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Research Report

Aquaporin expression in the cerebral cortex is increased atearly stages of Alzheimer disease

Esther Péreza, Marta Barrachinaa, Agustín Rodrígueza, Benjamín Torrejón-Escribanod,Mercé Boadab,c, Isabel Hernándezb, Marisa Sánchezb, Isidre Ferrera,e,⁎aInstitut de Neuropatologia, Servei Anatomia Patològica, Hospital Universitari de Bellvitge, carrer Feixa Llarga sn,08907 Hospitalet de Llobregat, SpainbFundació ACE, Institut Català de Neurociències Aplicades, Barcelona, SpaincServei de Neurologia, Hospital Universitari de la Vall d'Hebron, SpaindServeis Cientificotecnics, Campus Bellvitge, SpaineUniversitat de Barcelona, Hospitalet de Llobregat, Spain

A R T I C L E I N F O

⁎ Corresponding author. Institut de Neuropat08907 Hospitalet de Llobregat, Spain.

E-mail address: 8082ifa@comb.es (I. Ferre

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.09.109

A B S T R A C T

Article history:Accepted 26 September 2006Available online 22 November 2006

Abnormalities in the cerebral microvasculature are common in Alzheimer disease (AD).Expression levels of the water channels aquaporin 1 and aquaporin 4 (AQP1, AQP4) wereexamined in AD cases by gel electrophoresis andWestern blotting, and densitometric valuesnormalized with β-actin were compared with corresponding values in age-matchedcontrols processed in parallel. In addition, samples of cases with Pick disease (PiD) wereexamined for comparative purposes. A significant increase in the expression levels of AQP1was observed in AD stage II (following Braak and Braak classification). Individual variationswere seen in advanced stages which resulted in non-significant differences between ADstages V–VI and age-matched controls. No differences in AQP1 levels were observedbetween familial AD cases (FAD, all of them at advanced stages) and corresponding age-matched controls. Immunohistochemistry showed increased AQP1 in astrocytes at earlystages of AD. Double-labelling immunofluorescence and confocal microscopy disclosedAQP1 immunoreactivity at the cell surface of astrocytes which were recognized with anti-glial fibrillary acidic protein antibodies. No differences in the levels of AQP4were observed inAD, FAD and PiD when compared with corresponding controls. These results indicateabnormal expression of AQP1 in astrocytes in AD, and they add support to the idea thatabnormal regulation of mechanisms involved in the control of water fluxes occurs at earlystages in AD.

© 2006 Elsevier B.V. All rights reserved.

Keywords:Alzheimer diseasePick diseaseAquaporinsWater channel

1. Introduction

Alzheimer disease (AD) is characterized by the progressiveaccumulation of senile plaques, neurofibrillary tangles (NFTs)

ologia, Servei Anatomia P

r).

er B.V. All rights reserved

and neuropil threads in the brain (Duyckaerts and Dickson,2003). Themain component of senile plaques is amyloid-β (βAor βA4), a peptide derived from the amyloid protein precursor(APP) through β- and γ-secretase activity (Herreman et al.,

atològica, Hospital Universitari de Bellvitge, carrer Feixa Llarga sn,

.

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2000; Zhang et al., 2000). Presenilin-1 (PS-1) and presenilin-2(PS-2) are crucial components of the secretase machinery.Early onset familial AD is caused bymutations inAPP, PS-1 andPS-2 (Bertram, 2003). NFTs are mainly composed of hyper-phosphorylated tau that aggregates as paired helical fila-ments. As well as in NFTs, hyper-phosphorylated tau is foundin neuropil threads and in dystrophic neurites surroundingsenile plaques (Duyckaerts and Dickson, 2003). AD is consid-ered a systemic disease progressively affecting not only theentorhinal cortex, limbic structures and neocortex (Braak andBraak, 1999), but also the basal nucleus of Meynert (which isthe main source of the cortical cholinergic innervation),thalamus and brain stem.

