 days,
and processed as previously described (Marin et al.
2006). Animals were anesthetized with diethylether
and killed by decapitation. Animal procedures com-
ply with the Animal Care and Use Local Committee
at La Laguna University. Septal and hippocampal
tissues were dissected out in RSB buffer (10 mM
Tris-HCl, pH 8; 20 mM NaCl; 25 mM EDTA pH
8) with complete proteases inhibitor cocktail.
Detergent-free caveolae extraction. SN56 and HT22
cell cultures were carried out following the proce-
dure described by Song et al. (1996) for purification
of caveolin-rich membrane fractions. The different
sucrose bands obtained were used for Western blot
experiments. As a control of caveolae enrichment,
dot-blotting was performed using 2 ml volume of
each band to bind onto Hybond-C membranes.
Membranes were pre-incubated with 1% BSA di-
luted in Tris-buffered saline (TBS) [25 mM Tris-
HCl, pH 7.4; 137 mM NaCl; 50 mM KCl] for 1 h at
room temperature, and then exposed to horseradish
peroxidase-conjugated cholera toxin subunit B for
45 min at room temperature. Specific spots related
to the relative abundance of glycosphingolipid-toxin
subunit complexes were revealed with the ECL
chemiluminescence kit.
Immunoprecipitation. Protein suspensions from the
different samples were resuspended in cold immu-
noprecipitation buffer [50 mM Tris-HCl, pH 7.4;
150 mM NaCl; 10% glycerol; 1% Nonidet-P 40;
1 mM phenyl methyl sulfonyl fluoride (PMSF);
complete proteases inhibitor cocktail]. Samples
were processed using dynabeads, following the
manufacturer instructions. Excess (5�10 mg of anti-
body/mg protein) of anti-ERa antibody was added to
the extracts, and samples were incubated overnight
at 48C. Immunoprecipitated proteins bound to the
beads were extracted with SDS-PAGE loading
buffer.
Immunoblotting. For Western blot analysis, pro-
tein samples were resuspended in loading buffer
(625 mM Tris-HCl, 1% sodium dodecyl sulphate,
10% glycerol, 5% b-mercaptoethanol and 0.001%
bromophenol blue, pH 6.8), and boiled for 5 min.
Proteins were electrophoresed on 12.5% SDS-
PAGE. Proteins on gels were transferred to Hy-
bond-P membranes, and incubated with the differ-
ent primary antibodies used in this study, i.e., anti-
ERa (1:200), anti-VDAC (1:2000), anti-caveolin-1
(1:200) and anti-flotillin (1:500) antibodies. As
controls of the purity of plasma membrane and
mitochondrial extractions, anti-Na��K�-ATPase
a1-subunit (1:5000), anti-Hsp90 (1:2500) and
anti-UCP (1:1000) antibodies were used to reblot
onto these same membranes. Antibody labelling was
revealed by incubation with horseradish peroxidase-
conjugated secondary antibodies (1:10,000), and
visualized with the ECL chemiluminescence kit
(Amersham).
Immunocytochemistry. SN56 and HT22 cultures were
fixed under unpermeabilized conditions in PBS, pH
7.4, containing 2% paraformaldehyde, 1% glutar-
aldehyde and 120 mM sucrose that have been
previously demonstrated to preserve plasma mem-
brane integrity, thereby avoiding intracellular anti-
body leaking. In another set of experiments, cells
were fixed in the presence of Nonidet P-40 (0.5%)
for 1 min, in order to permeabilize the plasma
membrane. When using Mitotracker Red 580, the
compound (5 mM diluted in culture medium) was
incubated in vivo for 15 min prior to cell fixation and
permeabilization. Immunocytochemical assays were
performed as previously described (Marin et al.,
2003b). MC-20 anti-ERa (1:50) and Ab-2 anti-
VDAC antibodies were diluted, respectively, at 1:50
and 1:400 in PBS with 1:200 normal serum. Then,
the corresponding secondary biotinylated anti-rabbit
antibody and cyanine-3-coupled anti-mouse anti-
body (1:200 in PBS) were incubated for 1 h at room
temperature. Staining was revealed by exposure to
cyanine-2 dye-conjugated streptavidin (1:500) for
30 min at room temperature. In experiments using
Mitotracker Red 580, after incubation with anti-
VDAC antibody, cells were exposed to secondary
cyanine-2 dye-conjugated anti-mouse antibody.
