Journal of Alzheimer’s Disease 40 (2014) 981–992DOI 10.3233/JAD-132030IOS Press

981

Synaptic and Sub-Synaptic Localization ofAmyloid-! Protein Precursor in the RatHippocampus

Diana I. Rodriguesa, Jessie Gutierresa, Anna Pliassovaa, Catarina R. Oliveiraa,b,Rodrigo A. Cunhaa,b and Paula Agostinhoa,b,!aCNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, PortugalbFMUC-Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Accepted 31 December 2013

Abstract. Amyloid-! protein precursor (A!PP) is a large transmembrane protein highly expressed in the central nervous systemand cleavage of it can produce amyloid-! peptides (A!) involved in synaptic dysfunction and loss associated with cognitiveimpairment in Alzheimer’s disease (AD). Surprisingly, little is known about the synaptic and sub-synaptic distribution of A!PPin different types of nerve terminals. We used total, synaptic, sub-synaptic, and astrocytic membrane preparations obtained fromthe hippocampus of adult rats to define the localization of A!PP, using two different antibodies against different A!PP epitopes.Western blot analysis revealed that A!PP was not significantly enriched in synaptosomal as compared to total membranes.Within synapses, A!PP immunoreactivity was more abundant in pre- (60 ± 4%) than post- (30 ± 5%) or extra-synaptic fractions(10 ± 2%). Immunocytochemical analysis of purified nerve terminals indicated that A!PP was more frequently associated withglutamatergic (present in 31 ± 4% of glutamatergic terminals) rather than with GABAergic (16 ± 3%) or cholinergic terminals(4 ± 1%, n = 4). We also observed a general lack of co-localization of A!PP and GFAP immunoreactivities in the hippocampusof sections of adult rat brain, albeit we could detect the presence of A!PP in gliosomes (vesicular specializations of astrocyticmembranes), suggesting that A!PP has a heterogeneous localization restricted to certain regions of astrocytes. These resultsprovide the first direct demonstration that A!PP is mostly distributed among glutamatergic rather than GABAergic or cholinergicterminals of the adult rat hippocampus, in remarkable agreement with the particular susceptibility to dysfunction and degenerationof glutamatergic synapses in early AD.

Keywords: Alzheimer’s disease, amyloid-! protein precursor, gliosomes, nerve terminals, sub-synaptic fractions, synaptosomes

INTRODUCTION

The amyloid-! protein precursor (A!PP) is a trans-membrane protein highly expressed throughout theneuroaxis with complex trafficking and metabolicdynamics [1, 2]. This protein is expressed in threedifferent major isoforms generated by alternative splic-ing, consisting of 695, 751, or 770 amino acids, withthe A!PP 695 isoform being the most abundant in thebrain [1, 3]. In spite of its abundance, the physiological

!Correspondence to: Paula Agostinho, Center for Neuro-sciences of Coimbra, University of Coimbra, 3004-517 Coimbra,Portugal. Tel.: +351 239820190; Fax: +351 239822776; E-mails:pmgagostinho@gmail.com or pagostinho@fmed.uc.pt.

function of A!PP remains elusive, with evidencepointing to a role in the control of cell-cell adhesion,neuronal migration, synaptogenesis, maintenance ofsynaptic stability, or regulation of synaptic morphol-ogy [3–7].

A!PP has received considerable attention becauseits proteolytic processing through an amyloidogenicpathway can produce amyloid-! (A!) peptides [8,9], which has been proposed to be a major culpritof Alzheimer’s disease (AD) [10, 11]. The earliestphases of AD are associated with a dysfunction andloss of synapses in temporal brain regions, namelyin the hippocampus [12–14], which is in tight agree-ment with the ability of A! peptides to accumulate

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982 D.I. Rodrigues et al. / A!PP Location in Glutamatergic Synapses

in central synapses [15, 16] and trigger synaptotoxic-ity and memory impairment [17]. However, it is stillnot known why only some particular synapses, namelylimbic cortical glutamatergic synapses [18–21], begindegenerating in early AD. One unexplored possibil-ity is that A!PP and the secretases responsible for theamyloidogenic pathway might be differentially locatedin different types of nerve terminals. As a first step toexplore this possibility, we now investigated if A!PPis more abundantly located in synapses and if it ispredominantly associated with any particular type ofsynapses in the rat hippocampus, a brain region under-going an early synaptic dysfunction and loss in mildcognitive impairment and early AD [14].

