Postsynaptic density protein PSD-95 expression in Alzheimer's disease and okadaic acid induced...

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Postsynaptic density protein PSD-95 expression in Alzheimer'sdisease and okadaic acid induced neuritic retraction

Geneviève Leuba,a,b,1 Claude Walzer,c André Vernay,a,b Béatrice Carnal,a,b Rudolf Kraftsik,d

Françoise Piotton,c Pascale Marin,c Constantin Bouras,c and Armand Saviozc,⁎,1

aCenter for Psychiatric Neuroscience, Department of Psychiatry, CHUV, Lausanne, SwitzerlandbService of Old Age Psychiatry, Department of Psychiatry, CHUV, Lausanne, SwitzerlandcDepartment of Psychiatry, University Hospital Geneva, Geneva, SwitzerlanddDepartment of Cell Biology and Morphology, Lausanne University, Lausanne, Switzerland

Received 20 October 2007; revised 18 January 2008; accepted 22 February 2008Available online 12 March 2008

In order to understand how plasticity is related to neurodegeneration,we studied synaptic proteins with quantitative immunohistochemistry inthe entorhinal cortex from Alzheimer patients and age-matchedcontrols. We observed a significant decrease in presynaptic synapto-physin and an increase in postsynaptic density protein PSD-95,positively correlated with β amyloid and phosphorylated Tau proteinsin Alzheimer cases. Furthermore, Alzheimer-like neuritic retractionwas generated in okadaic acid (OA) treated SH-SY5Y neuroblastomacells with no decrease in PSD-95 expression. However, in a SH-SY5Yclone with decreased expression of transcription regulator LMO4 (asobserved in Alzheimer's disease) and increased neuritic length, PSD-95expression was enhanced but did not change with OA treatment.Therefore, increased PSD-95 immunoreactivity in the entorhinal cortexmight result from compensatory mechanisms, as in the SH-SY5Y clone,whereas increased Alzheimer-like Tau phosphorylation is not related toPSD-95 expression, as suggested by the OA-treated cell models.© 2008 Elsevier Inc. All rights reserved.

Keywords: Alzheimer's disease; Dendritic plasticity; Synaptic proteins;Entorhinal cortex; PSD-95; Synaptophysin; LMO4; Immunohistochemis-try; SH-SY5Y; Okadaic acid

Introduction

In Alzheimer's disease (AD), extensive neuronal lesions areknown to occur in tandem with plasticity (synaptic and/or dendriticsprouting) (Flood et al., 1985; Mckee et al., 1989; Bertoni-Freddariet al., 1990; Flood, 1994; Terry and Katzman, 2001; Price et al.,

2001). Dendritic proliferation were noted in the entorhinal cortex(EC) in AD patients together with dendritic deteriorations(Scheibel and Tomiyasu, 1978; Ihara, 1988; Mckee et al., 1989;Arendt et al., 1995, 1998). In order to better understand the relativecontribution of compensatory versus degenerative mechanisms inAD, we studied the immunohistochemical expression of specificsynaptic proteins (e.g. synaptophysin, α-synuclein) together withsome cytoskeletal proteins and AD markers (e.g. phosphorylatedneurofilaments and Tau) in the human EC of elderly versus ADpatients. As dendritic markers, we were interested by proteins im-plicated in spine morphology, e.g. the postsynaptic density proteinof 95 kDa PSD-95 (El-Husseini et al., 2000; Kim and Sheng, 2004;van Zundert et al., 2004; Boeckers, 2006) and spinophilin (Feng etal., 2000; Sarrouilhe et al., 2006). PSD-95mediated plasticity couldalso engage proteins linked to cytoskeleton (Kuriu et al., 2006; Chenget al., 2006). PSD-95 has not been studied previously by im-munohistochemistry in the aging human brain but was shown todecrease either in synaptosome fraction from AD association cortex(Gylys et al., 2004) or in the superior temporal gyrus ofADpatients byELISA (Love et al., 2006). However, no significant changes in PSD-95 were shown either by real-time PCR in the prefrontal and occipitalAD cortex (Dracheva et al., 2001), or by Western blot in the frontalcortex of oldest-old (Head et al., 2007). Spinophilin immunoreactivitywas recently related to neurofibrillary tangle (NFT) staging in thehuman hippocampus and frontal cortex (Akram et al., 2007).

Furthermore, we investigated in a cell model, where we can induceneuritic outgrowth or retraction, if and how the main changes inprotein expression (mainly PSD-95) observed in the AD entorhinalcortex are related to compensatory sprouting or dendritic degenera-tion. We used human SH-SY5Y neuroblastoma cells that are com-monly employed to induce Tau phosphorylation as an early stage ofneurofibrillary tangles formation in tauopathies (Caillet-Boudin andDelacourte, 1996; Shea and Fischer, 1996). SH-SY5Y cells weretreated with okadaic acid (OA), an inhibitor of phosphatases PP1 and

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⁎ Corresponding author. Fax: +41 022 305 53 50.E-mail address: armand.savioz@hcuge.ch (A. Savioz).