Fluorescence signals were monitored using a laser
scanning confocal imaging Fluoview 1000 system.
Statistical procedures
Differences between sample means were assessed by
one-way analysis of variance (ANOVA) followed by
either Student-Newman-Keuls t-test or post hoc
Tukey HSD test or Bonneferroni’s t-test where
appropriate. Numerical results are expressed as
mean9/SEM.
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Results
Exposure to specific antibodies directed to VDAC reduces
SN56 and HT22 cell death during Ab-induced injury
As we have previously demonstrated for SN56 cells
(Marin et al. 2003a), exposure of cultures to 5 mM
Ab1�40 for 24 h induced an elevated cell death
(�/ 80%) as compared to vehicle-treated cultures
(Figure 1A). In addition, HT22 cells were shown
here to die in a similar percentage when exposed to
5 mM Ab25�35 (Figure 1B). In contrast with SN56
cell line, Ab1�40 produced a lower toxic effect in
HT22 cells (data not shown). Pre-incubation of
SN56 and HT22 cultures with anti-VDAC antibody
(Ab-2) directed to the C-terminal region of human
porin significantly reduced amyloid-induced cell
death to less than 30% (Figure 1). Strikingly,
statistical analyses showed that this antibody sig-
nificantly reduced Ab1�40-induced toxicity in SN56
cells and Ab25�35-induced toxicity in HT22 cells
(pB/ 0.01, Tukey’s HSD test). A lower percentage of
cell mortality reduction, but still statistically signifi-
cant, was obtained when cultures were exposed to
another antibody directed to N-terminal region (Ab-
1). However, the vehicle of Ab-1 antibody contains
azide (0.02%), a potent cell toxic agent which
caused considerable cell mortality (63 and 81%
for, respectively, HT22 and SN56 cells), therefore
making its addition unable in amyloid beta toxicity
treatments. Conversely, the vehicle of Ab-2 anti-
VDAC antibody did not show any significant
changes in cell viability as compared to controls
(B/0.1%).
VDAC is localized at the plasma membrane domain of
both SN56 and HT22 cells
To study the putative extramitochondrial localiza-
tion of VDAC in SN56 and HT22 cell lines, we
performed plasma membrane isolations using the
cationic silica technique (Figure 2). Western blot
analyses of membrane fractions (MF) or whole cell
lysates (W) probed with anti-VDAC Ab-2 antibody
revealed a single band of 35 kDa, which is the
expected Mw for this protein. Two bands of 80 and
67 kDa corresponding to a membrane-related ERa-
like (mER) were also shown in these cells when
reprobing membranes with a specific anti-ERapolyclonal antibody (MC-20), as previously de-
scribed by our group (Marin et al. 2003b, 2005).
Potential contamination of membrane isolates with
cytosolic components was tested with an antibody
directed to intracellular chaperonin Hsp90 which
recognized a 90-kDa band in cytosolic extracts only,
whereas an antibody against the a1 subunit of Na�/
K� ATPase revealed a characteristic 120-kDa band
specifically in the membrane, but not cytosolic
fractions. Also, an antibody directed to a mitochon-
drial marker namely uncoupling protein 1 (UCP-1)
was used to confirm that VDAC expression in
Figure 1. Specific anti-VDAC antibodies reduce amyloid bpeptide (Ab)-induced neuronal mortality. SN56 (A) and HT22
(B) cultures were pre-incubated with specific antibodies directed
to VDAC (Ab-1 and Ab-2, 10 nM) for 2 h at 378C prior to
treatment with 5 mM Ab1 �40 (SN56) and Ab25 � 35 (HT22) for 24
h. Another set of cultures was exposed to the peptide alone (Ab).
