Molecular and Cellular Neuroscience 42 (2009) 172–183

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Molecular and Cellular Neuroscience

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VDAC and ERα interaction in caveolae from human cortex is altered inAlzheimer's disease

Cristina M. Ramírez a,1, Miriam González b,1, Mario Díaz c, Rafael Alonso a, Isidre Ferrer d, Gabriel Santpere d,Berta Puig d, Gundela Meyer b, Raquel Marin a,⁎a Laboratory of Cellular Neurobiology, Department of Physiology, Faculty of Medicine and Institute of Biomedical Technologies, University of La Laguna, 38071 Sta. Cruz de Tenerife, Spainb Department of Anatomy, Faculty of Medicine and Institute of Biomedical Technologies, University of La Laguna, Tenerife, Spainc Laboratory of Animal Physiology, Department of Animal Biology, Faculty of Biology, and Institute of Biomedical Technologies, University of La Laguna, Tenerife, Spaind Institute of Neuropathology, IDIBELL-Universitary Hospital of Bellvitge, University of Barcelona, Hospitalet de Llobregat, and CIBERNED, Institute of Health Carlos III, Spain

Abbreviations: ER, estrogen receptor; VDAC, voltage-Alzheimer's disease.⁎ Corresponding author.

E-mail address: rmarin@ull.es (R. Marin).1 These authors contributed equally to this study.

1044-7431/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.mcn.2009.07.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 March 2009Revised 23 June 2009Accepted 1 July 2009Available online 10 July 2009

Keywords:Voltage-dependent anion channelMembrane estrogen receptorCaveolaeCaveolin-1Human brainAlzheimer's disease

Voltage-dependent anion channel (VDAC) is a mitochondrial porin also found in the neuronal membrane(pl-VDAC), where its function may be related to redox homeostasis and apoptosis. Murine models haveevidenced pl-VDAC into caveolae in a complex with estrogen receptor alpha (mERα), which participates inneuroprotection against amyloid beta (Aβ), and whose integration into this hydrophobic domain remainsunclear. Here, we have demonstrated in caveolae of human cortex and hippocampus the presence of pl-VDACand mERα, in a complex with scaffolding caveolin-1 which likely provides mERα stability at the plasmamembrane. In Alzheimer's disease (AD) brains, VDAC was accumulated in caveolae, and it was observed indystrophic neurites of senile plaques, whereas ERα was expressed in astrocytes surrounding the plaques.Together with previous data in murine neurons demonstrating the participation of pl-VDAC in Aβ-inducedneurotoxicity, these data suggest that the channel may be involved in membrane dysfunctioning observed inAD neuropathology.

© 2009 Elsevier Inc. All rights reserved.

Introduction

VDAC is a highly conserved large conductance anion channel thatprovides the major pathway for transmembrane fluxes of ions andmetabolites across the outer mitochondrial membrane (Baker et al.,2004). The channel is part of the permeability transition pore andits activation is associated with cytochrome c release and apoptosis(reviewed in Tsujimoto and Shimizu, 2007). More recently, thisporin has also been claimed to form part of a, still undefined,plasmalemmal chloride channel complex (Thinnes, 2009a), tocontrol different aspects of cellular homeostasis (Thinnes, 2007).In neurons, there are also some few evidences demonstrating thatVDAC is present at the plasma membrane (pl-VDAC) (Bahamondeand Valverde, 2003), as well as in isolated caveolae and caveolae-like domains (Bàthori et al., 1999; Marin et al., 2007) where it mayparticipate in the maintenance of redox homeostasis in normal cells(Elinder et al., 2005), and in apoptosis modulation (Akanda andElinder, 2006; Akanda et al., 2008), where the channel has been

dependent anion channel; AD,

l rights reserved.

suggested to be part of an extrinsic apoptotic cell death (Thinnes,2009b). Caveolae are microstructures of the plasma membrane witha particular lipid composition and a high density of lipid-anchoredproteins (Anderson, 1998), that are considered to be a specific formof lipid rafts (Simons and Ikonen, 1997). In line with this, we haverecently observed this channel in murine neuronal caveolae in acomplex with estrogen receptor alpha (mERα), where it participatesin the toxic effect provoked by Aβ exposure (Marin et al., 2007). Themodulation of pl-VDAC is largely unknown, and only a few data incellular models have demonstrated that, in general, Maxi Cl−

channels can be modulated by phosphorylation (Liberatori et al.,2004), G-proteins (Schwiebert et al., 1990; McGill et al., 1993;Mitchell et al., 1997), fatty acids (Riquelme and Parra, 1999),amyloid beta (Jalonen et al., 1997), and estrogens and anti-estrogens(Li et al., 2000; Diaz et al., 2001; Valverde et al., 2002), the lattersuggesting that estrogen receptors (ERs) may be involved in VDACfunctionality at the plasma membrane.

ERs are involved in multiple neurotrophic and neuroprotectiveactions in response to estrogen exposure (Garcia-Segura et al., 2001;Dhandapani and Brann, 2002; Brann et al., 2007) not only throughclassical mechanisms known to involve nuclear receptors, but alsoby alternative mechanisms of action in which the participation ofERs located in close contact with the plasma membrane (mERs) hasbeen evidenced (Levin, 2002; Beyer et al., 2003; Toran-Allerand,

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2004; Raz et al., 2008). Among these actions, ERs have been shownin neuronal cultures and animal models to be crucial in protectionagainst a variety of insults such as oxidative stress, traumatic braininjury and Aβ-induced toxicity (Marin et al., 2005; Singh et al.,2008). However, the mechanisms underlying for the neuroprotec-tive effects in humans are unknown, a fact that may explain theinconclusive results of estrogen replacement therapies (ERT)(Henderson, 2006; Simpkins and Singh, 2008; Sherwin and Henry,2003). Related to the nature of mERs, very little information hasbeen compiled about the structural molecular features that allowthe integration of these hydrophilic molecules, lacking transmem-brane domains, into the plasma membrane in order to rapidlyinteract with their natural molecular targets. It is noteworthy that avariety of proteins compartmentalized in lipid rafts appear to beimplicated in important functional events related to brain main-tenance (Bouillot et al., 1996; Huang et al., 1999 Toran-Allerand,2004). Therefore, it is conceivable that membrane ERs could bepresent in caveolae associated with some molecules which maymodulate, among others, estrogen activities related to neuronalpreservation and integrity (Toran-Allerand et al., 2002; Deecher etal., 2003). Our previous work in murine neurons has evidenced thatthe presence of ERα in caveolar domains is involved in neuropro-tection against Aβ-related toxicity (Marin et al., 2003, 2006).Furthermore, pl-VDAC and mERα were shown in these neurons tointeract with caveolin-1, the essential protein for the formation ofcaveolae that may act as an anchoring factor to provide additionalstability for the integration and functionality of these proteins(Marin et al., 2008). In this regard, caveolin is known to regulate avariety of key signalling elements, including G-proteins, Src tyrosinekinases, some components of PI3K and MAPK pathways, and theamyloid precursor protein (APP) (Couet et al., 1997; Kang et al.,2006; Arcaro et al., 2007; Boulware et al., 2007), the latterindicating that caveolae are implicated in the production of Aβ(Ehehalt et al., 2003). Related to neuropathologies such asAlzheimer's and Parkinson's diseases (AD and PD), the importanceof caveolar signalling is only starting to be elucidated (Michel andBakovic, 2007). Here, we have identified pl-VDAC and mERα incaveolar fractions of human cortex and hippocampus, in a complexwith caveolin-1. Interestingly, VDAC, but not ERα, was abundant incaveolae of AD brains. In addition, we observed an accumulation ofthe porin in dystrophic neurites of senile plaques.