Cerebral blood vessels are also involved in AD. Amyloid-βdeposition in the blood vessels of the meninges and encepha-lon (amyloid angiopathy) occurs in the majority of cases(Kalaria et al., 1996). In addition, AD is commonly associatedwith cerebrovascular pathology, abnormal cholinergic inner-vation of intracerebral blood vessels, and brain hypoperfusion(de la Torre, 2004; Farkas and Luiten, 2001; Jellinger, 2002;Kalback et al., 2004). Moreover, arterioles and capillaries areabnormal in AD. These abnormalities are morphologicallycharacterized by atrophy, swelling and increased pynociticvesicles in endothelial cells; atrophy and irregularities ofsmooth muscle fibres; thickening and local disruption ofbasement membranes; and occasional swelling of astrocyticend feet (Farkas and Luiten, 2001; Grammas et al., 2002;Iadecola, 2004). Recentbiochemical studiesadd further supportto the importance of themicrovasculature in the pathogenesisof AD, as already suggested many years ago (Scheibel et al.,1989). Endothelial cellmarkersaredown-regulated (KalariaandHedera, 1995), vascular smooth muscle actin is reduced (Ervinet al., 2004), and collagen IV, heparan sulphate proteoglycansand laminin are augmented in basement membranes in AD(Berzin et al., 2000; Kalaria and Pax, 1995; Verbeek et al., 1999).All these observations lend support to the idea that water andnutrient transport may be impaired in AD.

Aquaporins (AQPs) facilitate water flux (and glycerol, ureaand ions) through the plasma membrane and may controlparticular aspects of homeostasis in several cell types (Amyry-Moghaddam and Ottersen, 2003; Amyry-Moghaddam et al.,2003; Kimelberg, 2004; Neely et al., 1999; Verkman, 2002). AQP1,AQP4, AQP9 andAQP11are present in brain (Gomori et al., 2000;Lehman et al., 2004; Oshio et al., 2005b). However, AQP11 isfunctionally distinct fromother aquaporins asXenopus oocytesexpressing AQP11 fail to transport water, glycerol, urea or ions(Gorelick et al., 2006). AQP1 is expressed in apical membranesof the choroid plexus epithelium and probably in astrocyticend-feet. Yet, AQP1 may be expressed in astrocytes inparticular conditions as in brain oedema associated withbrain contusion (Suzuki et al., 2006). AQP4, which is thepredominant water channel in the brain, is localized inastrocytes and ependymal cells (Badaut et al., 2002; Frigeri etal., 1995; Nielsen et al., 1993, 1997). Aquaporin water channelshave an hourglass form, the narrowest segment of whichpermits the passage of water but not of protons and othercations bymeansof size restriction andelectrostatic repulsion;the channels aremodulated by several mechanisms (Agre andKozono, 2003; Gunnarson et al., 2004). Several pieces ofevidence have shown that AQPs are abnormally expressed in

brain oedema following traumatic injury (Kiening et al., 2002;Saadoun et al., 2002b; Sato et al., 2000; Sun et al., 2003), cerebralischaemia (Aoki et al., 2003; Badaut et al., 2002; Sato et al., 2000;Taniguchi et al., 2000; Xiao et al., 2004) and hyponatremia(Vajda et al., 2000). AQP4 role in brain oedema is supported bythe observation of reduced brain oedema formation followingAQP4deletion (Amyry-Moghaddamet al., 2003, 2004;Manley etal., 2000; Vajda et al., 2002). Abnormal AQP1 and AQP4expression occurs in brain tumours (Oshio et al., 2005a,b;Saadoun et al., 2002a,b). Further evidences about the role ofAQP4 in brain homeostasis has been revealed by the relation-ship between the distribution of neuromyelitis optica-IgGautoantibodies, which bind to AQP4 resulting in a loss ofAQP4 expression in astrocytes, and the typical lesions of thisdemyelinating disease (Lennon et al., 2005; Misu et al., 2006;Pittock et al., 2006).

