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Neuropharmacology 76 (2014) 51e56

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Neuropharmacology

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Predominant loss of glutamatergic terminal markers in a b-amyloidpeptide model of Alzheimer’s disease

Paula M. Canas a, Ana Patrícia Simões a, Ricardo J. Rodrigues a, Rodrigo A. Cunha a, b, *

a CNC e Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugalb Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal

a r t i c l e i n f o

Article history:Received 30 April 2013Received in revised form18 July 2013Accepted 28 August 2013

Keywords:b-amyloidAlzheimer’s diseaseNerve terminalSynaptosomesGlutamatergicCholinergic

* Corresponding author. Center for Neuroscience ofUniversity of Coimbra, 3004-517 Coimbra, Portugfax: þ351 239822776.

E-mail address: cunharod@gmail.com (R.A. Cunha

0028-3908/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2013.08.026

a b s t r a c t

Alzheimer’s disease (AD) is characterized phenotypically by memory impairment, neurochemically byaccumulation of b-amyloid peptide (such as Ab1e42) and morphologically by an initial loss of nerveterminals in cortical and hippocampal regions. However, it is not knownwhat nerve terminals are mostlyaffected in early AD. We now used a mouse model of AD, based on the intra-cerebral administration ofsoluble Ab1-42, that leads to memory impairment and loss of nerve terminal markers within 2 weeks, toinvestigate which type of hippocampal nerve terminals was mostly affected in the hippocampus.Western blot analysis revealed a decrease of the density of vesicular glutamate transporters type 1(vGluT1, a marker of glutamatergic terminals; �20.1 � 3.6%) and of vesicular acetylcholine transporters(vAChT, a marker of cholinergic terminals; �27.2 � 0.9%) but not of vesicular GABA transporters (vGAT, amarker of GABAergic terminals) in the hippocampus of Ab-injected mice. Immunocytochemical analysisof single hippocampal nerve terminals revealed that the decrease of the density of vGluT1 reflects areduction of the number of vGluT1-immunopositive nerve terminals (�10.6 � 3.6%), while no significantchanges in the number of vAChT- or vGAT-immunopositive nerve terminals were observed. This pilotstudy shows that, in this Ab-based model of AD, there is an asymmetric loss of different synaptic markerswith a predominant susceptibility of glutamatergic synapses.

This article is part of the Special Issue entitled ‘The Synaptic Basis of Neurodegenerative Disorders’.� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Alzheimer’s disease (AD) is clinically characterized by an initialdeterioration of memory performance that evolves over time. Themost consistent morphological feature related to memory impair-ment in early AD is the loss of synapses (Scheff et al., 1990, 2006;Terry et al., 1991) and of synaptic markers (Masliah et al., 1989;Sze et al., 2000), which is also found in different animal modelsof AD (Mucke et al., 2000; Richardson et al., 2003; Rutten et al.,2005; Jacobsen et al., 2006; Canas et al., 2009). This is in accor-dance with the observation that different conditions that affectmemory performance (stress, depression, diabetes, sleep depriva-tion, epilepsy) also involve the dysfunction and/or loss of synapses,mainly in hippocampal regions (Cunha et al., 2006; Alhaider et al.,2010; Cognato et al., 2010; Duarte et al., 2012; Batalha et al., 2013).

Coimbra, Faculty of Medicine,al. Tel.: þ351 239820190;

).

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Albeit the cause of AD still remains to be firmly identified, theabnormal production of soluble forms of b-amyloid peptides (Ab),such as Ab1e42, have been proposed as a major culprit in AD(reviewed in Hardy and Selkoe, 2002). Ab can impair synapticfunction, typified by its ability to affect synaptic plasticity(reviewed in Venkitaramani et al., 2007; Selkoe, 2008), and triggerevents leading to a loss of viability of synapses (Mattson et al., 1998;Shankar et al., 2007; Canas et al., 2009). Accordingly, the exposureto Ab is correlated (Lue et al., 1999; Näslund et al., 2000; Fagan et al.,2007) and leads to memory impairment (reviewed in Selkoe, 2008)and the intracerebroventricular administration of Ab has beenproposed as a model of AD (reviewed in Lawlor and Young, 2010;Van Dam and De Deyn, 2011). This has allowed us to show thatthe pharmacological or genetic modification of presynaptic neu-romodulation systems able to control synaptic function andviability, such as adenosine A2A receptors, can prevent memorydeterioration (Canas et al., 2009).