1 Authors who have contributed equally.Available online on ScienceDirect (www.sciencedirect.com).

0969-9961/$ - see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.nbd.2008.02.012

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PP2A (Goedert et al., 1992; Drewes et al., 1993; Harris et al., 1993;Tanaka et al., 1998) mimicking the decrease of phosphatase activityobserved in AD brains (Gong et al., 1995), particularly to induceretrograde degeneration of neurites. Following phosphorylation ofcytoskeletal proteins (Tau, neurofilaments) and disruption of micro-tubules, the cells round up and ultimately die through apoptosis (Sheaand Fischer, 1996; Tanaka et al., 1998; Nuydens et al., 1998).Furthermore, as we previously demonstrated that LMO4, a transcrip-tion regulator that was recently shown to promote neuron survivalfrom hypoxia (Chen et al., 2007), was not only consistently decreasedin Alzheimer EC (Leuba et al., 2004), but generated altered phos-phorylation of cytoskeletal proteins and changes in neuritic outgrowthin SH-SY5Y cells (Vu et al., 2003), we also used the SH-SY5Y celllines with inhibited LMO4 translation (SH−LMO4 clone) or LMO4overexpression (SH+LMO4 clone) as a model of neuritic outgrowthor retraction. The immunohistochemical study and the experimentalmodels suggest compensatory mechanisms contributing to thepostsynaptic changes observed in AD.

Materials and methods

Human brains

A total of 20 brains were examined, 11 AD cases including 2familial AD (FAD) cases (mean age=84.2 years±8.5), 7 old con-trols (mean age=81.2 years±6.5) and two young cases of 32 and48 years for comparison. Most AD and FAD cases were hos-

pitalized in the Service of Old Age Psychiatry in Lausanne, anddiagnosed following the DSM-IIIR criteria. A few cases came fromother sources. Clinical diagnosis was confirmed by neuropatholo-gical examination in the Department of Pathology. All controlswere without neurological disorder and old controls were age-matched with sporadic AD cases (see Table 1). The Ethics Com-mission of the CHUV gave agreement to collect brains.

Histology and immunohistochemistry

The brainswere removedwith a postmortem delay of less than 30 h(except for 5 cases) and stored shortly in buffered 4% formaldehydeuntil sampling. Samples of entorhinal cortex (EC) were then removedand prepared for paraffin embedding, then 20 µm thick sections weregenerated for immunohistostaining and densitometric measurement.Immunohistochemical staining was performed on contiguous sec-tions. After pretreatment for 10′ in methanol: H2O2 (97:3), 2 rinsingsfor 5′ in H2O Millipore (and only for β amyloid 5′ in formic acid), 2rinsings in PBS for 10′ and 30′ incubation in normal serum (rabbit orswine: dilution 1:10), sections were then incubated overnight at 4 °Cwith the specific primary antibody: β amyloid against human amyloid(mouse monoclonal 1:100, Dako M 0872); AD2 against Ser 396 and404 of PHF Tau (mouse monoclonal 1:250, BIO-RAD n° 56484);SMI-31 against phosphorylated neurofilament H and M epitopes(mouse monoclonal 1:15,000, Sternberger SMI-31); MAP2 againstMicrotubule-Associated Protein 2 (mouse monoclonal 1:100, CHE-MICON MAB3418); GFAP against Glial Fibrillary Acidic Protein,

Table 1Description of cases and semi-quantitative analysis in the entorhinal cortex

Case Sex Age PMdelay

Group Cause of death Amyloid AD2 SMI-31 MAP2 Synaptophysin α-synuclein

PSD-95

Spinophilin LMO4 GFAP

1 M 32 7 C Congenital heartdefect

0 0 ++ + ++++ +++ + ++++ − +++

2 M 48 10 C Myocardial infarct 0 0 + +++ + + + +++ ++++ +3 M 72 7 C Heart failure 0 0+ +++ ++ ++++ ++++ + +++ − +++4 F 77 19 C Pulmonary

embolism0 0+ ++ +++ +++ +++ + ++ ++++ ++

5 M 78 22 C Urinary bladdercarcinoma

0 0+ ++ +++ +++ +++ ++ + +++ ++

6 M 78 28 C Bronchopneumonia 0+ + + − +++ +++ + ++++ ++++ ++7 F 84 30 C Myocardial infarct 0+ ++ ++ +++ +++ ++++ ++ +++ ++ +++8 F 87 N30 C Uterus carcinoma 0 + ++ − − − − − ++++ −9 M 89 15 C Bronchopneumonia 0 ++ +++ +++ ++ ++ + +++ ++++ +++10 M 69 9 FAD Heart failure ++ ++++ ++++ ++ + ++++ ++++ + + +++11 M 71 − FAD Breathing failure +++ ++ ++ − + +++ ++ +++ ++ ++++12 F 80 21 AD Renal insufficiency + ++++ ++++ ++ ++ +++ +++ ++ + +++13 M 84 N30 AD Bronchopneumonia ++++ ++++ ++++ ++ ++ +++ ++++ ++++ + ++++14 M 84 31 AD Bronchopneumonia ++ ++++ ++++ ++ ++ ++ ++ ++ 0+ ++++15 M 86 8 AD Broncho-aspiration ++++ +++ ++++ + +++ +++ +++ + + +++16 F 87 N30 AD Cardiac