It is conceivable that AQPs are also abnormally expressedin other conditions in which preservation of homeostasis dueto microvascular abnormalities is at risk.

Starting from these observations, this study is focused on theexpression of AQP1 and AQP4 in the frontal cortex in AD cases atdifferent stages of disease. AQP9 is not a focus in the presentstudy because preliminary studies have shown poor results ofthe antibodies tested. CombinedWestern blotting and immuno-histochemistry have been carried out in a selected number ofsporadic and familial AD cases which were processed in parallelwith corresponding age-matched neuropathologically verifiedcontrol cases. In addition, cases of Pick disease (PiD), anotherneurodegenerative disease with abnormal hyper-phosphory-lated tau accumulation (Bergeron et al., 2003; Dickson, 1998)have been examined for comparative purposes. The presentobservations have shown increased AQP1 expression in astro-cytes of the frontal cortex in the entorhinal stages of AD,suggesting early abnormalities in the regulation of AQPs in AD.

2. Results

2.1. General findings of AQP1 and AQP4 antibodies

The anti-AQP1 antibodies recognized a band of 28 kDa incontrol and diseased cases as well as a band of about 35 kDawhich was particularly abundant in early stages of AD. Similarobservations were found with the anti-AQP1 (Chemicon) andanti-AQP1 (Abcam) antibodies (Figs. 1A and B). The upper bandwas abolished following de-glycosylation at 35 °C. Both bandsdisappearedwhen the samplewasheated at 95 °C (Fig. 1C). Theanti-AQP4 antibody recognized a single band of about 38 kDa.No differences in the expression levels of AQP1 and AQP4werefound in samples mimicking progressive post-mortem delaysof 0, 3, 6 and 22 h (Figs. 1D and E). Therefore, differencesobserved in the present series were considered to be free ofindividual variations resulting from post-mortem delay. AQP1and AQP4 bands disappeared when the samples were incu-bated with the secondary antibodies only (Figs. 1F and G).

2.2. AQP1 is over-expressed in AD

Western blot studies showed a significant increase in thelevels of AQP1 in the frontal cortex in sporadic AD cases.

Fig. 1 – A, B: AQP1 immunoreactivity in frontal cortex homogenates in controls (C) and sporadic Alzheimer's disease (AD)cases stage II. AQP1 is recognized as an inner band of about 28 kDa and a variable upper band of about 35 kDa representing thenon-glycosylated and glycosylated forms, respectively. β-Actin was used as a control of protein loading and recognized as aband of 45 kDa. Similar patterns are seen with Abcam (A) and Chemicon (B) antibodies. C: Western blots of the frontalcortex heated at 35 °C and non-treated (1), de-glycosylated and heated at 35 °C (2), and de-glycosylated and heated at 95 °C (3).The upper band disappears following de-glycosylation. Both bands are abolishedwhen the fresh samples are heated at 95 °C. D,E: AQP1 and AQP4 immunoreactivity (D and E, respectively) in tissue samples with increased artificial post-mortem delay.Note that AQP1 and AQP4 levels are maintained within the periods of post-mortem of the samples used in the present study.F, G: Incubation with secondary antibodies alone. No immunoreactivity is found in these lanes.

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Interestingly, increased AQP1 levels were manifested at earlystages of AD corresponding to stages IIA/B of Braak and Braak(Fig. 2). Moreover, increased protein levels corresponded to thenon-glycosylated and glycosylated bands. Although AQP1 wasalso higher in stages V–VIC of AD, differences were notsignificant when compared with levels in age-matchedcontrols (Fig. 2). Similarly, although AQP1 levels were appar-ently higher in FAD cases, differences were not significant

when compared with corresponding age-matched controls(Fig. 3).