Results

Presence of VDAC and ERα in caveolar domains of human cortical andhippocampal membranes

We investigated by Western blot analysis the putative presenceof, both, VDAC and ERα at the plasma membrane of human cortexand hippocampus. Thus, specific antibodies directed to thesemolecules were used to immunoblot with purified membranefractions from temporal cortex (Cx) and hippocampus (Hi) ofhuman brains (Fig. 1A, MF). Whole extracts from the same sampleorigins were also run as immunoblotting controls (W). Resultsrevealed that the anti-VDAC antibody recognized a highly abundantband at 35 kDa, the expected Mw of this channel, in membranefractions from both brain areas. VDAC detected at this level wasnot the result of mitochondrial fraction contamination, as con-firmed by Western blotting assays using mitochondrial markerUCP3 (uncoupling protein 3) (data not shown). In addition, anantibody directed to classical ERα recognized a membrane ERmigrating at 67 kDa, coinciding with ERα Mw, in membraneprotein extracts from cortical and hippocampal origins. A highermER migrating at approximately 80 kDa was also recognized. Thisband has also been observed in previous studies (Rao, 1998; Marinet al., 2005; González et al., 2007), and may be the result of the

duplication of two exons within the ERα gene, as previouslyreported in cancer cells (Pink et al., 1996). Although not shownhere, a battery of antibodies directed to distinct epitopes of ERαconsistently reproduced these results, indicating that this receptoris a high homologue to ERα, which is in agreement with previousstudies in immortalized cells and neuronal tissues (reviewed inMarin et al., 2009). The purity of membrane isolates was confirmedby testing the distribution of cytosolic Hsp90, only observed inwhole tissue extracts, and transmembrane α1 subunit of Na+/K+

ATPase highly abundant at the membrane fractions.We next isolated lipid rafts from the temporal cortex of different

subjects, following the method described by Mukherjee et al.(2003). Characterization of these membrane domains was carriedout by SDS-PAGE and Western blot, using mouse monoclonalantibodies directed to flotillin-1 (Flot) and prion protein (PrP), toidentify raft-enriched fractions, mostly represented in fractions 1and 2 (Fig. 1B). Mouse anti-Cu/Zn superoxide dismutase (SOD-1)antibody was used as a cytosolic marker, observing the presence ofthis protein in fractions 5 and 6. Fraction 1 isolated from humancortex was used to explore the putative concentration of VDAC andERα by immunoblotting with specific antibodies against theseproteins. The results showed that both proteins were present inthese membrane microdomains (Fig. 1C). In the case of ERα, onlythe band corresponding to 67 kDa was observed in lipid rafts (LR)whereas 67- and 80-kDa bands were present in whole extracts (W).Moreover, an antibody directed to caveolin-1 (Cav-1), the hallmarkof caveolar fractions, was also incubated on these same samplesobserving, as expected, its high concentration in lipid rafts. Inanother set of experiments, we investigated the putative presence ofERβ in caveolae. However, we did not detect any signal on Westernblot using specific antibodies to this receptor (data not shown), thusconcluding that ERβ was not present in the membrane micro-domains of these neuronal types.

VDAC and ERα are part of a protein complex with caveolin-1 inhuman neurons

Previous work in murine septal and hippocampal areas hasevidenced the physical interaction of VDAC and ERα, in a complexwith caveolin-1 (Marin et al., 2008). To elucidate whether thesemolecular interactions might also be present in human neurons,we performed immunoprecipitation of caveolin-1 in membraneextracts from temporal cortex and hippocampus (Fig. 1D, Cx and Hirespectively), that resulted in the co-precipitation of VDAC and67 kDa-ERα in both brain regions (IPMF). Total membranefractions (MF) were run as a control of protein amount.Furthermore, ERα and VDAC were also shown to co-precipitatewith caveolin-1 in lipid rafts (IPLR), thus demonstrating theirassociation in these domains. Similar results were obtained inalternative immunoprecipitation assays using an antibody againstERα, where the co-precipitation of VDAC and caveolin-1 was alsodetected (data not shown).

Immunohistochemistry and co-localization of VDAC, ERα and caveolin-1in human cortical and hippocampal areas

We next illustrated by immunohistochemistry and confocalmicroscopy the co-localization of these protein markers in humancortex and hippocampus. Immunoreactivity of ERα and ERβ in thehuman brain has been previously illustrated (González et al., 2007).Therefore, we investigated the distribution of VDAC and caveolin-1 inhuman temporal cortex and hippocampus by incubation with specificantibodies on the corresponding cortical and hippocampal tissuesections. In the case of VDAC, immunoreactivity was strong in allcortical layers, except in layer I (data not shown). Immunolabellingwas particularly intense in pyramidal neurons (Fig. 2A), where it was

Fig. 1. ERα and VDAC localization and interaction at the plasma membrane and caveolar fractions of human cortical and hippocampal areas. (A) Proteins from total membranefractions (MF) were loaded on 12.5% SDS-PAGE for Western blot analysis with antibodies directed to either VDAC or ERα proteins. As a control of membrane samples purity, PVDFmembranes were re-blotted with, both, a polyclonal antibody directed to the Na+-K+-ATPase α1-subunit (α1ATPase) and a monoclonal antibody to Hsp90. Whole protein extracts(W) were also run for a comparison purpose. (B) Lipid rafts extracted from human cortex (Cx) were obtained as described in Mukherjee et al. (2003). As a control of lipid raftisolation, aliquots of equal volume from the different fractions (1–6) were loaded on SDS-PAGE forWestern blot with antibodies against flotillin-1 (Flot), prion protein (PrP) and Cu/Zn superoxide dismutase (SOD-1). (C) Fraction 1 corresponding to lipid raft isolates was used to immunoblot with anti-ERα, anti-VDAC and anti-Caveolin-1 (Cav-1) antibodies. (D)Total membrane fractions and lipid rafts were used to immunoprecipitate with polyclonal caveolin-1 (Cav-1) (IPMF and IPLR, respectively). The resultant precipitated proteinimmunocomplexes were run on SDS-PAGE, and immunoblotted with antibodies directed to ERα and VDAC. As immunoblotting control, membrane fractions (MF) and lipid rafts (LR)were also run.

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evident at the cell cytoplasm and plasmamembrane of cell bodies, andapical processes. Previous results have also evidenced a similarpattern of VDAC localization in cortical cells (Ménard et al., 1994). Inthe hippocampus, VDAC staining was present mainly at the cytoplasmand processes of pyramidal cells of Ammon's horn (CA1–CA3) (Fig.2B) and in hilus (H) cells (Fig. 2C), whereas a weak immunostainingwas observed in dentate gyrus (DG) cells. In the case of caveolin-1,staining was moderate but widely distributed throughout corticallayers II–VI, showing immunolabelling at the cytoplasm and apicalprocesses of pyramidal neurons (Fig. 2A′). In turn, staining intensity inthe hippocampus was particularly strong in pyramidal cells ofAmmon's horn (Fig. 2B′) and dentate gyrus cells (Fig. 2C′, DG),where the spot-like pattern may be compatible with caveolarstructures distributed along cell dendrites. No immunoreactivity wasobserved in the absence of primary antibodies (data not shown). Nodifferences in the pattern of distribution of these two molecules wereobserved for the different subjects of this study.

Furthermore, the co-localization in these brain areas of VDAC, ERαand caveolin-1 was analysed by double-labelled immunofluorescenceusing confocal microscopy. In the temporal cortex (Fig. 3), VDAC waspredominantly localized at the cytoplasm and neuritic processes ofcortical pyramidal neurons where it was shown to partially co-localizewith, both, caveolin-1 (Cav-1) (56.7% co-localization) and ERα (36%).Some cortical cells exhibited ERα-positive nuclei (lower panel), aspreviously evidenced (González et al., 2007), therefore not observingsignificant co-localization with VDAC in this case. In the hippocampus(Fig. 4), numerous pyramidal cells of different areas such as Ammon'shorn, hilus and dentate gyrus showed partial co-localization of VDACat the cytoplasm level as well as in neuronal processes with, both,caveolin-1 (respectively, 34.5%, 24.5% and 42.3%) and ERα (78%, 27%,and 38%). The observed immunofluorescence at the cell surface is inline with a membrane-related presence of these protein markers. Noimmunosignals were detected when experiments were performed inthe absence of primary antisera, confirming immunofluorescence

specificity (data not shown). These results are in concordance withthe immunoprecipitation data of VDAC, ERα and caveolin-1 demon-strated before.

VDAC amount is increased in caveolar domains of AD brains

Lipid rafts appeared to exhibit aberrant lipid composition in ADbrains as compared to healthy ones of similar age (Söderberg et al.,1992; Prasad et al., 1998), a fact that may affect the functionality andinteractions of signalling proteins present in these domains (Martin etal., in press). In order to study the presence of pl-VDAC and mERα inbrain areas showing AD pathology, we performed some immunopre-cipitation experiments in membrane fractions (MF) and lipid rafts(LR) from temporal cortex of AD brains (Fig. 5A). Equivalent healthyaged brains (64–78 years old) were used as controls (C). Immuno-precipitation with anti-caveolin-1 antibody in AD samples demon-strated that, as compared with control samples, VDAC co-precipitatedin a higher amount, in particular in lipid rafts. This was quantified bycalculating the densitometric results from immunosignal values of co-precipitated VDAC relative to total immunoprecipitated caveolin-1 foreach sample, observing approximately a twofold increase of co-precipitated VDAC in AD samples as compared to controls (Fig. 5B). Inanother set of experiments, we used the anti-ERα antibody forimmunoprecipitation of AD samples, observing that the amount of theco-precipitated VDAC did not show any significant variation ascompared with ER immunoprecipitations from healthy brains (notshown). These results suggest that VDAC, but not ERα, is over-expressed in caveolin-1 enriched lipid rafts in the cortex of AD brains.