The development of strategies targeting the protection of nerveterminal dysfunction in early AD might be better directed by acharacterization of an eventual differential susceptibility of thedifferent synaptic terminals to Ab. Indeed, the cholinergic

Fig. 1. Western blot quantification of different vesicular transporters in hippocampal

P.M. Canas et al. / Neuropharmacology 76 (2014) 51e5652

hypothesis of AD assumes a particular susceptibility of cholinergicneurons (reviewed in Bartus et al., 1982; Auld et al., 2002),reflecting a primary affection of cholinergic terminals in corticalregions early in AD (Hu et al., 2003). More recently, several studieshave suggested a particular vulnerability of glutamatergic synapses(Bell et al., 2003; Kirvell et al., 2006; Kashani et al., 2008;Minkeviciene et al., 2008; Proctor et al., 2010), whereas otherstudies have instead reported that it is GABAergic neurons that areinitially affected in early AD (Agarwal et al., 2008; Baglietto-Vargaset al., 2010; Verret et al., 2012).

As an initial attempt to compare the relative susceptibility ofdifferent types of nerve terminals in early AD, we used a previouslyvalidated AD mouse model based on an intracerebroventricularadministration of Ab1e42 to compare the change of density ofdifferent markers of glutamatergic (vesicular glutamate transportertype 1, vGluT1), GABAergic (vesicular GABA transporter, vGAT) andcholinergic (vesicular acetylcholine transporter, vAChT) nerve ter-minals in the hippocampus.

synaptosomes from control (water-injected) and Ab1e42-injected mice. Panel (A) e

Representative images of Western blot analysis for the different vesicular transporterstested, namely glutamate transporter 1 (vGluT1), glutamate transporter 2 (vGluT2),GABA transporter (vGAT) and acetylcholine transporter (vAChT). Panel (B) e Bar graphsummarizing the average density of the different vesicular transporters, expressed asthe ratio between the density of different vesicular transporters and of synaptophysinthat stains the whole synaptosomal population (fold change in relation to controlmice). There is a decrease of vGluT1 and vAChT in Ab1e42-injected animals. The dataare mean � SEM of n ¼ 4e8, *P < 0.05.

2. Materials and methods

2.1. Animals and drug administration

Male c57Bl6 mice (10e12 weeks) were acquired from Charles River and main-tained under controlled environment (23 � 2 �C; 12 h light/dark cycle) with adlibitum access to food and water. The b-amyloid (1e42) peptide fragment (Ab;Bachem, Bubendorf, Switzerland) was dissolved in water at a concentration of2.25 mg/ml and 2 nmol in 4 ml was administered intracerebroventricularly, as pre-viously described (Canas et al., 2009). This Ab solution was constituted by oligomersup to 4 monomers and circa 5% of the injected amount was present in the hippo-campus after 15 days, without forming aggregates (Canas et al., 2009). Control an-imals were intracerebroventricularly infused with a similar volume of water. Thesacrificewas performed 15 days after the administration. All studies were conductedin accordance with the principles and procedures outlined as “3Rs” in the guidelinesof European Union guidelines (86/609/EEC), FELASA and ARRIVE, and wereapproved by the Animal Care Committee of the Center for Neuroscience and CellBiology of Coimbra.

2.2. Western blot analysis of synaptic markers

After sacrifice, one of the hippocampi from each mouse was collected forpreparation of synaptosomes using sucrose and Percoll differential centrifugations,as previously described (Canas et al., 2009; Cognato et al., 2010). The hippocampalsynaptosomal extracts were solubilized in 5% sodium dodecyl sulfate (SDS; Bio-Rad)supplemented with 2 mM DTT and 100 mM PMSF and rapidly sonicated. Afterdetermining the amount of protein using the bicinchoninic acid method (Pierce),1/6volume of 6� SDS-PAGE sample buffer was added and electrophoresis was carriedout using a 10%-concentration SDS-PAGE. The proteins were then transferred topolyvinylidene difluoride (PVDF)membranes (GE Healthcare). Themembraneswereblocked for 1 h at room temperature (RT) with 5% low-fat milk in Tris-buffered salinemedium pH 7.6, and containing 0.1% Tween 20 (TBS-T). The membranes were thenincubated overnight at 4 �C with the primary antibodies (vGAT, 1:1000 from Cal-biochem; vGluT1, 1:10,000, vGluT2, 1:10,000 and vAChT, 1:500, from Millipore).After three washing periods of 15 min with TBS-T, the membranes were incubatedfor 2 h at RT with the appropriate alkaline phosphatase-tagged secondary antibody(Invitrogen) diluted in TBS-T containing low-fat milk. After three 15-min washeswith TBS-T, the membranes were incubated with Enhanced Chemi-Fluorescence(ECF) kit (GE Healthcare) and visualized in a VersaDoc 3000 imaging system withthe assistance of Quantity One software (Bio-Rad). To determine the ratio betweeneach vesicular transporter and synaptophysin, the membranes were re-probed forsynaptophysin (1:20,000, from SigmaeAldrich) as previously described (see Canaset al., 2009; Cognato et al., 2010). This normalization is essential to define if thereis a loss of any particular type of terminal compared to the global loss of nerveterminals that we have previously shown to occur in this model (Canas et al., 2009).Thus, if the ratio of any particular transporter compared to synaptophysin, anestablished global synaptic marker in Alzheimer’s disease (see Masliah et al., 1993)which staining reflects synaptic densities within the rodent hippocampus (e.g. Toggaet al., 1996; Cunningham et al., 2003), is decreased, then it can be concluded that thistype of terminals undergoes a loss greater than the average loss of nerve terminalsand hence in particularly susceptible. The values are presented as mean� S.E.M. of nexperiments (i.e. in preparation obtained from different mice) and the comparisonsbetween groups were carried out using an unpaired Student’s t test and a family-wise of 95% confidence level (P < 0.05) was considered.