These differences were not related with the levels of GFAPas GFAP was clearly more elevated in AD stages V–VIC and inFAD (Figs. 2 and 3).

Western blots to AQP4 did not reveal differences betweenAD cases and controls (Fig. 4). Similar results were obtained inFAD cases (Fig. 4).

Fig. 2 – A: AQP1 levels (Abcam antibody) in controls (C) and sporadic Alzheimer's disease (AD stages IIA/B and stagesV–VIC). AQP1 is recognized as an inner band of about 28 kDa and a variable upper band of about 35 kDa representingthe non-glycosylated and glycosylated forms, respectively. β-Actin was used as a control of protein loading andrecognized as a band of 45 kDa. Cases 1–4 in Table 1 were used as age-matched controls. Graphics represent the valuesof AQP1 normalized for β-actin in all cases (4 controls; 5 AD stages IIA/B; 9 stages V–VIC). A significant increase in AQP1expression is found in AD stages IIA/B, but not in stages V–VIC. Note the large individual variation in cases withadvanced AD. ANOVA with post hoc LSD test *p<0.05. B: Glial fibrillary acidic protein (GFAP) levels in controls (C) andsporadic Alzheimer's disease (AD stages IIA/B and stages V–VIC). Graphics represent the values of GFAP normalized forβ-actin in all cases (4 controls; 5 AD stages IIA/B; 9 stages V–VIC). A significant increase in GFAP expression is found inAD. ANOVA with post hoc LSD test *p<0.05.

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No differences in AQP1 levels were observed in the frontalcortex in PiD when compared with controls (Fig. 5). Similarnegative findings were observed for AQP4 in PiD (data notshown).

2.3. Increased AQP1 in AD is localized in astrocytes

Although both AQP1 antibodies were raised against the sameconsensus of AQP1 the AQP1antibody Abcam showed betterimmunostaining than the AQP1 antibody Chemicon and forthis reason was used in subsequent studies. Immunohisto-chemical studies showed AQP1 immunoreactivity in astro-cytes (and choroid plexus) in control and AD cases.Increased AQP1 immunoreactivity was found in corticalastrocytes in AD cases (Figs. 6B, D, and H). Curiously,increased AQP1 immunoreactivity was constantly observedin cases of AD corresponding to stages IIA/B, whereasindividual variations occurred in AD cases at stages V–VIC(Figs. 6E and H).

AQP4 immunoreactivity was localized in astrocytes incontrol and diseased brains. Although the number ofastrocytes was increased in AD, as revealed in sectionsstained with anti-GFAP antibodies (Figs. 6A, D, and G), noincrease in AQP4 expression occurred in these cells (Figs. 6 C,F, and I).

Lack of relationship between AQP1 expression andnumber of astrocytes was further demonstrated in PiD.Although the number of astrocytes was markedly increasedin the cerebral cortex, AQP1 immunoreactivity, as well asAQP4 immunoreactivity, did not markedly differ fromcontrols (Fig. 7).

2.4. Double-labelling immunofluorescence and confocalmicroscopy

Samples of frontal cortex immunolabelled with anti-AQP1and anti-GFAP antibodies revealed punctate AQP1 immu-noreativity at the periphery of astrocytic cell bodies, per-

Fig. 3 – A: AQP1 levels (Abcam antibody) in controls (C) andfamilial Alzheimer's disease (FAD) cases. Cases 5–8 in Table 1were used as age-matched controls. Graphics represent thevalues of AQP1 normalized for β-actin in all cases (4 controlsand 5 FAD). Although the band corresponding to AQP1(28 kDa) appears higher in FAD, no significant changes wereseen between control and FAD cases, probably due to thelarge individual variations in FAD. B: Glial fibrillary acidicprotein (GFAP) expression levels in controls (C) and FADcases. A significant increase in GFAP expression is found inAD. ANOVA with post hoc LSD test *p<0.05.