Localization of VDAC and ERα in human brains with AD pathology

We next explored by immunofluorescence and confocal micro-scopy the pattern of expression of VDAC and ERα in AD brains. Apunctuate VDAC immunoreactivity was found in the cytoplasm of

Fig. 2. Detection of VDAC and caveolin-1 (Cav-1) by DAB staining method in human cerebral cortex and hippocampus. Immunoreactivity was obtained at the cytoplasm andprocesses of cells in layers II–VI of cerebral cortex (A, A′), pyramidal cells of hippocampus (B, B′), dentate gyrus (DG) and hilus (H) (C, C′). Scale bar: 40 μm.

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neurons with and without neurofibrillary tangles in control anddiseased cases (Fig. 6). In addition, strong VDAC immunoreactivityoccurred in cellular processes surrounding amyloid plaques, as seen indouble-labelled sections to VDAC and amyloid beta (Aβ) (Figs. 6A–B).Moreover, double-labelling immunofluorescence to VDAC and phos-pho-tau AT8 revealed partial co-localization of these two molecules,thus indicating the localization of VDAC in dystrophic neuriticprocesses of senile plaques (Figs. 6C–D). In contrast, VDAC did notco-localize with CD68 used as a microglial marker, or with GFAP usedas astrocytic marker (data not shown). No immunosignals weredetected in the absence of primary antibodies (data not shown).

In the case of ERα, a discrete immunoreactivity for this proteinwasseen in the cytoplasm of neurons and glial cells but, in the context ofAD, strong ERα immunoreactivity was only observed in reactiveastrocytes, especially those surrounding amyloid plaques (Figs. 6E–F).Increased ERα immunoreactivity was not found in dystrophic neuritessurrounding amyloid plaques.

Discussion

VDAC is traditionally known as a large conductance anion channelimportant in mitochondrial function, providing the main transmem-brane transport of ions, ATP and other metabolites through the outermitochondrial membrane (Baker et al., 2004). However, severalevidences have indicated the presence of a plasmalemmal VDAC (pl-VDAC) highly homologous to its mitochondrial counterpart indifferent cell types (Thinnes et al., 1989; Reymann et al., 1995;Buettner et al., 2000), including astrocytes (Dermietzel et al., 1994)and neurons (Bahamonde and Valverde, 2003; Elinder et al., 2005;Marin et al., 2007). pl-VDAC has also been proposed to play a role incell volume regulation as a chloride channel complex, to mediate ATPtranslocation across the plasma membrane, to induce mitochondrial-independent apoptosis as a factor of extrinsic apoptotic pathway, andeven to function as a redox enzyme capable of reducing NAD+ (DePinto et al., 2003; Okada et al., 2004; Elinder et al., 2005; Thinnes,

Fig. 3. Co-localization of VDACwith caveolin-1 and ERα in pyramidal cells of cerebral cortex. Cerebral cortex sections were incubated with specific antibodies directed to VDAC, ERαand caveolin-1 (Cav-1). After washing, sections were exposed to corresponding secondary rabbit biotinylated anti-mouse antibody, and anti-rabbit antibody labelled to cyanine 5.VDAC staining was revealed by incubationwith Alexa Fluor® 488 dye-conjugated streptavidin. Panels on the right illustrate co-localization of the digital merged of fluorescent signalscontaining both green and red colour distributions. The overlapped pixels are indicated by black spots. Scale bar: 20 μm.

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2007, 2009b). There are also some few evidences demonstrating thatVDAC may be present in isolated caveolae and caveolae-like domains(Bàthori et al., 1999; Marin et al., 2007). Caveolae are specialisedmicrodomains of the plasma membrane containing a high density ofkey molecules (Okamoto et al., 1998; Patel et al., 2008) that areinvolved in the regulation of different dynamic events, such asendocytosis, lipid trafficking, and signal transduction (Schlegel et al.,1998). In these domains, it has been suggested that VDAC mayparticipate in the maintenance of redox homeostasis in normal cells(Elinder et al., 2005), forming part of a plasmalemmal chloridechannel complex (Thinnes, 2007), in apoptosis modulation (Akandaand Elinder, 2006; Akanda et al., 2008), and in the induction of Aβtoxicity (Marin et al., 2007). These findings reveal the potentialimportance of this porin in the mechanisms of neurotoxicity, whichremains largely unexplored. In the present work, pl-VDAC was foundfor the first time to be present in lipid rafts of human temporal cortexand hippocampus. In cortex, VDAC was shown to co-precipitate withERα and caveolin-1, considered as themain protein responsible for the

Fig. 4. Co-localization of VDAC with caveolin-1 and ERα in hippocampal cells. Hippocampalantibodies directed to VDAC, ERα and caveolin-1 (Cav-1). Then, sections were washed, andrabbit antibody labelled to cyanine 5. VDAC staining was revealed by incubation with Alexathe digital merged of fluorescent signals containing both green and red colour distributions

induction of caveolae formation (Li et al., 2000), a finding that is inagreement with our previous experiments demonstrating the inter-action of pl-VDAC, ERα and caveolin-1 in murine cortical andhippocampal neurons (Marin et al., 2007, 2008). In contrast, no co-precipitation was found in these samples with flotillin, anotherhallmark of lipid rafts in neurons (data not shown). Moreover, ERβwas not represented in these lipid rafts, although it is known to behighly abundant inwhole extracts from these brain areas (González etal., 2007). Together with our previous observations, the interaction ofpl-VDAC with ERα in human healthy brains suggests that the porinmay participate in ER-related neuronal responses.

Estrogen signalling mechanisms have been demonstrated inneurogenesis, neuronal differentiation, synaptic plasticity, and neu-roprotection mainly in brain areas involved in mood and cognition(Dhandapani and Brann, 2002; Toran-Allerand, 2004; Marin et al.,2005; Raz et al., 2008). An important aspect of estrogen actions is thepreservation of human brain against some neurological disorders suchas Alzheimer's disease (AD), cerebral ischemia and Parkinson's

tissue sections of Ammon's horn, hilus and dentate gyrus were incubated with specificexposed to corresponding secondary rabbit biotinylated anti-mouse antibody, and anti-Fluor® 488 dye-conjugated streptavidin. Panels on the right illustrate co-localization of. The overlapped pixels are indicated by black spots. Scale bar: 20 μm.

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Fig. 5. Association of caveolin-1 with ERα and VDAC at the plasma membrane and lipidrafts of human cerebral cortex of AD brains. (A): Total membrane fractions and lipid raftswere used to immunoprecipitate with polyclonal caveolin-1 (Cav-1) (IPMF and IPLR,respectively). The resultant precipitated protein immunocomplexes were run on SDS-PAGE, and immunoblotted with antibodies directed to ERα and VDAC. As immunoblot-ting control, membrane fractions (MF) and lipid rafts (LR) were also run. Figureillustrates a representative immunoblot assay after the incubation with the differentantibodies. (B): Relative fold increase of co-precipitated VDAC relative to the amount ofimmunoprecipitated caveolin-1, quantified by the densitometric values from immuno-signals of each sample. ⁎pb0.05 versus control. Three assays per group.

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disease (PD) (Brann et al., 2007). However, very little is known aboutthe dynamics of ERs in the human brain, and that may partiallyexplain the controversial results about the beneficial impact ofestrogen replacement therapies (ERT) against these neurologicaldisorders (Henderson, 2006; Simpkins and Singh, 2008). In line withthis hypothesis, a distinct spatio-temporal intracellular distributionfor ERs has been reported in cognitive areas of the adult brain(Österlund et al., 2000; Taylor and Al-Azzawi, 2000; González et al.,2007) which is altered during menopause (Ishunina and Swaab,2007). In the present work, we have confirmed a distinct cellularlocalization of ERα in adult human cortex and hippocampus,observing the presence of the receptor in the cytoplasm and nucleiof human frontal cortex whereas it was exclusively cytoplasmic in thepyramidal cells of Ammon's horn, hilus and dentate gyrus of thehippocampus.