As a control of viability, cells were concomitantly exposed to
peptide or antibody vehicles only. O.D. represents absorbance
units measured at 450 nm relative to vehicle ones, as a direct
correlation with the number of viable cells in the culture. *** p B/
0.001 vs. Ab; ** p B/0.01 vs. Ab; �/ p B/0.05 vs. Vehicle; �/�/ p B/
0.01 vs. Vehicle; �/�/�/ p B/0.001 vs. Vehicle. Seven assays per
group.
VDAC-ER association at nerve plasma membranes 151
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plasma membrane fractions was not due to the
presence of mitochondrial proteins in these particu-
lar fractions.
To confirm the membrane-associated presence of
VDAC, we performed immunocytochemical assays
in non-permeabilized cells incubated with Ab-2
antibody (Figure 3A). Some immunosignals at the
cell surface were observed by confocal microscopy,
and were also noticed in neurites in a spot-like
pattern (arrows), therefore corroborating a mem-
brane-related labelling of anti-VDAC antibody.
VDAC is present in mitochondrial sub-populations of
these cell lines
We next validated the expected presence of this
porin at the mitochondria of both SN56 and HT22
cell lines using immunocytochemistry and confocal
microscopy (Figure 3B). Mitochondria of cell cul-
tures were identified with MitoTracker Red 580 (red
colour), prior to fixation under detergent-permeabi-
lizing conditions and incubation with mouse mono-
clonal anti-VDAC antibody (Ab-2), followed by
incubation with secondary antibody tagged to cya-
nine 2 (green colour). Co-localization of both dyes
was indicated by overlay of the red and green
channels (third panel). Confocal microscopy analy-
sis revealed a high co-localization of both mitochon-
drial markers in the two cell lines studied
(respectively, 76% for SN56 and 78% for HT22),
confirming the presence of this porin in mitochon-
drial subpopulations mainly located at perinuclear
areas. Such differential VDAC localization in mito-
chondria has been previously observed in other cell
types (Bahamonde & Valverde 2003).
VDAC is concentrated in caveolar fractions
Some previous studies in neurons have evidenced
the presence of VDAC and ER in caveolar-like
microdomains (Bathori et al. 1999, Toran-Allerand
et al. 2002). Based upon these observations, we
explored in SN56 and HT22 cell lines whether
either VDAC or mER may be integrated in these
plasma membrane microstructures. Using a sodium
carbonate detergent-free method for purification of
caveolae membranes, we tested for the presence of
VDAC and mERa. Caveolae fractions were obtained
by discontinuous sucrose-gradient centrifugation,
which resulted in ten fractions and the pellet. Equal
aliquots of these fractions were loaded on 12.5%
SDS-PAGE and proteins were stained with Coo-
massie blue (Figure 4A). Proteins resolved in 12.5%
SDS-PAGE were transferred to Hybond-P mem-
branes for Western blotting, and incubated with Ab-
2 and MC-20 antibodies. Data showed that both
proteins were present at low density fractions 5�7
corresponding to caveolae (F5�F7, Figure 4A)
although, as compared to VDAC, a higher amount
(more than two fold) of caveolar protein extract was
required to detect the presence of ERa. Thus,
VDAC was particularly abundant in these fractions
whereas mERa was observed to be only partially
present. As immunoblotting control, whole cell
protein extracts were also used to incubate with
Figure 2. Presence of VDAC in plasma membrane fractions of
SN56 and HT22 cell lines. Proteins from plasma membrane
purifications by the cationic silica method were loaded on 12.5%
SDS-PAGE for Western blot analysis with antibodies directed to
either VDAC or ERa proteins. As a control of plasma membrane
purification, membranes were reblotted with, both, a polyclonal
antibody directed to the Na��K�-ATPase a1-subunit and a
monoclonal antibody to Hsp90. As a control of the potential
contamination with mitochondrial proteins, an antibody directed
to the mitochondrial marker UCP-1 was also used. MF and CF
represent, respectively, samples of plasma membrane fractions
and equivalent cytosols. Whole protein extracts (W) were loaded
for comparison purpose.
