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: rmarin@ull.es

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).

154 R. Marin et al.

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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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