insufficiency++ ++ ++ ++ ++ ++ +++ ++ 0+ +++

17 F 88 7 AD Cardio-resp.insufficiency

++ +++ ++ ++ ++ +++ ++ + + +++

18 M 92 20 AD Heart failure + ++++ +++ + + + ++++ ++++ + ++++19 F 95 16 AD Myocardial infarct ++ +++ +++ ++ ++ ++ ++++ ++++ + +++20 F N90 − AD Heart failure +++ ++++ ++++ + + +++ ++++ + 0+ +++

M: male; F: female; C: control case; AD: Alzheimer's disease; FAD: Familial AD; PM: postmortem delay in hours.Antibodies used for semi-quantification and non parametric statistical analysis: see Materials and methods, Results.Semi-quantitative analysis: 0: no staining; 0+: very few elements stained; +: weak staining; ++: medium staining; +++: strong staining; ++++: very strong staining.LMO4 data were taken from a previous paper for 6 controls and 5 AD cases; Tab. II in (Leuba et al., 2004).

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principal intermediate filaments of astrocytes (polyclonal rabbit anti-human 1:1500, Dako Z0334); synaptophysin (polyclonal rabbit anti-human synaptophysin 1:100, Dako n° 0010) and α-synuclein againstthe corresponding presynaptic proteins (mouse monoclonal 1:50,ZYMED 18-0215); spinophilin against the corresponding spinespecific protein (rabbit polyclonal 1:500, CHEMICON AB5669);PSD-95 against postsynaptic density protein 95 (mouse monoclonal1:100, Upstate 05-494 andmousemonoclonal 1:75, ABRMA1-045);for LMO4 against LIM only protein LMO4 (Goat polyclonal anti-LMO4/S17 1:100, Santa Cruz sc-11120) data were taken for halfof the cases from Table 1 in (Leuba et al., 2004). Microwaveamplification (5′ at 800 W) was used for MAP2, synaptophysin,α-synuclein, spinophilin and PSD-95 before serum incubation.After 2 rinsings in PBS for 10′, sections were then incubated withsecondary antibodies for 2 h at room temperature. Followingsecondary antibodies were used: swine anti-rabbit immunoglo-bulin 1:300 (Dako E0353), rabbit anti-mouse 1:300 (Dako E0413)and rabbit anti-goat 1:100 (Dako P0449). After incubationwith the secondary antibody, sections were rinsed again twicefor 10′ in PBS and incubated in ABC Complex/HRP (Dako K0355) for 1 h. After rinsing twice in PBS, staining was revealedfor 10′ in 0.03% 3, 3′-diaminobenzidine (DAB; Sigma D 5637)

and 0.015% H2O2/PBS. Omission of primary or secondary anti-bodies resulted in no immunoreactivity. All sections were mountedwith pertex.

Semi-quantitative immunohistochemical analysis

Immunopositive signals were estimated semi-quantitatively underbright field illumination using a Zeiss Axioplan microscope, at amagnification of 100× and 400×, in the EC. The screening was per-formed, using 5–10 fields on 1–2 different sections in each brain by twoindependent persons (blind assessment). Estimations, taking intoaccount both the amount of stained elements and the intensity of thestaining, were comparable: they varied from 0 (no staining) to + (weakstaining), ++ (medium staining), +++ (strong staining) and ++++ (verystrong staining). See Table 1 and Figs. 1 and 2A. Data were analyzedwith the help of the statistical analysis system SAS (SAS Institute Inc.,1989). A non parametric test (Kruskal–Wallis) was used to compareestimations of the different immunostainings, in control and AD groupsand a non parametric correlation analysis (Spearman) was performedbetween them in each group. For statistics, semi-quantitative estimationssuch as 0+, +, ++, +++, ++++,were graded as 0.5, 1, 2, 3, 4. Thismethodwas successfully used in two previous papers (Leuba et al., 2004, 2005).

Fig. 1. Photomicrographs of 20 µm paraffin sections in the entorhinal cortex (EC), showing immunostaining for human βAmyloid (mouse monoclonal 1:100, Dako),for PHF Tau (AD2 mouse monoclonal 1:250), for postsynaptic density protein 95 (PSD-95 mouse monoclonal 1:100, Upstate), for synaptophysin (polyclonal rabbit1:100, Dako). For details, seeMaterials andmethods. C: control case n° 3; AD: AD case n° 12; superior line showing the greatest part of EC at amagnification of 2–5×;inferior line showing layers 2–3 (β amyloid, AD2, PSD-95) and 5–6 (synaptophysin) at a magnification of 10×. For description, see Results. Scale=200 µm.