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fectly delineating in most instances the glial cell surface(Fig. 8). However, the presence and amount of AQP1differed from one cell to another in control and diseasedbrains. Some astrocytes were not decorated with anti-AQP1antibodies.

Fig. 4 – AQP4 levels in controls (C) and sporadic Alzheimer'sdisease (AD stages IIA/B and stages V–VIC). AQP4 isrecognized as a band of about 38 kDa. β-Actin was used as acontrol of protein loading and recognized as a band of 45 kDa.Cases 1–4 in Table 1 were used as age-matched controls.Graphics represent the values of AQP1 normalized forβ-actinin all cases (4 controls; 5 AD stages IIA/B; 9 stages V–VIC). Nodifferences between control and diseased brains areobserved. Similarly, no differences in AQP4 levels areobserved between FAD cases and controls.

3. Discussion

The present results have shown increased AQP1 expression incortical astrocytes in AD. Interestingly, these changes occur atearly stages of AD (stage IIA/B of Braak and Braak). Yet nosignificant increase is observed in advanced stages of AD(stages V–VIC) when compared with age-matched controls,although there is still a tendency toward increased AQP1expression in Western blots and individual variations whenexamined with immunohistochemistry. Therefore, modifica-tions in AQP1 do not correlatewith tau hyper-phosphorylationand amyloid deposition. No differences in AQP1 levels are

seen in FAD cases, although no entorhinal stages of familialcases were available for study.

AQP1 immunohistochemistry has shown the presence ofAQP1 in astrocytes in control and diseased brains. However,double-labelling immunofluorescence and confocal micro-scopy has disclosed several important points. The first one isthe localization of AQP1 at the cell surface of astrocytes ascould be expected for a membrane protein. The apparent

Fig. 5 – A: AQP1 levels in controls (C) and cases with Pickdisease (PiD). β-Actin was used as a control of proteinloading. No differences in AQP1 protein levels are foundbetween control and diseased cases. B: Glial fibrillary acidicprotein (GFAP) levels in controls (C) and PiD cases. Asignificant increase in GFAP levels is found in PiD. ANOVAwith post hoc LSD test *p<0.05.

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cytoplasmic localization of AQP1 in thick sections processedfree-floating is therefore a false image of AQP1 localization.The second important aspect is the lack of uniformity of AQP1expression when comparing one astrocyte to another. Someastrocytes were not decorated with anti-AQP1 antibodies,whereas AQP1 expression was marked in others. This mayexplain the lack of relationship between the number ofastrocytes, as revealed with anti-GFAP antibodies, and theexpression of AQP1 in individual cases. Although not exam-ined in detail, similar findings are probably applicable to AQP4.

The present findings showing increased AQP1 expressionin selected astrocytes may represent a link between well-known arteriolar and capillary abnormalities (Ervin et al., 2004;Farkas et al., 1999; Farkas and Luiten, 2001; Grammas et al.,2002; Iadecola, 2004; Kalaria and Hedera, 1995) and specificresponses to water transport in perivascular processes ofastrocytes in AD. Furthermore, all these observations addsupport to the idea that water and nutrient transport may beimpaired at early stages in AD. No similar scenario seems tooccur in PiD although the disease in all PiD cases wasadvanced, and no cases of early PiD pathology were availablefor study.

It is worth stressing that hypoperfusion and oxidativestress have been proposed as crucial contributors to neuronaldegenerative pathology in AD (Aliev et al., 2003a, b). Oxidativestress has been demonstrated to occur at early stages of thedisease before the appearance of tau hyper-phosphorylation

and massive amyloid-β deposition (Zhu et al., 2004a,b). Thepresent findings further support the idea that other abnorm-alities, including abnormal regulation of mechanismsinvolved in the control of water (and ion) fluxes, occur atearly stages in AD. The recognition of events that precedehallmark lesions of AD is crucial for a better understanding ofmechanisms that may favour the development and progres-sion of the disease.