MATERIAL AND METHODS

Animals

Male Wistar rats (Charles River, Barcelona, Spain)were used throughout this study and were handledaccording to the principles and procedures outlinedas “3Rs” in the guidelines of European Union guide-lines (86/609/EEC), FELASA and ARRIVE, and wereapproved by the Animal Welfare Committee of theCenter for Neuroscience and Cell Biology of Coimbra.The rats were maintained under controlled environ-ment (23 ± 2"C, 12 h-light/dark cycle, ad libitumaccess to food and water) until 8–10 weeks old. Theanimals were sacrificed by decapitation, after anesthe-sia with halothane atmosphere, and the hippocampiwere rapidly isolated to perform total, synaptic, sub-synaptic, and astrocytic membrane preparations.

Synaptosomes and total membranes

Total membranes and synaptosomes were preparedfrom the same animals, essentially as previouslydescribed and validated [22]. Briefly, the hippocampifrom one rat were homogenized at 4"C in sucrose solu-tion (0.32 M sucrose, 1 mM EDTA, 10 mM HEPES,1 mg/mL BSA - bovine serum albumin; pH 7.4),supplemented with a protease inhibitor, phenylmethyl-sulfonyl fluoride (PMSF 0.1 mM), a cocktail ofinhibitors of proteases (CLAP 1%, Sigma-Aldrich,Sintra, Portugal) and the antioxidant dithiothreitol(DTT, 1 "M). The homogenate was centrifuged at3,000# g for 10 min at 4"C, and the resulting super-natant was then divided to prepare total membranesand synaptosomes.

To prepare the total membranes, the supernatant wasfurther centrifuged at 25,000 # g for 60 min at 4"C.

The supernatants were discarded and the pellet, corre-sponding to total membranes from different brain celltypes, was resuspended in 5% (w/v) sodium dodecylsulfate (SDS).

To prepare synaptosomal membranes, the super-natant was further centrifuged at 14,000 # g for 12 minat 4"C. The resulting pellet (P2 fraction) was resus-pended in 1 mL of a 45% (v/v) Percoll solution inHEPES buffer (140 mM NaCl, 5 mM KCl, 25 mMHEPES, 1 mM EDTA, 10 mM glucose; pH 7.4) (see[22]). After centrifugation at 14,000 # g for 2 min at4"C, the white top layer was collected (synaptosomalfraction), resuspended in 1 mL HEPES buffer and fur-ther centrifuged at 20,800 # g for 2 min at 4"C. Thesupernatants were discarded and the pellet was resus-pended in 5% (w/v) SDS supplemented with 0.1 mMPMSF and CLAP (1%), for western blot analysis.

Sub-synaptic fractionation

To obtain enriched membrane fractions represen-tative of the different sub-cellular components ofsynapses, such as the pre-synaptic, post-synaptic, andextra-synaptic fractions, we used a method previouslydescribed by Philips and colleagues (2001) with somemodifications (see [22, 23]); this sub-synaptic fraction-ation method allows an over 90% effective separationof the pre-synaptic active zone (enriched in SNAP-25), post-synaptic density (enriched in PSD95) andnon-active zone fraction or extra-synaptic fraction(enriched in synaptophysin).

The sub-synaptic fractionation begins with thepreparation of synaptosomes, as described above.These synaptosomes were diluted 1:10 in cold 0.1 mMCaCl2 and an equal volume of 2 # solubilization bufferpH 6.0 (2% Triton X-100, 40 mM Tris, pH 6.0 set at4"C) was added to the suspension. The membraneswere incubated for 30 min on ice with mild agitationand the insoluble material (synaptic junctions) pel-leted (40,000 # g for 30 min at 4"C). The supernatant(extra-synaptic fraction) was decanted and proteinsprecipitated with six volumes of acetone at $20"Cand recovered by centrifugation (18,000 # g for 30 minat $15"C); this extra-synaptic fraction was solubi-lized in 5% SDS (with 1% CLAP, 0.1 mM PMSF, and1 "M DTT) and kept at $20"C for further westernblot analysis. The pellet, containing the synaptic junc-tions, was washed in solubilization buffer (pH 6.0)and resuspended in 10 volumes of a second solubiliza-tion buffer now at pH 8.0 (1% Triton X-100, 20 mMTris, pH 8.0 set at 4"C), to disrupt inter-synaptic con-tacts. This mixture was stirred softly for 30 min on

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ice and further centrifuged at 40,000 # g for 30 minat 4"C. The pellet contains the post-synaptic density(insoluble with the low amount of detergent used)and the supernatant corresponds to pre-synaptic activezone. This separation depends on pH and the amountof detergent used for solubilization (see [23]). Toconcentrate the pre-synaptic proteins, the supernatant(10 mL) was diluted 5x with cold acetone ($20"C)and kept overnight at $20"C, and further centrifugedat 18,000 # g for 30 min at $15"C. The obtained pellet(pre-synaptic fraction), as well as the pellets corre-sponding to post-synaptic density, were solubilizedwith 5% SDS containing 1% CLAP, 0.1 mM PMSF,and 1 "M DTT for further western blot analysis.