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Image J densitometry

Using the ImageJ analysis system (NIHUSA, public domain) on aCompacq computer, densitometric measurements were also per-formed for the diffuse staining observed with the synaptic markerssuch as synaptophysin, α-synuclein, spinophilin and PSD-95. In eachcase, color photographs were taken with the Zeiss Axioplan micro-scope, at a low magnification (1×) allowing to see the whole corticalplate, and at the same illumination for all captures, thanks to a con-trolled voltage (6.45 V) for the luminosity of the microscope. Colorphotographs were then transformed in grey level values anddensitometric measurements were performed on each photo, including3 columns along radial scans going perpendicularly from pia to whitematter. The width of the columns was 200 µm and the length cor-responded to the depth of the cortical plate. Measurements wereobtained in arbitrary units, assuming that 255 represents completeopacity (dark) and 0 complete transparency (white). Background wasmeasured on individual photos and automatically subtracted from thedensitometric measurements. Taking into account that the mean cor-tical depth varies from case to case and also sometimes from onesection to the other in the same case, the maximum thickness of EC inthe measured location was considered as 100% and divided into 3equal parts, corresponding grossly to superior, median and inferior EClayers. Amean densitometric value was calculated in each case and foreach staining in the 3 groups of layers. For statistical analysis, we usedthe statistical analysis package SAS (SAS Institute Inc., 1989) and useda twoway analysis of variance to compare the different values betweengroups and layers. We used a boxplot representation to show thesignificant data obtained for PSD-95 and for synaptophysin (Fig. 2B).

SH-SY5Y cell culture and okadaic acid treatment

Human SH-SY5Y neuroblastoma cells (European Collection ofAnimal Cell Cultures, Salisbury, UK) and the SH-SY5Y cell lines,transfected with LMO4 cDNA cloned in sense (SH+LMO4=clone13) (Vu et al., 2003) as well as anti-sense orientation in the vectorpIRESneo2 (SH−LMO4=clone 10) (Vu et al., 2003), were grown ina 37 °C incubator containing 5% CO2/95% humidified air in RPMI-1640 (Invitrogen) with 10% FCS, 100 U/ml penicillin G (Invitrogen)and 100 µg/ml streptomycin (Invitrogen). Cells were passaged every5–6 days. For this purpose confluent cells were released in DPBS(150 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 7.9 mM Na2HPO4

. 2-H2O, 0.1 mM EDTA; pH 7.4), centrifuged (1100 rpm, 5′ at roomtemperature) and resuspended in full medium at the desired dilution.200 µg/ml geneticin disulfate (BD/Clontech) was added to the RPMImedium for the transfected cells (clones SH−LMO4 and SH+LMO4(Vu et al., 2003)).

SH-SY5Y cells (at 28 passages) were treated or not with 50,100, 200, 500 nM okadaic acid (OA, Sigma; 25 µg/ml RPMI) for5 h and then collected for Western blot analysis. Representativepictures were taken with a phase-contrast microscope coupled to aNikon Coolpix 995 digital camera (Fig. 3C). Clone SH−LMO4was treated either 3 h, 3 h 45 min or 5 h with OA and analyzedsimilarly to SH-SY5Y cells.

Cell viability assay

For assessing the viability of SH-SY5Y neuroblastoma cells orclone SH−LMO4, grown in RPMI-1640 and 10% FCS, 50,000cells/well were treated or not with OA for 5 h (including 30′ toequilibrate the plate content to room temperature) in 2 ml medium.

After removal of 1.9 ml of medium an equal volume of CellTiter-Glo Reagent prepared according to the manufacturer (Promega)was added in each well. The content was mixed for 2′ to induce celllysis, incubated at RT for 10′ and luminescence (in relative lightunits) was recorded with a GloMax 20/20 luminometer (Promega).Medium without cells resulted in background luminescence. Val-ues are expressed in percentage with absence of OA treatment setat 100%. Experiments were repeated three times and statisticaldifferences were assessed using a Tukey's studentized range testwith significance Pb0.05.

Western blots

SH-SY5Y cells were scraped in DPBS and collected bycentrifugation (5′, 1000 rpm, 4 °C). After a washing step in DPBSand a second centrifugation, they were resuspended in lysis buffer(50 mM Tris, 2% SDS, 5 mM EDTA, 2 mM EGTA, pH 6.8) withprotease inhibitor cocktail (10 µl/ml, Sigma). Subsequently, soni-cation (Bandelin Sonopuls HD200, 1×10 s) was carried out andprotein concentration was determined by the BCA protein assay(Pierce). The proteins were denatured in Laemmli loading buffer(50 mM Tris–HCl, pH 6.8, 10% glycerol, 2.5% β-mercaptoethanol,2% SDS, 0.01% Bromophenol blue) at 98 °C for 6 min. Proteinsextracted from murine primary cortical neurons grown in Neu-robasal medium and B27 supplement (Invitrogen) were occasion-ally used as control.