2.3. Immunocytochemical analysis of single nerve terminals

Since aWestern blot analysis only evaluates relative changes of protein densitiesrather than actual a loss of elements endowed with these proteins, we next tookadvantage of an immunocytochemical procedure to quantify the number of plattednerve terminals endowed with each of the markers of the different nerve terminals,which allows disentangling changes occurring in different types of nerve terminalsirrespective of their relative densities in the hippocampus (Köfalvi et al., 2005;Rodrigues et al., 2008). Briefly, after sacrifice, the other hippocampus from eachmouse was collected for purification of nerve terminals with a discontinuous Percollgradient (Dunkley et al., 2008) presenting less than 2% of postsynaptic or glialcontaminations with the rest being stained with synaptophysin (Rodrigues et al.,2005, 2008). The nerve terminals were placed onto coverslips previously coatedwith poly-D-lysine (SigmaeAldrich), fixed with 4% paraformaldehyde (plus 4% su-crose in 0.9% NaCl) for 15 min and washed twice with 0.1 M phosphate bufferedsaline medium (PBS). The nerve terminals were permeabilized in PBS with 0.2%Triton X-100 for 10 min, blocked for 1 h in PBS with 3% bovine serum albumin (BSA)and 5% normal bovine serum to prevent non-specific binding, washed twice withPBS and then incubated with the primary antibodies against synaptophysin (syn-aptophysin 1:200) and against one out of the 4 different vesicular transporters(vGAT,1:1000; vGluT1,1:1000; vGluT2,1:1000; vAChT,1:500) in PBSwith 3% BSA for1 h at RT. After washing three times with PBS with 3% BSA, the nerve terminals wereimmunolabeled with Alexa Fluor 488-conjugated donkey anti-mouse and AlexaFluor 594-conjugated goat anti-guinea pig (1:200, Invitrogen) for 1 h at RT. It wasconfirmed that none of the secondary antibodies produced any signal in prepara-tions to which the addition of the corresponding primary antibody was omitted.Most importantly, it was confirmed that the individual signals in double-labeledfields are not enhanced over the signals under single-labeling conditions. Afterwashing and mounting on slides with Prolong Gold Antifade (Invitrogen), thepreparations were visualized in a Zeiss Axiovert 200 microscope, with Axiovisionsoftware 4.6 (PG-Hitec, Lisbon, Portugal). Each coverslip (three to four per experi-ment) was analyzed by counting three to four different fields analyzing a minimumof 50 individualized elements per field. The values are presented as the percentageof the total number of glutamatergic, GABAergic or cholinergic terminals identifiedby the immunolabeling of the different vesicular markers in relation to the totalpopulation of nerve terminals stained with synaptophysin, as mean � S.E.M. of nexperiments (i.e. preparations obtained from different mice). Comparisons betweengroups were carried out using an unpaired Student’s t test and a family-wise of 95%confidence level (P < 0.05) was considered.