Preparation of astrocytic membranes (gliosomes)

Gliosomes were prepared as previously describedand validated [24]. Briefly, the hippocampi werehomogenized as described above and centrifuged at3,000 # g for 10 min at 4"C. The resulting supernatantwas gently stratified onto a discontinuous Percollgradient (2, 6, 15, and 23% v/v of Percoll in amedium containing 0.32 M sucrose and 1 mM EDTA,pH 7.4). The mixture was centrifuged at 31,000 # gfor 5 min (without using the brake to avoid the dis-ruption of gradients; see [25]). The layers between 2and 6% of Percoll (gliosomal fraction) and between15 and 23% of Percoll (purified nerve terminals)were independently collected, each washed in 10 mLof Krebs-HEPES buffer (140 mM NaCl, 5 mM KCl,5 mM NaHCO3, 1.2 mM NaH2PO4, 1 mM MgCl2,10 mM glucose, and 10 mM HEPES, pH 7.4), and bothfurther centrifuged at 22,000 # g for 15 min at 4"C,to remove myelin components and postsynaptic mate-rial from the gliosomal and nerve terminal fractions,respectively. Both fractions were then resuspended in5% SDS with 1% CLAP, 0.1 mM PMSF, and 1 "MDTT for subsequent western blot analysis.

Western blot analysis

The protein content of membrane samples wasestimated using the bicinchoninic acid (BCA) assay(Pierce Technology, Rockford, IL, USA). Sampleswere then diluted 6x in SDS- buffer (0.5 M Tris,30% glycerol, 10% SDS, 0.6 mM DTT, and 0.012%of bromophenol blue) and denaturated at 95"C for5 min. These samples and the pre-stained molecu-lar weight markers (dual-color standards; BioRad,Amadora, Portugal) were loaded and separated bySDS-PAGE electrophoresis (in 7.5% polyacrylamide

resolving gels with 4% polyacrylamide stackinggels) under denaturating/reducing conditions, using abicine-buffered solution (20 mM Tris, 192 mM bicine,and 0.1% SDS; pH 8.3) at 80–100 mV. The proteinswere then transferred from the gel (by applying acurrent of 1 A for 1.5 h at 4"C under constant agita-tion) to polyvinylidene difluoride (PVDF) membranes(0.45 "m pore diameter; GE Healthcare, Bucking-hamshire, UK), previously activated in 100% methanoland hydrated in distilled water, using a CAPS [3-(cyclohexylamino)-1-propane-sulfonic acid]-bufferedsolution with methanol (10 mM CAPS and 10%methanol; pH 11). The membranes were then blockedfor 1 h at room temperature with 5% BSA in Tris-buffered saline (20 mM Tris, 140 mM NaCl; pH7.6) with 0.1% Tween 20 (TBS-T) and then incu-bated overnight at 4"C with the primary antibodiesdiluted in TBS-T with 3% BSA. After washing threetimes for 15 min each with TBS-T, the membraneswere incubated for 1 h at room temperature with thephosphatase-linked secondary antibodies, also dilutedin TBS-T with 3% BSA. Again, membranes werewashed three times for 15 min each with TBS-T, andthen incubated with enhanced chemifluorescence sub-strate (ECF; GE Healthcare) for varying times, up toa maximum of 5 min. Finally, proteins were detectedusing the Imaging system VersaDoc 3000 (Biorad) andanalyzed with Quantity One software (BioRad).

The following primary antibodies were used: rabbitanti-A!PP C-terminal (1:8,000; A8717 from Sigma),mouse anti-A!PP N-terminal (1:1,000; clone 22C11,MAB348 from Millipore, Madrid, Spain), rabbitanti-synaptophysin (1:20,000; Millipore), mouse anti-SNAP25 (1:40,000; Sigma), and mouse anti-PSD95(1:20,000; Sigma). The following secondary antibod-ies were also used: goat anti-rabbit IgG antibodyconjugated with alkaline phosphatase (1:20,000; GEHealthcare) and goat anti-mouse IgG antibody conju-gated with alkaline phosphatase (1:10000; Santa CruzBiotechnology, Heidelberg, Germany).