Equal amounts of proteins (20 µg), verified by GAPDH im-munostaining and membrane staining with Protogold (BBI interna-tional), were separated by SDS-polyacrylamide gel electrophoresis(10% acrylamide; Miniprotean II dual slab cell, Bio-Rad) and thenelectroblotted (overnight, 35V;Mini Trans-Blot electrophoretic transfercell, Bio-Rad) to a nitrocellulose membrane (Protran, Schleicher andSchuell). 10 µl Seeblue Plus 2 prestained standard (Invitrogen) as wellas 20 µg proteins from primary neurons were loaded as molecularweight standard and control, respectively.

After washing the membranes with H2O and 3×10′ PBS, non-specific binding sites were blocked by incubating the membrane in5% nonfat dry milk, 0.5% BSA, 0.2% Tween-20 in PBS 1× for 1 h.The membrane was then incubated for at least 1 h 30 with theprimary antibody diluted in blocking solution.

Antibodies for AD2 (dilution 1:300), SMI-31 (1:500), synap-tophysin (1:1000) and PSD-95 (1:1000; Upstate) were the same asthose used in immunohistochemistry. Other antibodies were fromChemicon: mouse monoclonal Tau-1 against non phosphory-lated Tau between amino acids (aa) 189–207 of the longestisoform of 441 aa (1:3000) and mouse monoclonal anti-GAPDH, clone 6C5 (1:10,000); from Innogenetics: mousemonoclonal AT-8 against phospho-Tau at Ser199, Ser 202 andThr 205 (1:3000), mouse monoclonal AT100 (1:1000) thatrecognizes PHF but none of the human central nervous systemTau proteins from postmortem physiologically aged brains orbiopsy-derived adult brain samples (Matsuo et al., 1994), mousemonoclonal AT180 against phospho-Tau at Thr231 and Ser235(1:1000); and finally from Santa Cruz: rabbit polyclonal anti-caspase-3, H277 (1:1000). The membrane was then washed inH2O and 3× 10′ in PBS, and incubated 10′ in blocking solution,then for 1 h with a horseradish peroxidase (HRP)-labeled anti-rabbit or anti-mouse antibody (Amersham Biosciences) diluted1:3000 in blocking solution. After a last standard washing stepand rincing in H2O specific bands were detected by chemilu-minescence (SuperSignal West Pico Cheminescent Substrate,

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Pierce) on film (Hyperfilm ECL, Amersham). Secondaryantibodies have been shown not to contribute to the observedsignals.

For the Western blots (Fig. 3B) with the rat monoclonal anti-LMO4 antibody (clone 20F8, dilution 1:1000; Sum et al., 2005), abis-Tris 13% gel was run in MOPS buffer (60 µg protein/slot) and

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the transfer was carried out according to NuPAGE technology(Invitrogen Life Technologies). As secondary antibody the poly-clonal HRP anti-rat, P0450 (Dako), was used.

Western blot densitometry

For the densitometric analysis, films were scanned with a GS-700 imaging densitometer (Bio-Rad). The results were expressedas optical density unit×area (mm2) using the molecular analystsoftware program (Bio-Rad). The ratios of antibody signal/GAPDH signal were used to compare clones and SH-SY5Y cellstreated or not with OA. For quantitative analysis Western blots

were repeated at least 3 times. Statistical differences were assessedusing a Tukey's studentized range test with significance Pb0.05.

Immunofluorescence cytochemistry

After removal of the cell medium, SH-SY5Y cells or SH−LMO4clone treated or not with 100 nM OA for 3 h were rinsed 5′ in PBS,fixed 30′ in paraformaldehyde 4% and then washed again 5′ in PBS.After 10′ permeabilisation in PBS with 0.2% Triton X-100, they werewashed twice as described above and incubated in PBS 90-120′ atroom temperature with the primary antibody against PSD-95 (dilution1:100, goat polyclonal anti-PSD-95, ab-12093,Abcam). Subsequently,