3. Results

Western blot analysis showed a decrease of the density ofvGluT1 (20.1 � 3.6%, n ¼ 5, P < 0.05) and vAChT (27.2 � 0.9%, n ¼ 4,

Fig. 2. Immunocytochemical quantification of hippocampal nerve terminals immu-nolabeled for different vesicular transporters, from control (water-injected) and Ab1e42-injected mice. Panel (A) e Representation of the percentage of nerve terminalsimmunopositive for the different vesicular transporters tested [namely glutamatetransporter 1 (vGluT1), glutamate transporter 2 (vGluT2), GABA transporter (vGAT)and acetylcholine transporter (vAChT)] in relation to the total nerve terminal popu-lation identified as synaptophysin-positive elements. Panel (B) e Representative im-ages of immunocytochemistry for the different vesicular transporters, in whichvesicular transporters are in red and synaptophysin is in green. There is a decrease ofvGluT1 and 2-immunopositive nerve terminals in Ab1-42-injected animals. The data aremean � SEM of n ¼ 4e6, *P < 0.05. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

P.M. Canas et al. / Neuropharmacology 76 (2014) 51e56 53

P < 0.05), and no difference of vGluT2 and vGAT between the Ab-injected and control animals (Fig. 1). This indicates that the Ab-induced loss of nerve terminal markersmostly corresponds to a lossof glutamatergic and cholinergic markers.

An immunocytochemistry approach was used to complementthe Western blot results, showing a decrease of glutamatergicnerve terminals (10.6 � 3.6%, n ¼ 5, P < 0.05) and no differences ofthe relative numbers of GABAergic and cholinergic nerve terminalsbetween the Ab-injected animals when compared to the controlanimals (Fig. 2). This indicates that Ab mostly induces a loss ofnerve terminals endowed with glutamatergic markers, a conclu-sion that should be additionally confirmed by future immunohis-tochemical studies in hippocampal brain sections.

4. Discussion

The present study shows that the intracerebroventricularadministration of Ab1e42 caused a predominant loss of gluta-matergic and cholinergic markers, the former reflecting a reductionof the number of glutamatergic nerve terminals in the hippocam-pus. In contrast, neither the density of vGAT nor the number ofGABAergic nerve terminals was altered. These preliminary datafocusing solely on the evaluation of neurochemical markers, sug-gest that glutamatergic synapses are the most susceptible todysfunction and damage in this animal model of Alzheimer’sdisease.

The main conclusion of this study, based on the reduced densityof the best glutamatergic terminal marker e vGluT1 (Takamoriet al., 2000), is in line with several studies that have suggestedthe involvement of glutamatergic dysfunction in early AD. Indeed, areduction of vGluT1 density in cortical regions was also observed inpost-mortem studies in AD patients (Bell et al., 2006; Kirvell et al.,2006; Kashani et al., 2008), as well as in animal models of AD (Bellet al., 2006; Minkeviciene et al., 2008). This might be related to thetight interplay between APP-derived formation of Ab and the ac-tivity of glutamatergic synapses (Cirrito et al., 2008) and to theaccumulation of Ab in glutamatergic nerve terminals in hippo-campal regions at early stages in AD models (Lacor et al., 2004;Sokolow et al., 2012). Furthermore, several studies in differentanimal models of AD have converged in the identification of earlydysfunctions of synaptic plasticity at cortical and hippocampalglutamatergic synapses (Hsia et al., 1999; Oddo et al., 2003;Trinchese et al., 2004; Jacobsen et al., 2006; Liu et al., 2008;Auffret et al., 2009), which result from a combined alteration ofthe set-up of AMPA and NMDA receptors in synapses (Almeidaet al., 2005; Hsieh et al., 2006; D’Amelio et al., 2011), and from adecreased density and efficiency of astrocytic glutamate trans-porters ( Masliah et al., 2000; Matos et al., 2012; Schallier et al.,2011) which is also observed in the afflicted brain regions of ADpatients (Hardy et al., 1987a; Westphalen et al., 2003). We nowobserved not only a modification of the density of vGluT1, which iscompatible with a dysfunction of glutamatergic synapses, but also areduction in the number of nerve terminals endowed with vGluT1,which is indicative of a particular vulnerability of glutamatergicterminals in early AD models. Notably, reductions of glutamatergicmarkers were also shown to accompany cognitive deficits uponstroke (Kirvell et al., 2010). This can probably reflects the particularassociation of modifications of glutamatergic terminals withcognitive deficits, but it remains to be elucidated if this is a causalrelation or just a consequence of the greater relative abundance ofglutamatergic terminals in cortical regions that make their alter-ations more evident upon memory impairment.