4. Experimental procedure

4.1. Brain samples

Brain tissues were obtained from the Institute of Neuropatho-logy and University of Barcelona/Clinic Hospital brain banksfollowing the guidelines of the local ethics committees.Clinically, nine patients had suffered from severe (Globaldeterioration scale) dementia of Alzheimer type; five caseswere considered familial AD (FAD) on the basis of theinvolvement of relatives with a similar disease and confirma-tion by genetic studies of APP, PS1 and PS2. Four cases wereclinically diagnosed as PiD because of sporadic fronto-temporal dementia and severe fronto-temporal lobar atrophyon neuroimaging studies (CT and MRI). Fourteen cases wereneurologically normal. The post-mortem delay was between 3and 20 h. Cases with and without clinical neurological diseasewere processed in the same way following the same samplingand staining protocols. At autopsy, half of each brainwas fixedin 10% buffered formalin, while the other half was cut incoronal sections 1 cm thick, frozen on dry ice and stored at−80 °C until use. In addition, 2-mm-thick samples of thefrontal cortex (area 8) were fixed with 4% paraformaldehydefor 24 h, cryoprotected with 30% sucrose, frozen on dry ice andstored at −80 °C until use.

Following neuropathological examination, five cases werecategorized as AD stage II of neurofibrillary degeneration andstage A or B of amyloid-β deposition, and nine cases werecategorized as AD stage VC or VIC (Braak and Braak, 1999). ADcases at intermediate stages were not included. The five caseswith familial AD corresponded to stage VIC. No cases with ADchanges and associated diffuse Lewy pathology were includedin the present series. The four cases with suspected PiDconformed to well-established neuropathological criteria forPiD (Bergeron et al., 2003; Dickson, 1998). The remaining eightcases did not have neuropathological abnormalities and wereconsidered as controls. A summary of the main clinical andneuropathological findings in the present series is shown inTable 1.

In addition, samples of the frontal cortex from one controlindividual were obtained at 3 h post-mortem and immediatelyfrozen (time 0), or stored at 4 °C for 3 h, 6 h or 22 h, and thenfrozen to mimic variable post-mortem delay in tissue proces-sing and its effect on protein preservation.

4.2. Gel electrophoresis and Western blotting

For Western blot studies, 0.1 g of frontal cortex from eachsample was added to 800 μl of protein lysis buffer andhomogenized using a plastic homogenizer. This buffer

Fig. 6 – GFAP (A, D, G), AQP1 (B, E, H) and AQP4 (C, F, I) immunoreactivity in control (A–C), AD stage IIB (D–F) and AD stage VIC(G–I). Increased AQP1 immunoreactivity is found in AD stage IIB. However, AQP1 immunoreactivity (number of astrocytes andintensity of the immunoreaction) in AD stage VIC does not differ from control. No differences in AQP4 immunoreactivity areseen between control and AD cases independently of the disease stage. GFAP immunostaining of corresponding areas showsmoderate astrocytic gliosis in advanced stages of AD. Cryostat sections without counterstaining. Bar in A, D, G=100 μm. Bar inB, C, E, F, H, I=50 μm.

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contained 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mMEDTA, 1 mM EGTA, 5 μg/ml sodium orthovanadate and amixture of protease inhibitors (1 mM DDT, 2 mM phenyl-methylsulfonyl fluoride, 1 μg/ml aprotinin, leupeptin and

pepstatin). All the samples were centrifuged at 5000 rpm for10 min. The supernatant was recovered and the proteinconcentration of total homogenate was determined with theBCA Protein Assay Kit (Pierce, Madrid) using bovine serum

Fig. 7 – GFAP (A), AQP1 (B) and AQP4 (C) immunoreactivity in PiD. No differences are seen between control and PiD cases.GFAP immunostaining of corresponding areas shows marked astrocytic gliosis in PiD. Cryostat sections withoutcounterstaining. Bar in A=100 μm. Bar in B, C=50 μm.