Together with ERα, VDAC and caveolin-1 were also localized in thecytoplasm and neuronal processes of different populations of humancortical and hippocampal cells. Previous data on caveolin-1 expressionin human brain had already demonstrated its presence in pituitary(Rotondo et al., 2004), and the vasculature of the leptomeninges andcortex (Virgintino et al., 2002; Van Helmond et al., 2007). Moreover,the immunofluorescent analysis by confocal microscope, a powerfultool that allows a high axial resolution, revealed a partial co-localization of these three proteins in cortex and hippocampus.These evidences are in agreement with the immunoprecipitation

Fig. 6. Panels A, B: Double-labelling immunofluorescence to VDAC (green) and Aβ (red) splaques. Nuclei stained with DRAQ 5 are visualized in blue. Panels: C, D: Double-labellinglocalization of VDAC accumulation and AT8 in dystrophic neurites of senile plaques (arrow(green) and Aβ (red) showing ER immunoreactivity in astrocytes surrounding amyloid plaqside. Bar in panels A, C, and E: 20 μm. Bar in panels B, D, and F: 40 μm.

results in cortex, demonstrating a protein–protein interactionbetween these three markers. We have further investigated theempirical interaction of VDAC and ERα with caveolin-1, usingbioinformatics tools (Gromiha et al., 2004; Bagos et al., 2004), tosearch in human VDAC and ERα sequences (Greene et al., 1986;Shanmugavadivu et al., 2007) for the existence of motifs susceptible ofbinding to caveolar scaffolding domain (CSD), known to be the site ofinteraction with different signalling proteins (Couet et al., 1997).Database search in ERα sequence resulted in the existence of aconsensus sequence ϕXϕXXXXHy, (ϕ, aromatic amino acid; Hy, bulkyhydrophobic amino acid; X, any amino acid) at 459–466 positions ofthe ligand binding domain (LBD) (sequence Y459TFLSSTL466). In VDAC,we also identified a sequence ϕXϕXXϕXHy susceptible of CSD binding(sequence Y62RWTEYGL69), presumably located in the intracellularloop between the third and fourth β-strands. This sequence isidentical to that previously identified in Mus musculus (Marin et al.,2008), and compatible with the consensus sequence susceptible ofbinding to CSD located at the second intracellular loop of the humanVDAC molecular structure (Bayrhuber et al., 2008). These analyses arein line with the possibility that caveolin-1 may be a docking proteinfor pl-VDAC and mERα in caveolae, as previously hypothesized inmouse (Marin et al., 2008). Although not explored in this work, otherproteins may be part of this complex. Previous data in differentneuronal types have reported the physical association of ERα withinsulin growth factor-1 (IGF-1) receptor, and with the downstreamproteins insulin receptor substrate-1 (IRS-1), and phosphatidil-inositol-3-kinase (PI3K) that, together with the demonstrated ERαinteraction with β-catenin, may represent different mechanisms ofestrogen to promote cell survival (Garcia-Segura et al., 2007; Marin etal., 2009).

The interaction of ERα with specific membrane proteins has beensuggested as a mechanism to integrate this hydrophilic molecule thatlacks transmembrane domains (Arvanitis et al., 2004; Toran-Allerandet al., 2002). In this order of ideas, caveolin-1 not only may conferstability for the receptor, but it may also contribute to functionalevents related to neuroprotection triggered at the plasma membrane.Thus, in neocortical explants, association of membrane steroidreceptor ER-Xwith caveolar microdomains settled the rapid activationof intracellular signalling pathways (Toran-Allerand et al., 2002) and,in hippocampal cells, caveolin proteins were found crucial formembrane ER activation of mGluR signalling (Boulware et al., 2007;Luoma et al., 2008). Also, mERα in association with caveolin-1 wascapable of modulating neuronal survival against Aβ toxicity (Marin etal., 2003, 2008), evoking the involvement of caveolins in neuroprotec-tion. In this order of ideas, it has been demonstrated that lipid rafts areinvolved in regulating Aβ generation (Ehehalt et al., 2003; Cordy et al.,2006), and caveolin-1 has been shown to be up-regulated in senescentcerebral neurons (Kang et al., 2006) and in AD brain (Gaudreault et al.,2004). In addition, mice deficient in caveolin-1 (CavKO mice)appeared to have a selective alteration in central cholinergic functions(Gioiosa et al., 2008).

It is now starting to be evidenced the existence of lipid alterationsin plasma membrane phospholipids and lipid rafts as a result of ADpathology (Söderberg et al., 1992; Prasad et al., 1998; Martin et al., inpress) that may have important consequences not only in themembrane physico-chemical properties, but also in the dynamicpartitioning and configuration of the resident proteins. In the presentwork, we have checked whether VDAC, ERα and caveolin-1 presenceat the plasma membrane may be altered in lipid rafts from corticalareas showing AD pathology at late stages (V–VI). The results obtained

howing that VDAC-immunoreactive cellular processes surround Aβ deposits in senileimmunofluorescence to VDAC (green) and phospho-tau AT8 (red) showing partial co-s), as seen in merge sections. Panels E, F: Double-labelling immunofluorescence to ERues, as seen in merge sections. In all cases, merged is represented in panels on the right

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Table 1Description of the different brain samples used in this study.

Case Age (years) Gender Post-mortem delay AD diagnosis Braak stage

1 40 F 9 h 15 min − 02 47 F 9 h 35 min − 03 58 F 4 h − 04 66 F 8 h − 05 64 F 5 h − 06 71 F 8 h 30 min − 07 78 F 3 h 40 min − 08 45 M 10 h − 09 52 M 7 h − 010 54 M 3 h − 011 59 M 6 h 25 min − 012 75 F 4 h 15 min + V/VI13 77 F 3 h 15 min + V/VI14 79 M 7 h 30 min + V/VI

F: female; M: male.

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by immunoprecipitation and immunoblotting demonstrated that pl-VDAC increased its co-precipitation with caveolin-1 in AD samples, ascompared with healthy brain extracts, whereas ERα levels remainedinvariable. ERα immunoreactivity was mainly observed in astrocytesof AD brains, in particular, surrounding amyloid plaques. This findingis in agreement with previous data where ERα was shown in thenucleus and cytoplasm of reactive astrocytes in either non-injured orAD hippocampus (Lu et al., 2003). Similar observations were found forERβ in this same brain area, observing some alterations for thisreceptor expression in AD (Savaskan et al., 2001). Other earlier studiesin animal models have also demonstrated that ERα expression inreactive astrocytes increases following an insult. Thus, an excitotoxicinjury to the hippocampus or a stab wound to the parietal cortexincreased immunoreactivity for ERα in astrocytes (García-Ovejero etal., 2002). Moreover, ERα expressionwas up-regulated in astrocytes ofthe primate brain following an inflammatory model of injury(Blurton-Jones and Tuszynski, 2001). In sum, the increased ERαexpression in astrocytesmay be a generalized response to brain injury,suggesting a role for this receptor in the involvement of estrogenprotective effects related to these glial cells.

Interestingly, immunofluorescence assays in AD brains revealedthat VDAC immunoreactivity was prominent in dystrophic neurites ofsenile plaques in AD, suggesting that the porin may be involved insome aspects of this neuropathology. These findings are furtherreinforced by previous observations where VDAC was shown to co-localize with altered mitochondria in autophagic processes, in whichcaveolin-1 was also abundant (Perez-Gracia et al., 2008). The presentdata indicate that VDAC may have some relevant activity at theneuronal membrane. Although very little is known about the role ofVDAC in the human nervous system, it should be mentioned that thischannel has been found to be present in a nitrated form inhippocampus of AD brains, as part of neuronal impairments presentin this neurological disease (Sultana et al., 2006), and VDAC levelsappeared to be deranged in brains of patients with AD and Downsyndrome (Yoo et al., 2001).

Additional studies on the dynamics of protein/protein interactionsin lipid rafts, and the identification of factors modulating local VDACand ERα functionality at the neuronal membrane would be worthy ofpursuing, in the search of new molecular targets for efficienttherapeutical approaches.