Fig. 3. SH-SY5Y cells as model for altered neuritic outgrowth (in A and B) and AD-like neuritic retraction with okadaic acid (OA) treatment (in C). In A,immunocytochemistry carried out with the 20F8 anti-LMO4 antibody (magnification 20×). The second raw of pictures is identical to the first one, except for nuclearstaining (DAPI). SH-SY5Y cells (SH) show stronger LMO4 nuclear immunoreactivities compared to the SH−LMO4 clone. The clone grows longer neurites due toinhibition (−LMO4) of the transcription cofactor LMO4. The SH-SY5Y cells show weaker nuclear immunoreactivities compared to the SH+LMO4 clone whichgrows shorter neurites due to overexpression (+LMO4) of LMO4 (Vu et al., 2003). Phalloidin stainingwas used to visualize F-actin and neuritic length. In B,Westernblot showing LMO4 expression in SH+LMO4 or SH−LMO4 clones compared to the SH-SY5Y cells. In C, the OA untreated SH-SY5Y cells or SH−LMO4 cloneare compared by phase-contrast microscopy (magnification 10×) with the treated SH-SY5Y cells or SH−LMO4 clone, respectively. Neuritic retraction can beobserved particularly in the clone between 0 nM to 100 nMOA (5 h). Both, SH-SY5Y cells and the clone start to round up between 100 nM to 200 nMOA.At 500 nMOA (not shown) SH-SY5Y cells and the SH−LMO4 clone are phenotypically identical to the cells treated with 200 nM OA. Note that the SH+LMO4 clone hasalready retracted neurites (Vu et al., 2003) and was therefore not treated with OA.

Fig. 2. Panel A: Photomicrographs of 20 µm paraffin sections, showing details of presynaptic protein synaptophysin and postsynaptic protein PSD-95immunostaining in superior (I), median (II) and inferior (III) layers of the entorhinal cortex (EC) of control (C) and Alzheimer (AD) cases. (C): control case n° 5;(AD): AD case n° 12; Scale=200 µm. The fourth row shows enlarged magnification (40×/immersion) in inferior layers. Scale=50 µm. Note in AD the increase inpunctate and fibrillary immunostaining of PSD-95, and the decrease in diffuse immunostaining of synaptophysin; in the latter healthy neurons are clearly unstainedin controls, while in AD there are less visible neurons. Panel B: Boxplot representation of densitometric measurements of presynaptic protein synaptophysin andpostsynaptic protein PSD-95 in superior, median and inferior layers of the entorhinal cortex (EC) of control (C) and Alzheimer (AD) cases.Within the boxplot, thebox itself represents the interquartile range (inferior quartile range=25%; superior quartile range=75%), the horizontal line, the median value and the cross, themean; the vertical lines (whiskers) indicate values laying outside the box within a range of 1.5× that of interquartiles. A marked and significant increase wasobserved in AD compared to control group for PSD-95 protein (Pb0.0001), without significant difference between layers. On the contrary, a significant decreasewas observed between control and AD groups for synaptophysin (P=0.04). In addition a significant difference was observed between layers (Pb0.001), with adecrease in densitometric density of synaptophysin from superior to inferior layers and a greater difference between groups in inferior layers.

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the cells were washed twice, incubated one h at room temperature withthe secondary antibody (donkey anti-goat Alexa 488 IgG, MolecularProbes, Oregon, USA), rinsed once again 5′ in PBS and colored 5′ in0.1% DAPI. Finally, the cells were mounted in fluorosave. Omissionof the primary antibody resulted in no immunopositive signal overbackground in control tests. Slideswere analyzedwith a Zeiss axioplanmicroscope equipped with epifluorescence. For the LMO4 immuno-cytochemistry, rat monoclonal anti-LMO4, clone 20F8 (1:100) (Sumet al., 2005) and goat anti-rat Alexa 488 IgG (Molecular Probes) wereused. F-actin was visualized after cell fixation with 4% paraformalde-hyde in PBS for 20 min directly by using Alexa Fluor 488 phalloidinaccording to the manufacturer's instructions (Molecular Probes).

Results

Human entorhinal cortex (Figs. 1–2, Table 1)

We first compared the different immunostainings betweencontrol and AD brains with a qualitative and semi-quantitativemethod, focusing mainly on pre- and postsynaptic proteins togetherwith AD pathological markers. A new and striking differenceappeared in sections immunostained with the anti-PSD-95 antibodywhich revealed a manifest increase in AD compared to controls (seeTable 1 and Figs. 1–2A). This increase was statistically significant(P=0.002; Kruskal–Wallis non parametric analysis). PSD-95 is apostsynaptic protein involved in NMDA and AMPA receptorssignaling and its distribution comes out as a mostly diffuse one, withmainly a granular (small dots) but also filamentous (fine fibers)appearance (Fig. 2A).We observedmore dots with stronger staining,together with very fine fibers, mostly horizontal in superior layersand vertical in inferior layers (Figs. 1–2A). On adjacent sectionsstained with an antibody against the presynaptic protein synapto-physin, appearing as a diffuse staining in the neuropile (Fig. 2A), weobserved on the contrary a significant decrease in AD casescompared to controls (P=0.004). There was also a marked decreasein synaptophysin from superior to inferior layers, with a greaterdifference between controls and AD cases in inferior layers. Fol-lowing corresponding sections stained with antibodies against thepresynaptic protein α-synuclein and against spinophilin, a marker ofdendritic spines, revealed no statistically significant differencebetween AD and controls, while the staining against MAP2, amarker of microtubule protein associated to the somato-dendriticpart of neurons, revealed a significant decrease between control andAD cases (P=0.004). Values from LMO4 protein, a transcriptionregulator used in the SH-SY5Y study (see further) decreased sig-nificantly in EC with AD (Pb0.001). Spearman non parametriccorrelation coefficients were significant between, on the one hand,the increase in PSD-95 protein, and on the other hand, the decreasein synaptophysin (r=−0.61, P=0.009), in LMO4 (r=−0.69,P=0.003) and in MAP2 (r=−0.66, P=0.007). The decrease inLMO4was also significantly correlated with that of MAP2 (r=0.73,P=0.003).