Apart from the putative dysfunction and the loss of gluta-matergic synapses, the present study also identified a decreaseddensity of vAChT in the hippocampus of mice challenged with Ab.There is ample evidence for a cholinergic dysfunction associatedwith memory impairment (Bartus et al., 1982), in particular withAD (Auld et al., 2002), as heralded by the observations that Ab candecrease the evoked release of acetylcholine from hippocampal

P.M. Canas et al. / Neuropharmacology 76 (2014) 51e5654

slices (Kar et al., 1996) and that there is a decreased ability to triggeracetylcholine release in cortical samples from demented in-dividuals (Sims et al., 1983). Several studies in humans and in an-imal models of AD showed that the modification of the cholinergicsystem is an early event, already measurable at pre-symptomaticstages of AD (Watanabe et al., 2009; Potter et al., 2011; Zhanget al., 2012; but see Davis et al., 1999). However, there is somedebate as to whether the changes are compensatory or part of thedisease mechanism. Besides, the activity of choline acetyltransfer-ase (ChAT), used as a cholinergic marker, was reported to beincreased in MCI and decreased with evolution of dementia(Ikonomovic et al., 2003). This is probably the reason why somestudies reported a decrease of vAChT density (Hu et al., 2003; Bellet al., 2006), whereas others found no modification or increasedvAChT density in AD models (Wong et al., 1999; Gau et al., 2002). Itthus appears that the timing of observation might be crucial sinceAb affects cholinergic function in a time- and concentration-dependent manner (Perez et al., 2007; Nunes-Tavares et al.,2012). The present results add now one important novel observa-tion: at early AD in the used mouse model of Ab-induced memoryimpairment, there is a dysfunction of the cholinergic system (loss ofvAChT density) without a measurable loss of cholinergic terminals(no loss of vAChT-containing nerve terminals). This may occurlatter in the evolution of the disease based on the observation thatthe cholinergic degeneration in AD models is secondary to a glu-tamatergic excitotoxicity (Harkany et al., 2000).

In parallel with the dysfunction of glutamatergic and cholinergicterminals and the loss of glutamatergic terminals in this model ofearly AD, we also observed an apparent preservation of the pre-synaptic GABAergic marker, vGAT. It is important to emphasize thatthe lack of change of the ratio between vGAT and synaptophysinlevels between control and Ab-treated mice does not necessarilymean that there is no loss of GABAergic terminals; in fact, this resultmeans that a putative loss of vGAT is not more prominent than thegeneral loss of nerve terminals in the hippocampus of Ab-treatedmice sincewe have previously shown that there is a global decreaseof synaptophysin density in Ab-treated mice (Canas et al., 2009).This lack of evident changes in GABAergic terminals is in line withthe recent report showing that the number of GABAergic neurons isnot altered in early phases of AD (Krantic et al., 2012) being onlymodified at later stages of AD (Hardy et al., 1987b; Bell et al., 2003,2006; León-Espinosa et al., 2012). However, this finding should nothinder the evidence that there are early changes in the activity ofGABAergic neurons in early AD (Baglietto-Vargas et al., 2010; Verretet al., 2012), typified by a desynchronization of hippocampal cir-cuits (reviewed in Palop and Mucke, 2010) which correction withlevetiracetam results in the preservation of memory performance(Sanchez et al., 2012). Since this is proposed to result from thedysfunction of particular populations of interneurons (Baglietto-Vargas et al., 2010; Verret et al., 2012), it is possible that putativechanges in the presynaptic density and function of these particularpopulations may not be measurable with a global marker ofGABAergic terminals such as vGAT.

In conclusion, the present study revealed a particular suscepti-bility of glutamatergic nerve terminals in the hippocampus in amodel of Ab-induced memory impairment whereas cholinergicterminals seem affected but are not lost and no significant changeswere observed in GABAergic terminals. Although the present studyonly investigated neurochemicalmarkers rather than histological orfunctional features, it further emphasizes thenotionhinted fromtheexploration of transgenicmodels of early-staged amyloid pathologyand frompost-mortem studies in patientswith AD, that the amyloidpathology progresses in a neurotransmitter-specific manner(reviewed in Bell and Cuello, 2006). This asymmetric affection ofnerve terminals in early AD may be due to different metabolic

demands, to alterations of the levels of trophic factors, susceptibilityto glial dysfunction or alternatively to a particular APP-derived Abproduction in glutamatergic versus cholinergic and GABAergicsynapses (Wurtman, 1992; Counts and Mufson, 2005; Cirrito et al.,2008; Agostinho et al., 2010; de la Monte, 2012). The elucidationof the molecular mechanisms underlying this differential suscepti-bility of different terminals may pave the way for the developmentof novel therapeutic strategies to arrest the evolution of AD.

Conflict of interests

None of the authors has any conflicts of interest.

Acknowledgments

This study was sponsored by Fundação para a Ciência e Tecno-logia (PTDC/SAU-NMC/114810/2009 and PEst-c/SAU/LA0001/2011).

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