Immunocytochemical analysis of purified nerveterminals

Nerve terminals were purified through a discontin-uous Percoll gradient initially described by Dunkleyand adapted by our group (see, [25, 26]). In brief,the hippocampi were homogenized in 0.25 M sucroseand 10 mM HEPES with 1% CLAP, 0.1 mM PMSF,and 1 "M DTT (pH 7.4 at 4"C) and then centrifugedat 9,500 # g for 13 min at 4"C. The resulting pelletwas placed onto a Percoll discontinuous gradient, con-

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taining 23, 10, and 3% Percoll made up in a sucrosesolution (0.32 M sucrose, 1 mM EDTA, and 0.25 MDTT), and centrifuged at 25,000 # g for 11 min at4"C. Purified nerve terminals were collected betweenthe 10 and 23% bands and diluted in Krebs-HEPESbuffer, then centrifuged at 22,000 # g for 11 min at4"C. The pellet of nerve terminals was resuspended inKrebs-HEPES buffer and layered over coverslips pre-viously coated with poly-D-lysine and left to adherefor 1 h. Then, these nerve terminals plated on cov-erslips were fixed with 4% paraformaldehyde for15 min and washed twice with phosphate-bufferedsaline (PBS, composed of 140 mM NaCl, 3 mM KCl,20 mM NaH2PO4, 15 mM KH2PO4, pH 7.4).

The immunocytochemical analysis of hippocampalnerve terminals was performed essentially as previ-ously described [21, 30]. The nerve terminals werepermeabilized in PBS with 0.2% Triton X-100 for10 min and then blocked for 1 h in PBS with 3%BSA and 5% normal horse serum. The nerve ter-minals were then washed twice with PBS with 3%BSA and incubated in the same medium with rabbitanti-A!PP C-terminal (1:1,000; Sigma), together witheither guinea pig anti-vesicular glutamate transporter 1(vGluT1; 1:1,000; Synaptic System, Goettingen, Ger-many), guinea pig anti-vesicular GABA transporter(vGAT; 1:500; Synaptic System), guinea-pig anti-vesicular acetylcholine transporter (vAChT, 1:300;Abcam, Cambridge, UK) or mouse anti-synaptophysin(1:200; Sigma) for 1 h at room temperature. Afterwashing three times with PBS with 3% BSA, the nerveterminals were incubated for 1 h at room tempera-ture with AlexaFluor-594-labeled (red) goat anti-rabbitIgG antibody and AlexaFluor-488-labeled (green) goatanti-guinea-pig or anti-mouse IgG antibodies (1:200for each; Invitrogen, Carlsbad, CA, USA). We con-firmed that none of the secondary antibodies producedany signal in preparations to which the additionof the corresponding primary antibody was omitted.After the incubation with the secondary antibodies,the coverslips with the nerve terminals were washedand mounted on slides with Prolong Gold Antifade(Molecular Probes, Leiden, The Netherlands). Thepreparations were visualized in a Zeiss ImagerZ2 fluorescence microscope equipped with a Plan-ApoChromat 63x/1.4 and analyzed with AxiovisionSE64 4.8.2 software (Carl Zeiss, Gottingen, Germany).Five images were randomly taken from each coverslip(2-3 per experiment), and a minimum of 90 individ-ualized elements per field were analyzed using theImage J (NIH, USA) program and a customized macroto count group of pixels with a defined size, being

the synaptosomes defined between 2-15 pixels (see[21]). The quantitative estimation of co-localized pro-teins in immunocytochemical studies was performedusing another Image J-based macro calculating the co-localization coefficients from the two color-channelscatter plots based on the Pearson’s correlation coef-ficient, a standard procedure for matching one imagewith another in pattern recognition, upon determina-tion of the threshold intensity for each color channel.

Immunohistochemistry of rat brain sections

The preparation of brain coronal sections wascarried out as previously described [27]. Briefly,the rats were transcardiacally perfused with a 4%paraformaldehyde solution in 0.15 mM sodium phos-phate buffer (pH 7.4) under deep anesthesia. Thefixed brains were frozen and sectioned (30 "m coronalslices) using a cryostat (Leica CM3050 S). Immunohis-tochemistry was performed on free-floating sections aspreviously described by our group [28]. The sectionswere washed with PBS and exposed to a 10 mM citricacid solution (pH 6.0) during 20 min at 60"C for anti-gen retrieval. After washing, the sections were blockedin PBS containing 5% normal horse serum and 0.25%Triton X-100 for 2 h at 25"C before incubation in thissame solution with the primary antibodies (overnightat 4"C): rabbit anti-A!PP C-terminal (1:1,000; Sigma)or mouse anti-A!PP N-terminal (1:100; Millipore)together with either mouse anti-NeuN (1:100; Mil-lipore), rabbit anti-GFAP (1:1,000; Dako, Glostrup,Denmark), or mouse anti-GFAP (1:1,000; Sigma).Excess of antibody was rinsed twice for 10 min withPBS and sections were then incubated for 2 h at 25"Cwith AlexaFluor 488-labeled donkey anti-mouse andAlexaFluor 594-labeled donkey anti-rabbit secondaryantibodies (1:400; Invitrogen) made up in PBS.