Experimental methods

Human brain samples

Post-mortem human brains without neurological, psychological,or neuropathological disorders, and brains with severe dementia ofAlzheimer type were obtained from autopsies performed at the

Institute of Neuropathology and University of Barcelona/ClinicHospital Brain Banks, and from donations to the Department ofAnatomy supervised by Ethics Committee of the University of LaLaguna. The post-mortem interval ranged was between 2 and 10 h.The different cases are described in Table 1. The experiments wereundertakenwith the approval of the Ethics Committee of University ofBarcelona and University of La Laguna University, and the Code ofEthical Principles for Medical Research Involving Human Subjects ofthe World Medical Association (Declaration of Helsinki).

Antibodies

The primary rabbit polyclonal anti-caveolin-1 (N-20), and rabbitpolyclonal anti-ERα (MC-20) antibodies were from Santa CruzBiotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti-ERαantibody (AB10) directed to the ligand binding domain of the receptorwas from Neomarkers (Fremont, CA, USA). Mouse monoclonal anti-VDAC, directed to human porin (Ab-1) was from Calbiochem (La Jolla,CA, USA), monoclonal anti-Na+-K+-ATPase α1-subunit was pur-chased from Upstate (Millipore Iberica, Madrid, Spain), and mono-clonal anti-heat shock protein 90 (Hsp 90) from Stressgen (BioNovaCientífica, Madrid, Spain). To identify raft-enriched fractions, mousemonoclonal anti-flotillin antibody (BD Biosciences, Madrid, Spain),and mouse monoclonal anti-prion protein (PrP) antibody (3F4, DAKO,Glostrup, Denmark) were used. Mouse anti-superoxide dismutase 1(SOD-1) antibody, raised against a prokaryotic recombinant fusionprotein corresponding to N-terminal domains I to V of the Cu/Znsuperoxide dismutase was purchased from Novocastra Laboratories(Newcastle uponTyne, UK).Mouse anti-Aβ a, anti-glial fibrillary acidicprotein and anti-CD68 antibodies were from DAKO, mouse anti-phosphor-tau AT8 from Innogenetics (Gent, Belgium). The rabbit anti-ERα and anti-VDAC antibodies in immunofluorescent assays were,respectively, from Santa Cruz and Quimigen (Madrid, Spain).Alexa555 anti-rabbit or anti-mouse were, both, from MolecularProbes (Leiden, Netherlands). DRAQ 5 was purchased from MolecularProbes, and Immuno-Fluore Mounting medium was from Sigma(Madrid, Spain),

Isolation of mitochondrial and microsomal fractions

Human brain tissue was dissected out, and immediately homo-genized in RSB buffer containing 10mMTris–HCl, pH 8.0, 20mMNaCl,25 mM EDTA and complete protease inhibitor cocktail (RocheDiagnostics, Barcelona, Spain), using a Teflon-glass homogenizergrinder. The whole procedure was carried out at 4 °C. The tissuehomogenate was centrifuged at 900×g for 15 min to avoid nuclei andtissue debris. The obtained supernatant was centrifuged at 10,000×gfor 15 min to sediment the mitochondrial fraction. A second super-natant was collected, and centrifuged at 100,000×g at 4 °C, followingstandard protocols to sediment microsomal fractions. Precipitatedfractions corresponding to mitochondrial and microsomal fractionswere resuspended in SDS loading buffer (625 mM Tris–HCl pH 6.8; 1%SDS, 10% glycerol, 5% β-mercaptoethanol; 0.01% bromophenol blue),boiled at 95 °C for 5 min, and proceeded for Western blotting.

Isolation of lipid rafts

Lipid raft isolation was performed as previously described(Mukherjee et al., 2003), with slight modifications. Briefly, 0.1 g offrontal cortexwas homogenized in 8 volumes of buffer A (50mMTris–HCl pH 8.0, 10 mM MgCl2, 20 mM NaF, 1 mM Na3VO4, 5 mM β-mercaptoethanol, 1 mM PMSF) and protease inhibitor cocktail,containing 1% Triton X-100 and 5% glycerol, in a glass homogenizergrinder. The tissue was then centrifuged at 500×g for 5 min, and thesupernatant was collected and mixed in an orbital rotor for 1 h at 4 °C.About 800 μl of sample was mixed with an equal volume of 80%

181C.M. Ramírez et al. / Molecular and Cellular Neuroscience 42 (2009) 172–183

sucrose in buffer A, and overlaid with 7.5 ml of a 35% sucrose solutionand 2.7 ml of a 15% sucrose solution in buffer A, in 10 mlultracentrifuge tubes (Ultraclear, Beckman Coulter, Madrid, Spain).The sucrose gradients were centrifuged at 150,000×g for 18 h at 4 °Cin a Beckman SW 41Ti rotor. Fractions of 2 ml were collected from thetop to the bottom. Characterization of lipid rafts was carried out bySDS-PAGE and Western blot, using mouse monoclonal anti-flotillin-1and anti-PrP antibodies, both diluted 1:1000, to identify lipid raftfractions. Mouse monoclonal anti-SOD-1 antibody was used at 1:1600as a cytosolic marker.

Immunoblotting

For Western blot analysis, protein samples were quantified bybicinchoninic acid assay (Bio-Rad Laboratories, Madrid, Spain),resuspended in SDS loading buffer, and boiled for 5 min at 95 °C.Equal amounts of protein extracts were loaded on 12.5% SDS-PAGE,transferred to Hybond-P membranes, and incubated with primaryantibodies directed to the different proteins analysed: ERα (diluted1:200), VDAC (diluted 1:2000), and caveolin-1 (diluted 1:200). Ascontrols of the purity of plasma membrane and mitochondrialfractions, anti-Na+-K+-ATPase α1-subunit antibody (diluted1:5000), and anti-Hsp 90 antibody (1:2500) were used to re-blotonto these same membranes. Antibody labelling was revealed byincubation with horseradish peroxidase-conjugated secondary anti-bodies (1:10,000), and visualized with ECL chemiluminescence kit(Amersham, Madrid, Spain).

Immunoprecipitation

Protein suspensions from microsomal fractions and lipid raftsobtained as described above were resuspended in cold immunopre-cipitation buffer containing 50 mM Tris–HCl, pH 7.4; 150 mM NaCl,10% glycerol, 1% Nonidet-P 40, 1 mM phenyl methyl sulfonyl fluoride(PMSF) and protease inhibitor cocktail. For immunoprecipitation oflipid rafts, these fractions were previously solubilized by adding 2%octyl β-D-glucopyranoside (Bio-Sigma, Tenerife, Spain),1mMNa3VO4,1 mM EDTA. Samples were then processed using sheep anti-rabbit oranti-mouse IgG dynabeads (Dynal, Oslo, Norway), following themanufacturer's instructions. Excess of primary antibody (5–10 μg ofantibody/mg protein) was added to the extracts, and samples wereincubated overnight at 4 °C with gentle rotation. Following addition of30 μl dynabead suspension/1 mg protein, protein–dynabead com-plexes were washed in PBS, extracted with SDS loading buffer, andelectrophoresed on 12.5% SDS-PAGE. For Western blot analyses,proteins were blotted with primary anti-VDAC (1:1000), anti-ERαAB-10 (1:500) and anti-caveolin-1 (1:500) antibodies, followed byincubation with either anti-mouse or anti-rabbit horseradish perox-idase-conjugated secondary antibodies (diluted 1:10,000). Visualiza-tion of specific immunosignals was obtained with ECLchemiluminescence kit.

Immunohistochemistry

The middle temporal gyrus (T2), frontal cortex and hippocampuswere identified according to the atlas by Dubernoy (1991) anddissected out. The blocks were fixed in Bouin's fixative (24 h at roomtemperature), embedded in paraffin, cut into 10 μm thick coronalsections, and mounted on xilane-coated slides. After rehydration,sections were boiled in 10 mM citrate buffer pH 6 at 100 °C for 20 minfor antigen retrieval, rinsed in 0.05 M Trizma-buffered saline pH 7.6(TBS), rinsed in TBS with 1:20 normal serum for 20 min, andincubated overnight at room temperature with anti-VDAC (1:1000)and anti-caveolin-1 (1:200) in a humidified chamber. Sections werewashed in TBS and exposed to biotinylated secondary goat anti-rabbitantibody (1:150, DAKO, Glostrup, Denmark) for 30 min. After several

washes in TBS, sections were incubated with avidin–biotin complex(ABC, DAKO) in TBS for 30 min and developed using a TBS solutioncontaining 0.04% 3, 3′-diaminobenzidine (DAB), 0.04% nickel sulphateand 0.03% hydrogen peroxide. Afterwards, the immunoreactedsections were dehydrated, cleared in xylene and cover-slipped withEukitt (Freiburg, Germany). Negative controls were performed in theabsence of primary antibodies.