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MC-20 antibody (WCE). To verify caveolae distri-
bution, an anti-caveolin-1 antibody was used to
reblot onto these same membranes, observing ca-
veolin-1 enrichment in F5�F7 fractions, thereby
indicating at least a partial localization of VDAC
and mERa in caveolae-like domains (Figure 4B).
Flotillin-1, another protein known as a lipid raft
marker was also found in these same fractions
whereas clathrin, a plasma membrane associated
protein known to be absent of caveolae, was not
present in caveolin-enriched fractions. The purity of
the caveolae-like fractions was also tested by dot-blot
assays using extracts from the different sucrose-
gradient centrifugation fractions to incubate with
Figure 3. Patterns of immunofluorescent staining of VDAC in SN56 and HT22 neurons. Cell cultures fixed under either non-
permeabilized (A) or detergent-permeabilized (B) conditions were labelled with anti-VDAC antibody. In the case of detergent-treated cells,
in vivo cultures were previously exposed to Mitotracker Red 580 for 15 min prior to fixation and permeabilization. After primary antibody
incubation, cells were exposed to secondary goat biotinylated anti-mouse antibody and cyanine 2 dye-conjugated streptavidin. Phase
contrast images are also shown to visualize cell shape. (A) Notice the staining in neurites (arrows). (B) Panel on the right illustrates
colocalization of the digital imaged composed by fluorescent signals containing both green and red colour distributions obtained with
FluoView 1000 software. The overlap spots are indicated by black spots. Bar: 50 mm. This figure is reproduced in colour in Molecular
Membrane Biology online.
p
VDAC-ER association at nerve plasma membranes 153
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horseradish peroxidase conjugated to cholera toxin bsubunit, a lipid raft marker which binds to ganglio-
sides (Figure 4C). The toxin was found to bind
preferentially to caveolin-enriched F5�F7 fractions,
thus confirming the caveolae-like enrichment parti-
cularly in these fractions. To ensure the purity of
caveolar fractions free of mitochondria contamina-
tion, additional western blot assays were performed
on both, caveolin-enriched and mitochondrial frac-
tions, using anti-VDAC antibody and anti-UCP-1
antibody (Figure 4D). Although UCP-1 was present
exclusively in mitochondria, the porin was observed
in either caveolar or mitochondrial fractions. Alto-
gether, these results demonstrate that both, VDAC
and ERa proteins are observed in caveolae micro-
domains.
VDAC associates with mER at the plasma membrane of
SN56 and HT22 cells
Since VDAC and mER are both observed to be
present at the plasma membrane, we explored
whether these two proteins may be interacting at
this level. In protein extracts from either whole
lysates or purified plasma membrane fractions,
immunoprecipitation of ERa using MC-20 antibody
resulted in the co-precipitation of the porin in both
neuronal lines. Figure 5 illustrates a representative
Western blot obtained with SN56 cultures. Similar
results were obtained with HT22 cultures (data not
shown). Immunoprecipitation with non-immune
antisera did not result in either the precipitation or
co-precipitation of these proteins (Figure 5, lane C).
As a control of immunoprecipitation specificity, an
antibody against Hsp90, a protein previously ob-
served to associate with cytosolic ER in SN56 cells
(Marin et al. 2001), was also used to immunoblot
observing, as expected, its co-precipitation with
canonical ER in whole lysates (IPW), but not in
membrane fractions (IPMF). An anti-caveolin-1
antibody was also shown to immunoblot with the
immunoprecipitated samples, indicating that caveo-
lin-1 also associates with mER in these purified
fractions.