AD pathological markers exhibited a significant increase for βamyloid protein (Pb0.001), phosphorylated Tau protein/AD2(Pb0.001), phosphorylated neurofilaments/SMI-31 (P=0.019)and glial fibrillary protein GFAP (P=0.011). β amyloid proteinindicated a correlated increase with phosphorylated Tau protein(r=0.69, P=0.002), phosphorylated neurofilaments/SMI-31(r=0.50, P=0.04) and GFAP (r=0.57, P=0.02). The increase inPSD-95 was significantly correlated with that of amyloid (r=0.67,P=0.003), AD2 (r=0.77, Pb0.001) and SMI-31 (r=0.58,

P=0.014), while the decrease in synaptophysin was correlated withthe increase in amyloid (r=−0.53, P=0.03) and AD2 (r=−0.67,P=0.003). The decrease in LMO4 was correlated with the increasein β amyloid (r=−0.72, P=0.001), AD2 (r=−0.76, Pb0.001),SMI-31 (r=−0.61, P=0.001), and GFAP (r=−0.51, Pb0.04). Bothdecrease in LMO4 andMAP2were significantly correlated (r=0.73,P=0.003). On the whole, the increase in postsynaptic PSD-95 andMAP2, and the decrease in presynaptic synaptophysin were sig-nificantly associated to AD pathological changes.

The semi-quantitative estimation of synaptic markers was com-pleted by a quantitative analysis using densitometric measurementfrom pia to white matter and dividing the cortical depth in 3 arbitraryparts corresponding grossly to superior, median and inferior layers(see methods). The statistical analysis of densitometric data betweengroups and layers confirmed significant differences for synapto-physin and PSD-95 proteins, the data being expressed with a boxplotrepresentation (Fig. 2). The analysis by groups of layers confirmedthat there was a significant decrease in synaptophysin from superiorto inferior layers and that the decrease in AD was more pronouncedin the inferior layers, while there was no such difference betweenlayers for PSD-95.

SH-SY5Y neuroblastoma cells (Figs. 3–5)

SH-SY5Y neuroblastoma cells were treated or not with 50, 100,200 and 500 nMOA for 5 h. OA treatment induces a retraction of theneurites (Fig. 3). At 100 nM OA neurites of SH-SY5Y cells areretracted, the cells start to round up but they show almost no decreasein viability (b5%; Fig. 4A). Thus, treatments of 50 to 100 nM OAcan primarily model AD-like neuritic retraction. At 200 nMOA SH-SY5Y cells round up and a 40% decrease in cell viability is detected.At 500 nM OA SH-SY5Y cells show a drastic reduction in viability(78%). Similar results were observed with clone SH−LMO4 eitheror not treated with 50 to 500 nM OA: a loss of viability is absent upto 100 nM OA and significant only at 500 nM OA. Western blotswere carried out with several antibodies (Figs. 4B, 5A). Increasedactivated caspase-3, shown by 17 and 19 kDa fragments, could notbe detected by Western blotting carried out on 50 to 200 nM, butonly on 500 nMOA-treated cells (Fig. 4B), indicating that for lowerOA treatments cells are modeling early neuritic degeneration ratherthan death. We observed faint changes in phosphorylation of cyto-skeletal proteins Tau and neurofilaments NF-H and -M at 50 nMOAwith antibodies AT-8 and SMI-31, but not with AD2, AT-100 andAT-180 (Fig. 4B). At 500 nM OA all antibodies showed signalalterations including the AD-specific antibodies AD2, AT-100 andAT-180. Tau-1 signal had drastically decreased, indicating an in-creased phosphorylation of Tau. At 500 nM OA, contrary to lowerconcentrations, AD-specific changes in Tau phosphorylation aretherefore observed. Tau-1 and AD2 immunoreactive signals start todisappear or to increase at 100 nM OA, respectively. Triplicatedexperiments, using anti-GAPDH and Protogold staining to verifyequal protein loading between samples (Fig. 4B), were carried outwith the postsynaptic marker PSD-95. They showed no significantchanges in immunoreactivity with increased OA stimulation at leastup to 200 nM OA (Fig. 5A), suggesting no relation to increased Tauphosphorylation, neuritic degeneration and even early decreasedviability following inhibition of PP2A and PP1 in SH-SY5Y cells.