Immunofluorescence and confocal microscopy

For double immunofluorescence of healthy brains, tissue sectionswere obtained as described above. Anti-ERα MC-20, anti-VDAC oranti-caveolin-1 antibodies were diluted (1:100, 1:150 and 1:100,respectively) in phosphate-buffered saline (PBS) with 1:200 normalserum, and incubated overnight at 4 °C. Then, the correspondingsecondary biotinylated anti-rabbit antibody (1:500, Amersham,) andAlexa Fluor® 488-labelled anti-mouse IgG antibody (1:1000, Mole-cular Probes, Invitrogen, Eugene, OR, USA) were incubated for 1 h atroom temperature, followed by incubation with cyanine-5 dye-conjugated streptavidin (1:500, Amersham) for 30 min at roomtemperature. After washing in PBS, sections were exposed to“Autofluorescence Eliminator Reagent” (Chemicon, BioNova, Madrid,Spain). Then, the immunofluorescent sections were cover-slippedwith DABCO (1%) and glycerol-PBS (1:1). Negative controls wereperformed in the absence of primary antibodies. Fluorescenceimmunosignals were processed using Fluoview 1000 laser scanningconfocal imaging system (Olympus Optical, Barcelona, Spain), and co-localization percentage was calculated according to Fluoview 1000software.

Double-labelling immunofluorescence of AD sections was per-formed by, first, de-waxing 5 μm-thick sections, that were stainedwith a saturated solution of Sudan black B (Merck, Barcelona, Spain)for 10min to block the autofluorescence of lipofuscin granules presentin nerve cell bodies. Theywere then rinsed in 70% ethanol, andwashedin distilled water. The sections were incubated at 4 °C overnight with acombination of the primary antibodies to VDAC (1:100), Aβ (1:25),phosphor-tau AT8 (1:50), ERα (1:100), VDAC (1:2000), glial fibrillaryacidic protein (1:250) and CD68 (1:100). After washing in PBS, thesections were incubated in the dark for 45 min at room temperaturewith the cocktail of secondary antibodies diluted in the same vehiclesolution as the primary antibodies. Secondary antibodies wereAlexa488 anti-rabbit or anti-mouse and used at a dilution of 1:400.Nuclei were stained with DRAQ 5 diluted 1:2000. Some sections wereincubated only with the secondary antibodies. These sections wereconsidered as negative controls. After washing in PBS, the sectionswere mounted in Immuno-Fluore Mounting medium, sealed, anddried overnight. Sections were examined with a Leica TCS-SL confocalmicroscope.

Statistical analysis

Quantitative data were expressed as a mean±SEM, and wereanalysed by one-way ANOVA followed by Tukey's post-hoc test tocompare between groups. Statistical significance is indicated in thefigure.

Acknowledgments

This work was supported by grants SAF2007-66148-C02-01;SAF2007-66148-C02-02; FIS PI080582; and BFV2005/07608-BFI.

References

Akanda, N., Elinder, F., 2006. Biophysical properties of the apoptosis-inducing plasmamembrane voltage-dependent anion channel. Biophys. J. 90, 4405–4417.

182 C.M. Ramírez et al. / Molecular and Cellular Neuroscience 42 (2009) 172–183

Akanda, N., Tofighi, R., Brask, J., Tamm, C., Elinder, F., Ceccatelli, S., 2008. Voltage-dependent anion channels (VDAC) in the plasma membrane play a critical role inapoptosis in differentiated hippocampal neurons but not in neural stem cells. CellCycle 7, 3225–3234.

Anderson, RG., 1998. The caveolae membrane system. Annu. Rev. Biochem. 67, 199–225.Arcaro, A., Aubert, M., Espinosa del Hierro, M.E., Khanzada, U.K., Angelidou, S., Tetley, T.D.,

Bittermann, A.G., Frame,M.C., Seckl, M.J., 2007. Critical role for lipid raft-associated Srckinases in activation of PI3K-Akt signaling. Cell Signal. 19, 1081–1092.

Arvanitis, D.N., Wang, H., Bagshaw, R.D., Callahan, J.W., Boggs, J.M., 2004. Membrane-associated estrogen receptor and caveolin-1 are present in central nervous systemmyelin and oligodendrocyte plasma membranes. J. Neurosci. Res. 75, 603–613.

Bagos, G.P., Liakopoulos, D.T., Spyropoulos, I.C., Hamodrakas, S.J., 2004. PRED-TMBB: aweb server for predicting the topology of β-barrel outer membrane proteins.Nucleic Acids Res. 1, 32–35.

Bahamonde, M.I., Valverde, M.A., 2003. Voltage-dependent anion channel localises tothe plasma membrane and peripheral but not perinuclear mitochondria. PflugersArch. 446, 309–313.

Baker, M.A., Lane, D.J., Ly, J.D, De Pinto, V., Lawen, A., 2004. VDAC1 is a transplasmamembrane NADH-ferricyanide reductase. J. Biol. Chem. 279, 4811–4819.

Bàthori, G., Parolini, I., Tombola, F., Szabò, I., Messina, A., Oliva, M., De Pinto, V., Lisanti,M., Sargiacomo, M., Zoratti, M., 1999. Porin is present in the plasma membranewhere it is concentrated in caveolae and caveolae-related domains. J. Biol. Chem.274, 29607–29612.

Bayrhuber, M, Meins, T., Habeck, M., Becker, S., Giller, K., Villingers, S., Vonrhein, C.,Griesinger, C., Zweckstetter, M., Zeth, K., 2008. Structure of the human voltage-dependent anion channel. Proc. Natl. Acad. Sci. U. S. A. 105, 15370–15375.

Beyer, C., Pawlak, J., Karolczak, M., 2003. Membrane receptors for oestrogen in the brain.J. Neurochem. 87, 545–550.

Blurton-Jones, M., Tuszynski, M., 2001. Reactive astrocytes express estrogen receptors inthe injured primate brain. J. Comp. Neurol. 433, 115–123.

Bouillot, C., Prochiantz, A., Rougon, G., Allinquant, B., 1996. Axonal amyloid precursorprotein expressed by neurons in vitro is present in a membrane fraction withcaveolae-like properties. J. Biol. Chem. 271, 7640–7644.

Boulware, M.I., Kordasiewicz, H., Mermelstein, P.G., 2007. Caveolin proteins are essentialfor distinct effects of membrane estrogen receptors in neurons. J. Neurosci. 27,9941–9950.

Brann, D.W., Dhandapani, K., Wakade, C., Mahesh, V.B., Khan, M.M., 2007. Neurotrophicand neuroprotective actions of estrogen: basic mechanisms and clinical implica-tions. Steroids 72, 381–405.

Buettner, R., Papoutsoglou, G., Scemes, E., Spray, D.C., Dermietzel, R., 2000. Evidence forsecretory pathway localization of a voltage-dependent anion channel isoform. Proc.Natl. Acad. Sci. U. S. A. 97, 3201–3206.

Cordy, J.M., Hooper, N.M., Turner, A.J., 2006. The involvement of lipid rafts in Alzheimer'sdisease. Mol. Membr. Biol. 23, 111–122.

Couet, J., Li, S., Okamoto, T., Ikezu, T., Lisanti, M.P., 1997. Identification of peptide andprotein ligands for the caveolin-scaffolding domain. Implications for the interactionof caveolin with caveolae-associated proteins. J. Biol. Chem. 272, 6525–6533.

Deecher, D.C., Swiggard, P., Frail, D.E., O'Connor, L.T., 2003. Characterization of amembrane-associated estrogen receptor in a rat hypothalamic cell line (D12).Endocrine 22, 211–223.

De Pinto, V., Messina, A., Accardi, R., Aiello, R., Guarino, F., Tomasello, M.F., Tommasino,M., Tasco, G., Casadio, R., Benz, R., De Giorgi, F., Ichas, F., Baker, M., Lawen, A., 2003.New functions of and old protein: the eukaryotic porin or voltage dependent anionselective channel (VDAC). Ital. J. Biochem. 52, 17–24.

Dermietzel, R., Hwang, T.K., Buettner, R., Hofer, A., Dotzler, E., Kremer, M., Deutzmann,R., Thinnes, F.P., Fishman, G.I., Spray, D.C., 1994. Cloning and in situ localization of abrain-derived porin that constitutes a large-conductance anion channel inastrocytic plasma membranes. Proc. Natl. Acad. Sci. U. S. A. 91, 499–503.