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albumin (BSA) as a standard. All the samples were storedfrozen at −80 °C until use.

Thirty micrograms of proteins were mixed with loadingbuffer (20% glycerol, 4% 2-mercaptoethanol, 4% sodiumdodecyl sulfate, 0.002% bromophenol blue and 125 mM Tris–HCl, pH 6.8) and heated for 3 min at 37 °C (Manley et al., 2000).Parallel studies were carried out heating the samples to 95 °C.Some samples were deglycosylated with N-Glycosidase F⁎

Fig. 8 – Double-labelling immunofluorescence and confocalmicrovisualized in red. AC: AQP1 immunoreactivity occurs as punctateastrocytes (B). This is clearly seen in merge images (C). D, E and F:of one astrocyte in AC) showing punctate AQP1 immunoreactivit

recombinant (Roche) (12 U added to 20 μg) and incubatedovernight at 37 °C. Gel electrophoresis was carried out insodium dodecyl sulfate (SDS)–polyacrylamide gels, composedof 4% stacking (0.5 M Tris–HCl, pH 6.8, 10% SDS) and 10%resolving (1.5 M Tris–HCl, pH 8.8, 10% SDS), using a mini-protean system (Bio-Rad, Madrid) with molecular weightstandards (Bio-Rad). Proteins were then transferred to nitro-cellulose membranes (1 h, 100 V) using an electrophoretic

copy to AQP1 (Abcam antibody) visualized in green and GFAPdeposits (A) apparently decorating the surface of someMerge images of several examples (D is a high magnificationy at the cell surface of astrocytes. AD stage IIB.

Table 1 – Summary of the cases included in the present series

Age Gender p.m. delay Clinical diagnosis Neuropathological diagnosis Braak stages Mutation

1 80 F 3 Normal No lesions 02 79 F 7 Normal No lesions 03 73 F 5 Normal No lesions 04 75 M 6 Normal No lesions 05 18 M 18 Normal No lesions 06 49 F 7 Normal No lesions 07 41 F 12 Normal No lesions 08 50 M 12 Normal No lesions 09 69 M 10 Normal AD IIB10 66 M 18 Normal AD IIA11 74 M 22 Normal AD IIA12 59 M 7 Normal AD IIB13 78 F 19 Normal AD IIA14 82 F 10 AD AD VC15 69 M 20 AD AD VC16 69 M 6 AD AD VC17 81 F 3 AD AD VC18 67 M 2 AD AD VIC19 67 F 4 AD AD VIC20 75 M 2 AD AD VIC21 69 M 3 AD AD VIC22 78 F 3 AD AD VIC23 64 M 14 FAD AD VIC M139T PS124 56 M 16 FAD AD VIC A713T APP25 54 M 3 FAD AD VIC E381G PS126 57 M 9 FAD AD VIC V89L PS127 66 F 6 FAD AD VIC A713T APP28 56 M 9 PiD PiD 029 68 M 6 PiD PiD 030 61 M 8 PiD PiD 031 74 M 74 7 PiD 0

AD: Alzheimer's disease; FAD: familial AD; PiD: Pick's disease; Braak stages: II, V and VI of neurofibrillary degeneration; stages A, B, C of senileplaques (Braak and Braak, 1999). F: female; M: male; p.m. delay: post-mortem delay.