Finally, the sections were rinsed three times, incu-bated with DAPI (1:5,000; Sigma) for 15 min, andfurther rinsed and mounted onto gelatinized micro-scope slides. The slides were dried at room temperatureand covered with fluorescence mounting medium(Dako). Images were acquired in a Zeiss Imager Z2fluorescence microscope with Plan Neofluar 20x/0.4and 40x/0.6 objectives and analyzed with the Axio-vision SE64 4.8.2 software. Some sections werefurther analyzed in a Zeiss LSM510 META confocallaser-scanning microscope using a Plan-ApoChromatobjective 63x/1.4 with the LSM510 software. It wasconfirmed that none of the secondary antibodies pro-duced any signal in sections that were not incubatedwith primary antibodies.

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Statistical data analysis

Data were presented as mean ± S.E.M. of the num-ber of experiments indicated. Statistical significancewas determined using unpaired Student’s t test forcomparison between 2 groups or one-way ANOVA fol-lowed Tukey post hoc tests for comparison of multiplegroups.

RESULTS

Synaptic and extra-synaptic localization of A!PP

We first compared by western blot analysis thelevels of A!PP in membranes of synaptosomes (puri-fied synapses), which represent between 1-2% ofhippocampal volume [29], and in total membranes(a mixture of membranes from neuronal, glial, andendothelial cells) from the hippocampus of adult rats.Since the antibodies against A!PP can recognize dif-ferent A!PP isoforms, mature and immature forms ofthis protein, the soluble A!PP (sA!PP) fragments orthe APLPs, we tested two different antibodies againstA!PP; one that recognizes an epitope located at theC-terminal (A!PP C-term, from Sigma, A8717) ofA!PP695, A!PP751, and A!PP770 [30] and anotherthat binds to amino acids 66–81 of the N-terminal(A!PP N-term, from Millipore, MAB348) of thesethree A!PP isoforms [31]. By western blot analysis, itwas observed that the two antibodies displayed a sim-ilar immunoreactivity pattern, labeling proteins with110 kDa that corresponds mainly to full-length A!PP(see representative blots of Fig. 1); thus, except whenotherwise stated, we used the A!PP C-term antibody.As shown in Fig. 1, the levels of A!PP (C-terminal)were about 1.2 x higher in synaptosomes than in totalmembranes (p > 0.05) for the same amount of non-saturating protein (5 and 10 "g), and this differencedisappeared as the signal becomes saturated with 20 "gof protein loaded into the gel. These data indicate thatA!PP was present, but not significantly enriched, inhippocampal synapses.

Sub-synaptic localization of A!PP

To further evaluate the sub-synaptic localizationof A!PP, we used a fractionation procedure thatallows the separation of the pre-synaptic active zone,the post-synaptic density, and the non-synaptic (orextra-synaptic) zone [23]. Western blot analysis ofmarkers of the pre-synaptic active zone (syntaxin),extra-synaptic zone (synaptophysin), or post-synaptic

Fig. 1. A!PP immunoreactivity in membranes from nerve terminals(synaptosomes) is slightly higher than in total membranes from therat hippocampus. The density of A!PP was evaluated in synaptoso-mal (Syn) and in total membranes (TM) by western blot analysis,by loading three different quantities of protein (5, 10, 20 "g) of totalor synaptic membranes of the same hippocampus in the same gel.The displayed representative blots were obtained with two antibod-ies against A!PP (110 kDa), one recognizing the A!PP N-terminal(A!PP N-term) and other the A!PP C-terminal (A!PP C-term). Thegraph displays the average percentage of A!PP immunoreactivity(obtained with anti-A!PP C-terminal antibody) for each amount ofloaded protein calculated relative to the maximum immunoreactivityobtained for each immunoblot membrane. Data are the mean ± SEMof four independent experiments (four different animals). n.s., no sta-tistical difference (Student’s t test, p > 0.05, comparing Syn versusTM for the same amount of loaded protein).

density (PSD-95) confirmed a fractionation efficiencyof about 90% (Fig. 2B), similar to that previouslyobtained by our group (see [22]). A!PP immunore-activity was more abundant (n = 3; p < 0.05) in thepre-synaptic active zone fraction (59.9 ± 4.3%) thanin post- (29.6 ± 4.7%) or extra-synaptic fractions(10.4 ± 2.5%), which indicates that A!PP was mainlylocalized in the pre-synaptic active zone but it was alsopresent in the post-synaptic density.