Dhandapani, K.M., Brann, D.W., 2002. Protective effects of estrogen and selectiveestrogen receptor modulator in the brain. Biol. Reprod. 67, 1379–1385.

Diaz, M., Bahamonde, M.I., Lock, H., Muñoz, F.J., Hardy, S.P., Posas, F., Valverde, M.A.,2001. Okadaic acid-sensitive activation of Maxi Cl− channels by triphenylethyleneantioestrogens in C1300 mouse neuroblastoma cells. J. Physiol. 536, 79–88.

Dubernoy, H., 1991. The Human Brain. Surface, Three-Dimensional, Sectional Anatomyand MRI. Springer-Verlag, New York.

Ehehalt, R., Keller, P., Haass, C., Thiele, C., Simons, K., 2003. Amyloidogenic processing ofthe Alzheimer beta-amyloid precursor protein depends on lipid rafts. J. Cell Biol.160, 113–123.

Elinder, F., Akanda, N., Tofighi, R., Shimizu, S., Tsujimoto, Y., Orrenius, S., Ceccatelli, S.,2005. Opening of plasma membrane voltage-dependent anion channels (VDAC)precedes caspase activation in neuronal apoptosis induced by toxic stimuli. CellDeath Differ. 12, 1134–1140.

García-Ovejero, D., Veiga, S., García-Segura, L.M., DonCarlos, L.L., 2002. Glial expressionof estrogen and androgen receptors after rat brain injury. J. Comp. Neurol. 450,256–271.

Garcia-Segura, L.M., Azcoitia, I., DonCarlos, L.L., 2001. Neuroprotection by estradiol.Prog. Neurobiol. 63, 29–60.

Garcia-Segura, L.M., Diz-Chaves, Y., Perez-Martin, M., Darnaudéry, M., 2007. Estradiol,insulin-like growth factor-I and brain aging. Psychoneuroendocrinol. 32, 557–561.

Gaudreault, S.B., Dea, D., Poirier, J., 2004. Increased caveolin-1 expression in Alzheimer'sdisease brain. Neurobiol. Aging 25, 753–759.

Gioiosa, L., Raggi, C., Ricceri, L., Jasmin, J.-F., Frank, P.G., Capozza, F., Lisanti, M.P., Alleva,E., Sargiacomo, M., Laviola, G., 2008. Altered emotionally, spatial memory andcholinergic function in caveolin-1 knock-out mice. Behav. Brain Res. 188, 255–262.

González, M., Cabrera-Socorro, A., Pérez-García, C.G., Fraser, J.D., López, F.J., Alonso, R.,Meyer, G., 2007. Distribution patterns of estrogen receptor α and β in the human

cortex and hippocampus during development and adulthood. J. Comp. Neurol. 503,790–802.

Gromiha, M.M., Ahmad, S., Suwa, M., 2004. Neural network-based prediction oftransmembrane beta-strand segments in outer membrane proteins. J. Comput.Chem. 25, 762–767.

Greene, G.L., Gilna, P., Waterfield, M., Baker, A., Hort, Y., Shine, J., 1986. Sequence andexpression of human estrogen receptor complementary DNA. Science 231,1150–1154.

Henderson, V.W., 2006. Estrogen-containing hormone therapy and Alzheimer's diseaserisk: Understanding discrepant inferences from observational and experimentalresearch. Neuroscience 138, 1031–1039.

Huang, C.S., Zhou, J., Feng, A.K., Lynch, C.C., Klumperman, J., DeArmond, S.J., Mobley,W.C, 1999. Nerve growth factor signalling in caveolae-like domains at the plasmamembrane. J. Biol. Chem. 274, 36707–36714.

Ishunina, T.A., Swaab, D.F., 2007. Alterations in the human brain in menopause.Maturitas 57, 20–22.

Jalonen, T.O., Charniga, C.J., Wielt, D.B., 1997. Beta-amyloid peptide-induced morpho-logical changes coincide with increased K+ and Cl− channel activity in rat corticalastrocytes. Brain Res. 746, 85–97.

Levin, E.R., 2002. Cellular plasma membrane estrogen receptors. Steroids 67, 471–475.Li, Z., Niwa, Y., Sakamoto, S., Chen, X., Nakaya, Y., 2000. Estrogen modulates a large

conductance chloride channel in cultured porcine aortic endothelial cells. J.Cardiovasc. Pharmacol. 35, 506–510.

Liberatori, S., Canas, B., Tani, C., Bini, L., Buonocore, G., Godovac-Zimmermann, J., Mishra,O.P., Delivoria-Papadopoulos, M., Bracci, R., Pallini, V., 2004. Proteomic approach tothe identification of voltage-dependent anion channel protein isoforms in guineapig brain synaptosomes. Proteomics 4, 1335–1340.

Lu, Y.-P., Zeng, M., Hu, X.-Y., Xu, H., Swaab, D.F., Ravid, R., Zhou, J.-N., 2003. Estrogenreceptor α-immunoreactive astrocytes are increased in the hippocampus inAlzheimer's disease. Exp. Neurol. 183, 482–488.

Kang, M.J., Chung, Y.H., Hwang, C.I., Murata, M., Fujimoto, T., Mook-Jung, I.H., Cha, C.I.,Park, W.Y., 2006. Caveolin-1 upregulation in senescent neurons alters amyloidprecursor protein processing. Exp. Mol. Med. 38, 126–133.

Luoma, J.I., Boulware, M.I., Mermelstein, P.G., 2008. Caveolin proteins and estrogensignaling in the brain. Mol. Cell Endocrinol. 290, 8–13.

Marin, R., Guerra, B., Morales, A., Díaz, M., Alonso, R., 2003. An oestrogen membranereceptor participates in estradiol actions for the prevention of amyloid-β peptide1–40-induced toxicity in septal-derived cholinergic SN56 cells. J. Neurochem. 85,1180–1189.

Marin, R., Guerra, B., Alonso, R., Ramírez, C.M., Díaz, M., 2005. Estrogen activatesclassical and alternative mechanisms to orchestrate neuroprotection. Curr.Neurovasc. Res. 2, 287–301.

Marin, R., Ramírez, C.M., González, M., Alonso, R., Díaz, M., 2006. Alternative estrogenreceptors homologous to classical receptor alpha in murine neural tissues.Neurosci. Lett. 395, 7–11.

Marin, R., Ramirez, C.M., Gonzalez, M., Gonzalez-Munoz, E., Zorzano, A., Camps, M.,Alonso, R., Diaz, M., 2007. Voltage-dependent anion channel (VDAC) participatesin amyloid beta-induced toxicity and interacts with plasma membrane estrogenreceptor alpha in septal and hippocampal neurons. Mol. Membr. Biol. 24,148–160.

Marin, R., Ramírez, C.M., Morales, A., González, M., Alonso, R., Díaz, M., 2008.Modulation of Aβ-induced neurotoxicity by estrogen receptor alpha and otherassociated proteins in lipid rafts. Steroids 73, 992–996.

Marin, R., Diaz, M., Alonso, R., Sanz, A., Arevalo, M.A., Garcia-Segura, L.M., 2009. Role ofestrogen receptor alpha inmembrane-initiated signaling in neural cells: Interactionwith IGF-1 receptor. J. Steroid Biochem. Mol. Biol. 114, 2–7.

Martin, V., Fabelo, N., Santpere, G., Puig, B., Marin, R., Ferrer, I., Diaz, M., in press. Lipidalterations in lipid rafts from Alzheimer's disease human brain cortex. J.Alzheimer's Dis.

McGill, J., Gettys, T.W., Basavappa, S., Fitz, J.G., 1993. GTP-binding proteins regulate highconductance anion channels in rat bile duct epithelial cells. J. Membrane Biol. 133,253–261.

Ménard, G., Evrard, B., Bureau, M., Trottier, S., 1994. Cerebral distribution of the B-36VDAC protein in rat, cow andman brain: immunocytochemical study. Cell Mol. Biol.(Noisy-le-grand) 40, 295–300.