Figure 4. Pattern of VDAC and ERa distribution and caveolin-1
co-expression in sucrose gradient fractions from SN56 and HT22
cells. (A) Caveolae-enriched fractions from SN56 and HT22 cells
were obtained as described in Materials and Methods. Aliquots of
equal volume from each sucrose-gradient fraction were loaded on
12.5% SDS-PAGE before staining with Coomassie blue. Mole-
cular weight standards are indicated. Most protein was detected in
fractions 9�11 (F9�F11). Protein samples from the different F1�F11 fractions were transferred to Hybond-P membranes for
Western blotting assays, using specific antibodies to VDAC and
ERa proteins. Pattern of the different mERa-like isoforms
visualized only when loading high amount (�/two fold) of caveolar
fractions has been magnified. As a control of antibody binding,
total protein extracts (W) were also loaded. (B) As controls of
caveolae isolation, antibodies directed to caveolin-1, lipid raft
marker flotillin and chlatrin were also used. (C) As an additional
control of caveolae enrichment, 2 ml aliquots of each fraction was
bound to Hybond-C membranes and exposed Cholera toxin B
subunit conjugated to horseradish peroxidase. Most glycosphin-
golipids were found in F5�F7. (D) To ensure caveolar purification
free from mitochondrial contamination, additional Western blots
using total protein extracts (W), and mitochondrial (Mt) and F6
caveolar fraction (C) were performed using antibodies directed to
VDAC and uncoupling protein 1 (UCP-1).
Figure 5. Association between VDAC and mERa at the plasma
membrane of SN56 and HT22 cells. Plasma membrane fractions
from both cell lines were used to immunoprecipitate with
polyclonal MC-20 anti-ERa antibody (IPMF), and the resultant
precipitated proteins were immunoblotted with the corresponding
antibodies directed to VDAC, caveolin-1 and Hsp90. As a control
of immunoprecipitation efficiency, total protein extracts were also
used to purify ERa protein (IPW). In lanes W and MF, whole
protein extracts (W) and membrane fractions (MF) were run as
controls. No bands were detected in the absence of primary
antibodies (C).
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We next performed immunocytochemical assays
under non-permeabilizing conditions to visualize by
confocal microscopy the potential VDAC-mER
colocalization at the plasma membrane. SN56 and
HT22 cells were fixed in the absence of detergent
and co-incubated with anti-VDAC Ab-2 and anti-
ERa MC-20 antibodies, followed by incubation with
corresponding secondary antibodies coupled to
either FITC or cyanine-3 fluorophores. Results
monitored by confocal microscopy showed some
immunosignals for both antibodies associated with
the cell surface and in neurites, thus confirming
the plasma membrane-related labelling of VDAC
and mER proteins (Figure 6). Examination of
double fluorescence overlapping due to the close
proximity of emitted signals corresponding to each
of the two antibodies revealed some colocalization
of both proteins at the plasma membrane level. The
quantitative degree of fluorophore colocalization was
measured in scatterplotting analysis using confocal
microscope software programs, obtaining coeffi-
cients of approximately 30�40%. No immunosignals
were detected in the absence of the primary antisera,
thus confirming immunofluorescence specificity
(data not shown). Therefore, VDAC appears to
interact with ERa-like at the plasma membrane level
of these neuronal lines, suggesting the interactive
functionality of these two proteins in this domain.
VDAC is localized in murine septal and hippocampal
microsomal fractions where it interacts with a membrane-
related ERa-like
In an attempt to validate the presence of VDAC at
the plasma membrane of neurons from the original
tissues in vivo, in another set of Western blot
experiments microsomal purified fractions from
mouse septum and hippocampus were also used to
incubate with both, Ab-1 and MC-20 antibodies
(Figure 7). Ab-1 antibody was observed to recognize
a 35 kDa band corresponding to VDAC protein in
either whole protein (W), microsomal (M) or
mitochondrial (Mt) extracts from both septal and
hippocampal tissues. Similarly to the results ob-
tained with immortalized cell lines, immunoblotting
with an anti-ERa antibody revealed membrane-
related ERs migrating at the expected 67 kDa, as
well as higher bands at 80 kDa and 97 kDa, thus
corroborating our previous observations (Marin et
al. 2006). As a control of microsomal purity and
possible contamination with cytosolic molecules,
antibodies directed to a1 subunit of Na�/K�
ATPase and Hsp90 were also incubated in these
samples. Furthermore, an antibody directed to
UCP-1 was used as a control of mitochondrial
protein extraction. These results confirm the exis-
tence of a membrane-related VDAC in murine septal
and hippocampal neurons.