Different association of A!PP with different typesof nerve terminals

A subsequent major goal was to study if A!PP is dif-ferentially associated with glutamatergic, GABAergic,and cholinergic nerve terminals of rat hippocampus.This was tackled using a double labeling immuno-cytochemistry analysis of purified nerve terminals

986 D.I. Rodrigues et al. / A!PP Location in Glutamatergic Synapses

Fig. 2. A!PP is enriched in the pre-synaptic active zone of rat hip-pocampal synapses. A) A!PP density was assessed by western blotof synaptosomal membranes (Syn, internal control) and in its frac-tioned sub-synaptic fractions representative of the presynaptic activezone (Pre), of the postsynaptic density (Post), and of the extra-synaptic regions (Extra). The graph bars represent the relative A!PPimmunoreactivity in percentage of total reactivity that corresponds tothe sum of the immunoreactivities detected in pre-, post-, and extra-synaptic fractions, for each immunoblot, and are the mean ± SEMof three independent experiments. *p < 0.05, **p < 0.01, one wayANOVA, Tukey post-hoc test. B) The purity of these sub-synapticfractions was warranted by the segregation of the pre-synaptic activezone marker (syntaxin), of the post-synaptic density marker (PSD-95) and of the extra-synaptic vesicular marker (synaptophysin), asillustrated by the representative immunoblots.

(plated on coverslips) from the rat hippocampus todetermine the percentage of A!PP that co-localizeswith the different markers of the different nerve ter-minals, namely vesicular glutamate transporter type1 (vGluT1), vesicular GABA transporter (vGAT),or vesicular cholinergic transporter (vAChT). Wehad previously shown that glutamatergic nerve ter-minals represent about 60% [26, 32], GABAergicterminals 35% [26, 33] and cholinergic terminals3% [34, 35] of the total number of hippocampalnerve terminals (i.e., synaptophysin immunopositive).As shown in Fig. 3, we first determined that lessthan half of the total number of nerve terminals(i.e., synaptophysin immunopositive) were endowedwith A!PP (38.3 ± 3.9%, n = 4). Moreover, A!PP

was differently located in different nerve terminalssince A!PP co-localized with 30.9 ± 4.3% (n = 4)of glutamatergic terminals (vGluT1-immunopositive),with 16.1% ± 2.8% of GABAergic terminals (vGAT-immunopositive), whereas a co-localization of only3.7 ± 1.0% was observed for cholinergic terminals(vAChT-immunopositive). Thus, the localization ofA!PP was significantly different in different nerveterminals (p < 0.001, ANOVA), being higher in glu-tamatergic than in GABAergic (p < 0.05, n = 4) orcholinergic (p < 0.01, n = 4) terminals (Fig. 3C).

Localization of A!PP in astrocytes

Since our initial data indicated that A!PP was notonly present in nerve terminals but also in other brainmembranes, we investigated if the non-synaptic A!PPwas only neuronal or if A!PP was also present inastrocytic membranes. We first carried out an immuno-histochemical analysis of rat brain sections to probe theco-localization of A!PP with a marker of mature neu-rons (Neu-N) and of astrocytes (glial fibrillary acidicprotein, GFAP) in the hippocampus. We observed thatA!PP was co-localized with Neu-N (Fig. 4A), whereasno co-localization was observed between the A!PPand GFAP, even using confocal microscopy (Fig. 4B).However, when we compared by immunoblot thedensity of A!PP in gliosomal preparations (astro-cytic membranes) and in synaptosomes (isolated nerveterminals), we observed that A!PP was present in glio-somes with a density similar to that found in nerveterminals (Fig. 5). This surprising observation leadus to re-check (cf. [24]) the enrichment and cross-contamination of our gliosomes and nerve terminalspreparations by determining the density of a synapticvesicle marker (synaptophysin) and of GFAP, a glialprotein marker (see representative blots of Fig. 5),confirming that synaptosomes and gliosomes indeedcorrespond to enriched fractions of nerve terminalsand of astrocytes, respectively. In fact, our prepara-tion of gliosomes was enriched in specific astrocyticproteins, and was de-enriched in markers of nerve ter-minals (see also [24]), albeit the exact percentage ofcontamination with nerve terminal membranes couldnot be rigorously determined. Moreover, gliosomeswere more enriched in glutamate transporters, GLT-I/EAAT-2 and GLAST/EAAT-1, than global astrocyticmarkers such as GFAP, suggesting that gliosomes maymainly correspond to astrocytic processes membranesand may probably provide a better signal-to-noiseto detect A!PP immunoreactivity in astrocytic pro-cesses, as compared with the brain slices. Therefore,

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Fig. 3. A!PP is mostly localized in glutamatergic nerve terminals from the rat hippocampus. A) Representative images (with a magnification of63x) of double immunocytochemistry analysis of A!PP (stained red) with markers of different types of nerve terminals (stained green), mainlyglutamatergic (vesicular glutamate transporter type 1, vGLUT1), GABAergic (vesicular GABA transporter, vGAT), and cholinergic (vesicularacetylcholine transporter, vAChT), as well as with a generic marker of nerve terminals, synaptophysin (synaptosomes population, Syn). Theright hand images are merged images illustrating the co-localization (in yellow) of A!PP with the different synaptic markers. The graph barsdisplay the average percentage of synaptophysin-positive terminals that are endowed with A!PP (B) and the percentage of each different typeof nerve terminals that are endowed with A!PP (C). The results are presented as mean ± SEM of four independent experiments. Statisticalanalysis by ANOVA, analyzing differences between group means, p = 0.0004. *p < 0.05, **p < 0.01, Tukey post-hoc test. Scale bars: A, 10 "m.