Michel, V., Bakovic, M., 2007. Lipid rafts in health and disease. Biol. Cell 99, 129–140.Mitchell, C.H., Wang, L., Jacob, T.J.C., 1997. A large-conductance chloride channel in

pigmented ciliary epithelial cells activated by GTPγS. J. Membr. Biol. 158, 167–175.Mukherjee, A., Arnaud, L., Cooper, J.A., 2003. Lipid-dependent recruitment of neuronal

Src to lipid rafts in the brain. J. Biol. Chem. 278, 40806–40814.Okada, S.F., O Neal, W.K., Huang, P., Nicholas, R.A., Ostrowski, L.E., Craigen, W.J.,

Lazarowski, E.R., Boucher, R.C., 2004. Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP release and cell volume regulation in murine cells. J. Gen.Physiol. 124, 513–526.

Okamoto, T., Schlegel, A., Scherer, P., Lisanti, M.P., 1998. Caveolins, a family of scaffoldingproteins for organizing “preassembled signalling complexes” at the plasmamembrane. J. Biol. Chem. 273, 5419–5422.

Österlund, M.K., Gustafsson, J.A., Keller, E., Hurd, Y.L., 2000. Estrogen receptor β (ERβ)messenger ribonucleic acid (mRNA) expression within the human forebrain:distinct distribution pattern to ERα mRNA. J. Clin. Endocrinol. Metab. 85,3840–3846.

Patel, H.H., Murray, F., Insel, P.A., 2008. Caveolae as organizers of pharmacologicallyrelevant signal transductionmolecules. Annu. Rev. Pharmacol. Toxicol. 48, 359–391.

Perez-Gracia, E., Torrejón-Escribano, B., Ferrer, I., 2008. Dystrophic neuritis of senileplaques in Alzheimer's disease are deficient in cytochrome c oxidase. ActaNeuropathol. 116, 261–268.

183C.M. Ramírez et al. / Molecular and Cellular Neuroscience 42 (2009) 172–183

Pink, J.J., Wu, S.Q., Wolf, D.M., Bilimoria, M.M., Jordan, V.C., 1996. A novel 80 kDa humanestrogen receptor containing a duplication of exons 6 and 7. Nucleic Acids Res. 24,962–969.

Prasad, M.R., Novell, M.A., Yatin, M., Dhillon, H., Markesbery, W.R., 1998. Regionalmembrane phospholipid alterations in Alzheimer's disease. Neurochem. Res.. 23,81–88.

Rao, B.R., 1998. Isolation and characterization of an estrogen binding proteinwhich mayintegrate the plethora of estrogenic actions in non-reproductive organs. J. SteroidBiochem. 65, 3–41.

Raz, L., Khan, M.M., Mahesh, V.B., Vadlamudi, R.K., Brann, D.W., 2008. Rapid estrogensignalling in the brain. Neurosignals 16, 140–153.

Reymann, S., Florke, H., Heiden, M., Jakob, C., Stadtmuller, U., Steinacker, P., Lalk, V.E.,Pardowitz, I., Thinnes, F.P., 1995. Further evidence for multitopological localizationof mammalian porin (VDAC) in the plasmalemma forming part of a chloridechannel complex affected in cystic fibrosis and encephalomyopathy. Biochem. Mol.Med. 54, 75–87.

Riquelme, G., Parra, M., 1999. Regulation of human placental chloride channel byarachidonic acid and other cis unsaturated fatty acids. Am. J. Obstet. Gynecol. 180,469–475.

Rotondo, F., Horvath, E., Kovacs, K., Bell, D.C., Lloyd, R.V., Scheithauer, B.W., 2004.Expression of caveolin-1 and caveolin-2 in the human pituitary: an immunohis-tochemical study. Endocr. Pathol. 15, 345–350.

Savaskan, E., Olivieri, G., Meier, F., Ravid, R., Müller-Spahn, F., 2001. Hippocampalestrogen b-receptor immunoreactivity is increased in Alzheimer's disease. BrainRes. 908, 113–119.

Schlegel, A., Volonte, D., Engelman, J.A., Galbiati, F., Mehta, P., Zhang, X.L., Scherer, P.E.,Lisanti, M.P., 1998. Crowded little caves: structure and function of caveolae. CellSignal. 10, 457–463.

Schwiebert, E.M., Light, D.B., Fejes-Toth, G., Fejes-Toth, N., Stanton, B.A., 1990. A GTP-binding protein activates chloride channels in a renal epithelium. J. Biol. Chem. 265,7725–7728.

Shanmugavadivu, B., Apell, H.J., Meins, T., Zeth, K., Kleinschmidt, J.H., 2007. Correctfolding of the beta-barrel of the human membrane protein VDAC requires a lipidbilayer. J. Mol. Biol. 368, 66–78.

Sherwin, B.B., Henry, J.F., 2003. Brain aging modulates the neuroprotective effects ofestrogen on selective aspects of cognition in women: a critical review. Front.Neuroendocrinol. 29, 88–113.

Simons, K., Ikonen, E., 1997. Functional rafts in cell membranes. Nature 387, 569–572.Simpkins, JW., Singh, M., 2008. More than a decade of estrogen neuroprotection.

Alzheimers Dement. 4, S131–S136.

Singh, M., Sumien, N., Kyser, C., Simpkins, J.W., 2008. Estrogens and progesterone asneuroprotectants: what animal models teach us. Front. Biosci. 13, 1083–1089.

Söderberg, M., Edlund, C., Alafuzoff, I., Kristensson, K., Dallner, G., 1992. Lipidcomposition in different regions of the brain in Alzheimer's disease/seniledementia of Alzheimer's type. J. Neurochem. 59, 1646–1653.

Sultana, R., Poon, H.F., Cai, J., Pierce, W.M., Merchant, M., Klein, J.B., Markesbery, W.R.,Butterfield, D.A., 2006. Identification of nitrated proteins in Alzheimer's diseasebrain using a redox proteomics approach. Neurobiol. Dis. 22, 76–87.

Taylor, A.H., Al-Azzawi, F., 2000. Immunolocalisation of oestrogen receptor beta inhuman tissues. J. Mol. Endocrinol. 24, 145–155.

Thinnes, F.P., Gotz, H., Kayser, H., Benz, R., Schmidt, W.E., Kratzin, H.D., Hilschmann, N.,1989. Identification of human porins. I. Purification of a porin from human B-lymphocytes (Porin 31HL) and the topochemical proof of its expression on theplasmalemma of the progenitor cells. Biol. Chem. Hoppe-Seyler 370, 1253–1264.

Thinnes, F.P., 2007. Modulator of plasmalemma standing VDAC revealed!? Mol. Genet.Metab. 91, 116–118.

Thinnes, F.P., 2009a. VDAC-cored CaCC, CF patients best friend — finding noveltherapeutics for CF. Mol. Genet. Metab. 97, 92–93.

Thinnes, F.P., 2009b. Neuroendocrine differentiation of LNCaP cells suggests: VDAC inthe cell membrane is involved in the extrinsic apoptotic pathway. Mol. Genet.Metab. 97, 241–243.

Toran-Allerand C.D., 2004. Minireview: A plethora of estrogen receptors in the brain:where will it end? 145, 1069–1074.

Toran-Allerand, C.D., Guan, X., MacLusky, N.J., Horvath, T.L., Diano, S., Singh, M.,Connolly Jr., E.S., Nethrapalli, I.S., Tinnikov, A.A., 2002. ER-X: a novel, plasmamembrane-associated, putative estrogen receptor that is regulated during devel-opment and after ischemic brain injury. J. Neurosci. 22, 8391–8401.

Tsujimoto, Y., Shimizu, S., 2007. Role of the mitochondrial membrane permeabilitytransition in cell death. Apoptosis 12, 835–840.

Valverde, M.A., Hardy, S.P., Díaz, M., 2002. Activation of Maxi Cl− channels byantiestrogens and phenothiazines in NIH3T3 fibroblasts. Steroids 67, 439–445.

Van Helmond, Z.K., Miners, J.S., Bednall, E., Chalmers, K.A., Zhang, Y., Wilcock, G.K.,Love, S., Kehoe, P.G., 2007. Caveolin-1 and -2 and their relationship to cerebralamyloid angiopathy in Alzheimer's disease. Neuropathol. Appl. Neurobiol. 33,317–327.

Virgintino,D.,Robertson,D., Errede,M.,Benagiano,V.,Tauer,U.,Roncali, L.,Bertossi,M.,2002.Expression of caveolin-1 in human brain microvessels. Neuroscience 115, 145–152.

Yoo, B.Ch., Fountoulakis, M., Cairns, N., Lubec, G., 2001. Changes of voltage-dependentanion-selective channel proteins VDAC1 and VDAC2 brain levels in patients withAlzheimer's disease and Down syndrome. Electrophoresis 22, 172–179.