Figure 6. Colocalization of VDAC and mERa at the plasma membrane of SN56 and HT22 neurons. Cells were fixed under detergent-free
non-permeabilized conditions, and incubated with either MC-20 anti-ERa or anti-VDAC antibodies. After washing, cultures were exposed
to corresponding secondary goat biotinylated anti-rabbit antibody, and anti-mouse antibody labelled to cyanine 3. MC-20 staining was
revealed by incubation with cyanine 2 dye-conjugated streptavidin. Panel on the right illustrates colocalization of the digital imaged
composed by fluorescent signals containing both green and red colour distributions. The overlapped pixels are indicated by black spots. Bar:
50 mm. This figure is reproduced in colour in Molecular Membrane Biology online.
VDAC-ER association at nerve plasma membranes 155
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Evidence of the presence of mERs in mouse
septum and hippocampus shown here and in
previous work (Clarke et al. 2000, Milner et al.
2001, Towart et al. 2003, Marin et al. 2006), led us
to explore the putative VDAC-mER association in
these neural tissues. Immunoprecipitation assays in
protein extracts from septal and hippocampal
microsomes using MC-20 antibody revealed the
co-precipitation of VDAC (Figure 8). Caveolin-1
was also shown to co-precipitate in these samples,
thus confirming the association of the protein with
this complex obtained with SN56 and HT22 cell
lines. The high signal obtained with MC-20 anti-
body may be due to the excess of ERa immuno-
precipitated with this antibody. However, the
alternative assay using anti-VDAC antibody to co-
precipitate ERa could not be performed, as no
antibodies directed to VDAC recommended for
immunoprecitation assays are presently available.
No electrophoretic bands were detected in micro-
somal extracts incubated in the absence of primary
antibodies, thus confirming immunoprecipitation
specificity (not shown). These results corroborate
that VDAC and ER are interacting at the plasma
membrane in both septal and hippocampal tissues,
suggesting that this association may be a general
phenomenon, at least in neurons.
Discussion
The voltage-dependent anion channel (VDAC) in
eukaryotes has been traditionally considered to
localize exclusively in mitochondria, as part of the
pore transition protein complex. However, besides
mitochondrial localization, several evidences have
indicated the presence of a plasmalemmal VDAC
protein highly homologous to the mitochondrial
porin in different cell types (Thinnes et al. 1989,
Reymann et al. 1995, Buettner et al. 2000), includ-
ing neuroblastoma cells (Bahamonde & Valverde
2003) and astrocytes (Dermietzel et al. 1994). In the
present report, we have demonstrated by immuno-
blotting of membrane purified fractions, and by
immunocytochemistry and confocal microscopy the
localization of VDAC within the plasma membrane
of septal SN56 and hippocampal HT22 cell lines, as
well as in microsomal fractions of mouse septal and
hippocampal areas which are particularly relevant in
cognitive processes. The presence of VDAC at the
plasma membrane of HT22 that is activated during
apoptosis has been also observed in other recent
studies (Elinder et al. 2005, Akanda & Elinder 2006)
although, to our knowledge, our data represent the
first report demostrating the presence of a VDAC
Figure 7. Presence of either VDAC or mERa-like molecules in
microsomal fractions of mouse septum and hippocampus. (A)
Protein extracts from microsomal (M) or mitochondrial (Mt)
fractions of murine septum and hippocampus were loaded on
SDS-PAGE and immunoblotted with polyclonal MC-20 anti-
body, and with an antibody directed to VDAC. As a control of
antibody immunoaffinity, protein extracts from whole septal and
hippocampal tissues (W) were also loaded. (B) As a control of
either microsomal or mitochondrial fraction purity, samples were
reblotted with anti-Na�/K� ATPase a-1 antibody, as a marker of
microsomal fractions, and with anti-uncoupling protein 1 anti-
body, as a marker of mitochondrial fractions.
Figure 8. Association between VDAC, mERa and caveolin-1 in
microsomal fractions of murine septum and hippocampus.