988 D.I. Rodrigues et al. / A!PP Location in Glutamatergic Synapses

Fig. 4. Double immunohistochemistry analysis showing that A!PP co-localizes with a neuronal marker (Neu-N), but not with an astrocyticmarker (GFAP) in the rat hippocampus. A) Double staining with A!PP C-terminal (red) and Neu-N (green) and their co-localization (in yellow inthe merged image). A.1) Greater amplification of the hippocampal CA3 region. B) The top raw of images shows the triple immunohistochemistrystaining of A!PP C-terminal (red), GFAP (green), and the merged image with DAPI to stain nuclei, which shows the absence of A!PP/GFAPco-localization; the bottom raw of images shows a similar triple immunohistochemistry staining of GFAP (red), A!PP N-terminal (green), andthe merged image with DAPI to stain nuclei, which confirms the absence of A!PP/GFAP co-localization. Images were obtained by widefield(A and A.1) and confocal (B) microscopy and are representative of three independent experiments. Scale bars: A, 2000 "m; A.1, 100 "m;B, 20 "m.

we conclude that A!PP, besides being present in nerveterminals, may also have a heterogeneous localizationrestricted to particular regions of astrocytes, probablyastrocytic processes.

DISCUSSION

The present study provides novel information onthe cellular and sub-cellular localization of A!PPin the rat hippocampus, by showing that A!PP ismostly located pre-synaptically and it is mainly asso-ciated with glutamatergic rather than with GABAergicor cholinergic terminals. In spite of the predomi-nant association of A!PP with neurons rather thanwith astrocytes, A!PP also seems to be associated

with discrete astrocytic compartments enriched in glio-somes preparations, probably representing astrocyticprocesses.

A!PP is expressed in three different major isoformsgenerated by alternative splicing, consisting of 695,751, or 770 amino acids, with the A!PP 695 iso-form being the most abundant in the brain [1]. A!PPcan form heteromers with the homologs amyloid-precursor-like proteins 1 and 2 (APLP1 and APLP2),which are expressed in the brain and affect many bio-chemical pathways in a manner similar to A!PP [3,36]. However, since only the proteolytic processingof A!PP by secretases can give rise to A!, which isinvolved in AD pathogenesis, we focus our attention inA!PP. It is important to stress that we cannot fully ruleout the possibility that the antibodies used might also

D.I. Rodrigues et al. / A!PP Location in Glutamatergic Synapses 989

Fig. 5. A!PP is present in astrocytic membranes (gliosomes). Thedensity of A!PP was estimated by western blot analysis in mem-branes from nerve terminals (synaptosomes) and from astrocytes(gliosomes), obtained from the hippocampus of the same adult Wis-tar rats, by loading three different quantities of protein (5, 15, 25 "g)in the same gel. The bar graph displays the average percentageof A!PP immunoreactivity (obtained with anti-A!PP C-terminalantibody) for each amount of protein calculated relatively to themaximum immunoreactivity obtained for each immunoblot mem-brane and the data represent the mean ± SEM of four independentexperiments such as that displayed below the bar graph (obtainedwith the anti-A!PP C-terminal antibody). The purity of the glio-somes and synaptosomes was confirmed by the enrichment ofsynaptophysin (38 kDa) and GFAP (50 kDa) in synaptosomes andgliosomes, respectively, as illustrated in the representative westernblots.

recognize APLPs [36, 69]; however this possibility isremote since we used two different antibodies againstdifferent A!PP epitopes, our western blot analysis didnot reveal additional bands, and these antibodies havebeen used an validated in heterologous expression sys-tems and in A!PP knockout mice as largely selectivefor A!PP [70–72].