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transfer system (Bio-Rad). The nonspecific binding sites in themembranes were blocked for 1 h with TBST (10 mM Tris–HCl,pH 7.4, 140 mM NaCl and 0.1% Tween-20) containing 5%skimmed milk, and then incubated with one of the primaryantibodies at 4 °C overnight. The rabbit polyclonal antibodyanti-Aquaporin 1 (Chemicon International, Barcelona) wasused at a dilution of 1:1000 in the TBST–5% skimmedmilk. Therabbit polyclonal anti-Aquaporin-1 (Abcam, Cambridge) wasused at a dilution of 1:500. The mouse monoclonal anti-Aquaporin 4 (Abcam) was used at a dilution of 1:500. Rabbitpolyclonal anti-glial fibrillary acidic protein (GFAP) antibodies(Dako) were used at a dilution of 1:250. After washing, themembranes were incubated with the corresponding anti-rabbit or anti-mouse IgG secondary antibodies (Dako) at adilution of 1:1000 for 45 min at room temperature. Themembranes were developed with the chemiluminescenceECL system (Amersham, Pharmacia, Barcelona) followed byexposure of the membranes to autoradiographic films. Themonoclonal anti-β-actin antibody (Sigma) was used at adilution of 1:5000 as a control of protein loading.

The anti-Aquaporin 1 antibody (Chemicon) was producedusing a 19 amino acid synthetic peptide from the cytosoliccarboxyl terminal domain of rat AQP1, which has totalhomology with human AQP1. The anti-Aquaporin 1 antibody(ab 15078 Abcam) recognizes identical sequence. The use of

these antibodies was justified because the quality of theimmunostaining in histological sections was sub-optimalwith one of these antibodies. The anti-Aquaporin 4 antibody(Abcam) recognizes an epitope specific for the amino terminusof the M1 isoform of rat AQP4. Western blots of brainhomogenates revealed a band of about 38 kDa.

4.3. Statistical methods

Cases 5–8 (Table 1) were used as age-matched controls for FADcases; cases 1–4 were used as controls in the remaining ADand PiD cases.

Densitometric studies were carried out with the TotalLabv2.01 programme. Data were processed with STATGRAPHICSPLUS 5.0. ANOVA with post hoc LSD test: *p<0.05 (95%confidence interval).

4.4. Aquaporin immunohistochemistry

Cryostat sections 14m thick were processed free-floating withthe EnVision+system peroxidase procedure (Dako). The rabbitpolyclonal antibody anti-Aquaporin 1 (Abcam) was used at adilution of 1:500. The monoclonal anti-Aquaporin 4 (Abcam)was used at a dilution of 1:250. The rabbit polyclonal anti-GFAP antibody (Dako) was used at a dilution of 1:250. The

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sections were incubated with EnVision for 1 h at roomtemperature. The peroxidase reaction was visualized withNH4NiSO4 (0.05 M) in phosphate buffer (0.1 M), 0.05% diami-nobenzidine, NH4Cl and 0.01% hydrogen peroxide (dark blueprecipitate). Some sections were incubated without theprimary antibody. No immunoreactivity was found in thesesamples.

4.5. Aquaporin-1 immunofluorescence and confocalmicroscopy

Some samples were stained with a saturated solution ofSudan black B (Merck) for 30min to block the autofluorescenceof lipofuscin granules, rinsed in 70% ethanol and washed indistilled water. The sections were incubated at 4 °C overnightwith the anti-aquaporin 1 (Abcam) antibody at a dilution of1:500 and monoclonal anti-GFAP antibody (Dako) used at adilution of 1:400. After washing in PBS, the sections wereincubated in the dark with anti-rabbit Alexa488 (green) andanti-mouse Alexa555 (red) (Molecular Probes) used at adilution of 1:400. After washing in PBS, the sections weremounted in Immuno-Fluore Mounting medium (ICN Biome-dicals), sealed and dried overnight. Sections were examinedwith a Leica TCS-SL confocal microscope.

Acknowledgments

This work was supported in part by Fundació ACE, InstitutCatalà de Neurociències Aplicades de Barcelona and to thedonations obtained by Ms. Cristina Alonso and other familiesfor the research in early Alzheimer Disease, and FIS grantsPI04-355 and PI05/1570, and supported by the EuropeanCommission under the Sixth Framework Programme (Brain-Net Europe II, LSHM-CT-2004-503039). We wish to thank T.Yohannan for editorial assistance.

R E F E R E N C E S

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