Microsomal fractions from both septum and hippocampus were
used to immunoprecipitate with MC-20 antibody (IPM). The
resultant precipitated fractions were loaded on SDS-PAGE to
immunoblot with specific anti-VDAC and anti-caveolin-1 anti-
bodies. Total microsomal protein extracts were run as controls
(W).
156 R. Marin et al.
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isoform associated with the plasma membrane in
septal neurons. Another piece of evidence corrobor-
ating an extramitocondrial VDAC form has been its
presence in caveolae-enriched fractions of sucrose-
gradient centrifugation where caveolin-1 and flotil-
lin, the neuronal homologue of caveolin, were also
abundant. Previous studies have also demonstrated
the presence of the porin within caveolae-like
microdomains from bovine brain extracts (Bathori
et al. 1999). Immunocytochemical methodology also
allowed us to confirm the localization of VDAC at
the plasma membrane of non-permeabilized cells.
However, the precise function of the plasma mem-
brane related form of this porin remains to be
elucidated. Some authors have suggested that it
may function as a NADH- (ferricyanide) reductase
at the plasma membrane level, exerting a dual role,
on the one hand, in maintenance of redox homeo-
stasis in normal cells and, on the other hand,
apoptosis modulation in response to cell require-
ments (Elinder et al. 2005). VDAC has also been
proposed to mediate ATP translocation across the
plasma membrane contributing to cell volume reg-
ulation (Okada et al. 2004).
A main interest of this work is the potential
participation of VDAC in Ab-induced cytotoxicity,
as evidenced here using specific anti-VDAC anti-
bodies to inactivate this porin. In particular in the
case of the antibody directed to the C-terminus of
VDAC, interaction of this antibody with the porin
was able to considerably reduce cell death whereas
the antibody directed to the N-terminus induced a
lower, but still significant, effect on cell survival.
Although still remains to be elucidated, these
differences may be explained by the putative con-
formation of this channel at the plasma membrane,
probably rendering the molecule somehow less
accessible to antibodies directed to N-terminal
epitopes. In agreement with this possibility, it has
been proposed that the N-terminal domain of fungal
VDAC resides in a groove inside the lumen of the
mitochondrial wall (Mannella 1998). This novel
evidence may raise new hints about the control of
Ab-induced toxicity triggered at the plasma mem-
brane. Thus, one could hypothesize that Ab inter-
acting with this domain might provoke in some
manner VDAC activity thereby contributing to cell
death through unknown mechanisms leading to
intracellular apoptosis. Consistent with this notion,
some evidences have demonstrated a global decline
in mitochondrial activity in AD brain (Ojaimi &
Byrne 2001).
Different ERa-like forms were also detected at
the plasma membrane of both, SN56 and HT22
cells (67- and 80-kDa mER), and in micro-
somal fractions of murine septum and hippocampus
(67-, 80- and 97-kDa mER), thus confirming our
previous observations (Marin et al. 2003b, 2006).
This is in agreement with several studies in
neuronal tissues that have reported ERa immunor-
eactive bands at a wide range of molecular masses
including 80�112 kDa (reviewed in Marin et al.
2005). Together with VDAC and caveolin-1, 80-
kDa and 67-kDa mERs were also found in F5�F7
sucrose-gradient centrifugation fractions of SN56
and HT22 cells, therefore indicating that these
proteins are also present in caveolar-like domains.
Only a proportion of the total amount of purified
mERs was detected in caveolar fractions, suggesting
that these receptors are also distributed in other
plasma membrane regions. Even though the pre-
sence of an ERa subpopulation in caveolae has
been established in endothelial cells (Chambliss et
al. 2000, Deecher et al. 2003), to our knowledge no
previous data has reported a similar caveolar ERalocalization in neurons. It is noteworthy that
caveolae not only have been implicated in func-
tional events triggered at the plasma membrane in
neurons (Toran-Allerand 2004), but appear to
participate in important aspects of brain mainte-
nance as indicated by accumulation of, among
others, amyloid protein precursor (Bouillot et al.
1996) and nerve growth factor (Huang et al. 1999).