Our immunohistochemical results confirm previousstudies supporting a widespread distribution of A!PPthroughout the neuroaxis [1, 4, 37–40]. As also notedby others [37–40], the predominant localization ofA!PP was in neurites and in cell bodies of neurons, inagreement with the observed co-localization of A!PPwith the neuronal marker Neu-N. In agreement withseveral studies that have searched and confirmed aparticular localization of A!PP in synapses [38, 41,57], we found that A!PP was slightly enriched inmembranes from hippocampal synapses. This clearlocalization of A!PP in synapses, without a significantenrichment over total membranes, probably reflects

the intense localization of A!PP in intracellular mem-branes, namely in the peri-nuclear regions of neuronsand throughout neurites [37–39, 41–43], as a resultof the intense and complex trafficking of A!PP to besorted to the plasma membrane ([44, 45]; reviewed in[1] and [2]). The synaptic localization of A!PP nowfound in the rat hippocampus is in agreement with thepredominant synaptic effects attributed to A!PP. Infact, A!PP has been proposed to be involved in synapseformation [38, 46], elimination [47], and stability [5,48], as well as in the trafficking of synaptic compo-nents [49, 57]. Furthermore, some studies reported atight association between the metabolism of A!PPand synaptic activity [50, 51] and localization of #-secretase [52] and $-secretase in synapses [53], furtherreinforcing the association of A!PP with synapses inthe central nervous system. Within synapses, we nowprovide a direct comparison of the relative abundanceof A!PP in the presynaptic [44, 54] and in the post-synaptic compartments [38, 54], and it was found thatA!PP was more abundantly located in the presynap-tic active zone. This finding is in agreement with arecent report proving clear evidence for the localiza-tion of A!PP at the presynaptic active zone [55], andwith the indication that A!PP is largely excluded fromsynaptic vesicles [44], displaying a predominant local-ization at the plasma membrane of stable synapses[43–45].

One of the major conclusions of this study was thatA!PP displays a heterogeneous distribution amongdifferent types of synapses in the hippocampus: thusless than half of the hippocampal synapses wereendowed with A!PP and this protein was mostabundantly localized in glutamatergic rather than inGABAergic or cholinergic synapses. This is of partic-ular interest when considering that A!PP distributionshould define the generation of A! peptides [8, 9],which is considered the major candidate culprit in AD[10, 11]. In fact, the loss of synaptic markers (e.g.,[15, 56]) and the loss of synapses (e.g., [12, 14]) aresome of the earliest changes associated with mild cog-nitive impairment and early AD. Notably, albeit thehippocampus and other cortical structures seem to beprimarily affected [14], not all synapses, but rather aselective group of synapses, seem to be damaged. Inview of the ability of A! peptides to target synapses[15, 16] and to cause their dysfunction and damage(reviewed in [11–13, 58]), the currently reported local-ization of A!PP in a limited subgroup of synapsesmakes it tempting to suggest that only those synapsesendowed with A!PP will be able to generate abnor-mal levels A! peptides to trigger their deterioration

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during early AD pathogenesis. This provocative work-ing hypothesis is further heralded by the notableagreement between the presently observed more abun-dant localization of A!PP in glutamatergic synapseswith the particular impact of A! peptides on gluta-matergic synapses (reviewed in [6, 17, 59]) and with theparticular susceptibility of glutamatergic synapses inearly AD [16, 18–21]. Further studies will be requiredto document the association of different secretaseswith glutamatergic terminals and eventual modifica-tions of A!PP density and metabolism in glutamatergicsynapses in aging and in animal models of AD.

Finally, our results also identify the presence ofA!PP in gliosomes, which are a membrane prepara-tion representative of astrocytes [24, 60, 61]. This wasin striking contrast with the lack of co-localization ofA!PP immunoreactivity with GFAP in brain sections.Given that A!PP is expressed in astrocytes (e.g., [62,63]) and located in specializations of astrocytic profiles[64], it is tempting to suggest that A!PP may have arestricted in the processes of these cells, which maybe over-represented in gliosomes, as nerve terminalsare over-represented in synaptosomes, a suggestionin agreement with the particular activity of uptakeand release of glutamate in gliosomes in comparisonto other astrocytic preparations [24, 60]. This wouldprompt proposing a particular role of A!PP in thecontrol of glutamate release and uptake by astrocytes,which is affected by A! peptides [61, 65–67] and inAD [19, 68]. Certainly further studies need to be under-taken to better understand the role of astrocytic A!PPin physiological conditions and in AD.

Overall, the results of obtained in the present studyindicate that A!PP is located both in nerve terminals(synaptosomes) and in gliosomes (astrocytic mem-branes) of adult rat hippocampus. This protein ismainly present in the pre-synaptic compartment ofhippocampal neurons, being more enriched in gluta-matergic terminals than in GABAergic or cholinergicterminals. These data contribute to understanding whysynapses, mainly pre-synaptic glutamatergic termi-nals, are particularly vulnerable in AD.

ACKNOWLEDGMENTS

This work was funded by the Portuguese Foun-dation for Science and Technology (PTDC/SAU-NMC/114810/2009 and PEst-C/SAU/LA0001/2011)and CAPES.

Authors’ disclosures available online (http://www.j-alz.com/disclosures/view.php?id=2089).

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