In agreement with this hypothesis, our previous
data have demonstrated that mERa of SN56
neurons, observed here to be present in caveolae,
participates in rapid estrogen neuroprotective ac-
tions to prevent Ab-induced toxicity through acti-
vation of MAPK signalling (Marin et al. 2003b,
Guerra et al. 2004).
A very interesting finding of the present work was
the interaction of both, VDAC and ERa-like, at the
plasma membrane of SN56 and HT22 cells as
evidenced by immunoprecipitation and immunocy-
tochemical assays. These results were corroborated
in microsomal fractions of the equivalent murine
septal and hippocampal tissues, suggesting that the
association of these two proteins at the plasma
membrane level may be a general phenomenon.
Caveolin-1 was also shown to coprecipitate in these
samples, supporting the presence of both VDAC
and mER in caveolar domains. Because of the
hydrophilic nature of ER molecule which lacks
transmembrane domains (Nadal et al. 2001),
associations with anchoring proteins such as caveo-
lins within the plasma membrane may be required
for the receptor to be integrated. Even though
numerous evidences strongly support a pivotal role
of non-classical ERs in the control of cell survival
and integrity, very little is known about the putative
molecules that may be interacting with the receptor
at the plasma membrane domain, and thereby
VDAC-ER association at nerve plasma membranes 157
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modulating its functionality at this level. In these
order of ideas, extranuclear ERa in the nervous
system has been shown to form a multiprotein
complex with insulin growth factor
1-receptor (IGF-IR) and p85 subunit of phospha-
tidil-inositol 3 kinase (PI3K) that is regulated by
estrogens (Mendez et al. 2005). Moreover, mem-
brane ERs bound to its hormone may couple with
G proteins to initiate intracellular signalling that
may regulate neuroprotective responses (Toran-
Allerand 2004). From the present results, we can
conclude that VDAC might also be a potential
candidate to facilitate ER integration at the plasma
membrane.
Apart from its interaction with mER, the crucial
roles of VDAC in apoptosis and its putative involve-
ment in Ab-related injury shown here suggest that
this channel may participate in estrogen actions
related to cell integrity and preservation. Although
not explored here, one could hypothesize that
estrogen binding to its receptor at the cell surface
might indirectly regulate mER interactions with
some potential modulators such as VDAC. In
SN56 cells, we have previously demonstrated the
specific binding of physiological doses of 17-bestradiol to an ERa-like localized at the plasma
membrane (Marin et al. 2003a), and that cell
exposure to the hormone for 24 h increased the
amount of ERa-like present in this structure (Marin
et al. 2003b) without apparently affecting mER-
VDAC complexes (data not shown). These evi-
dences may reflect the existence of different still
unknown pathways modulating this interaction. A
suggested mechanism for estrogens to control
VDAC activation may be via post-translational
modifications. In agreement with this, some studies
in neuroblastoma cells performed by members of
our group have evidenced estrogen inactivation of
MaxiCl- channel by promoting its phosphorylation
(Dıaz et al. 2001). Also, an example in epithelial
cells has suggested that estrogens can directly
modulate VDAC expression in correlation with
apoptosis (Tagaki-Morishita et al. 2003).
In conclusion, we have demonstrated for the first
time the involvement of VDAC in Ab-induced
neuronal mortality, and the association of this
channel with an ERa-like at the plasma membrane
of septal and hippocampal neurons, known to be
crucial in cognitive processes. Taking into account
the established participation of mER in neuropro-
tection, the association of these two proteins at the
cell surface may provide novel insights into the
complex signalling through alternative mechanisms
necessary to promote cell survival against Ab-related
neurotoxicity.
Acknowledgements
This work was supported by grants SAF-2004-
08316-C02-01, PI84/04, PI042640 and from the
Spanish Network of Neurological Research (CIEN).
RM and MC are fellows of the ‘‘Ramon y Cajal’’
Programme, and CR and MG held research fellow-
ships from, respectively, MEC and CIEN (Spain).
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This paper was first published online on iFirst on 6 February 2007.
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