Considering now the two SH-SY5Y cell models (Vu et al., 2003)with altered LMO4 expression (Figs. 3A, B), we made the followingobservations (Fig. 5). Caspase-3 was not significantly activatedcomparing these clones with the wild type SH-SY5Y cells. Tau-1 and

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AD2 immunoractivities were not detected in clone SH+LMO4,indicating that the decreased neuritic lengthwas not related toAD-likephosphorylation as observed in OA-treated SH-SY5Y cells, whereTau phosphorylation increases at the AD2 site. In clone SH−LMO4,Tau-1 immunoreactivity was increased and AD2 remained low. PSD-95 showed significantly increased values in clone SH−LMO4(Pb0.001) compared to clone SH+LMO4 and SH-SY5Ycells. Thus,

PSD-95 expression level increases with neuritic outgrowth anddecreases with neuritic retraction in these clones.

When clone SH−LMO4 was treated with 50, 100, 200 or500 nM OA, we observed, similarly to OA treated SH-SY5Y cells,a reduction in neuritic length (Fig. 3C). Cells rounded up and adrastic reduction of cell viability was measured at 500 nM OA only(Fig. 4A) without detectable activation of caspase-3 (Fig. 5B). Tau-

Fig. 5. Changes in expression of PSD-95 as a consequence of AD-likeokadaic acid (OA)-induced neuritic regression or neuritic outgrowth (SH−LMO4) and retraction (SH+LMO4) as shown byWestern blots (in A and B)or immunocytology (in C). In A, PSD-95 immunoreactive signal is eitherincreased in clone SH−LMO4 with inhibited LMO4 expression and longerneurites or decreased in clone SH+LMO4 with LMO4 overexpression andretracted neurites (Vu et al., 2003). In SH-SY5Y cells treated or not with 50to 200 nM OA, PSD-95 shows no changes in expression with increased OAtreatment. Immunoreactive signals in the SH−LMO4 clone are similar tothose in primary neurons (on the right side). Tau-1 and AD2 signals indicatethat Tau is progressively phosphorylated as neuritic retraction proceeds,whereas activated caspase-3 immunoreactive signal (arrow) does not in-crease with OA concentrations. Anti-GAPDH was used here to demonstrateequal protein loading. Only genuine sizes of immunoreactive signals areshown. In B, Western blots showing that PSD-95 immunoreactivity does notdiminish in SH−LMO4 clone with 5 h OA treatment despite AD-likeneuritic degeneration. As for SH-SY5Y cells, Tau-1 and AD2 signals re-spectively decreased or increased with OA concentrations, whereas caspase-3 was not activated (arrow). In C, PSD-95 immunostaining (green) is shownin the SH-SY5Y cells and in the SH−LMO4 clone, following or not 100 nMOA treatment (3 h), to illustrate neuritic (visible in SH−LMO4 clone only)and cytoplasmic (in all cases) localization. DAPI staining for nuclei (blue);magnification 40×.

Fig. 4. Effects of okadaic acid (OA) treatments on viability and apoptosisactivation of SH-SY5Y cells (SH) or the SH−LMO4 clone. In A, analysis ofcell viability of SH-SY5Y cells and SH−LMO4 clone treated with 0 to500 nM OA. Decreased cell viability starts at 200 nM OA. Reduction in cellviability is statistically different (as indicated by stars) if SH-SY5Y cells orthe SH−LMO4 clone without OA treatment are compared with SH-SY5Ycells (Pb0.005) or SH−LMO4 clone (Pb0.008) treated with 500 nM OA,respectively. In B, Western blots of SH-SY5Y cells treated with 50, 500 nMOA or not. Left: antibodies against protein Tau: Tau-1 immunoreactivitydecreases while AD2, AT-8, AT-100 and AT-180 immunoreactivity increaseswith Tau phosphorylation; right: against PSD-95 and the neurofilaments NF-H and -M (SMI-31). Activation of the pro-apoptotic caspase-3 can beobserved only with 500 nM OA (arrow) when Tau and NF epitopes aremassively phosphorylated in an AD-like manner. PSD-95 immunoreactivityremains unchanged at 50 nM OA and is slightly decreased (30%) at 500 nMOA. Anti-GAPDH and Protogold staining (pattern shown as example:proteins of 50 to 64 kDa) of the membranes from the Western blots wereused to demonstrate equal protein loading. Only genuine molecular weightsare shown.

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1 signal decreased and AD2 signal increased, whereas PSD-95immunoreactivity did not change when neurites retracted (Fig. 5B).Note that LMO4 expression changes significantly neither in theSH−LMO4 clone (background levels) nor in the SH-SY5Y cellsfollowing OA treatments (faint expression; Western blots notshown). In the OA-untreated SH−LMO4 clone, PSD-95 wasclearly localized both in the cytoplasm and neurites (Fig. 5C),whereas in the SH-SY5Y cells, and in the OA treated SH-SY5Ycells and SH−LMO4 clone with shorter neurites, PSD-95 is pre-dominantly cytoplasmic. The signal appears similar in intensitywhen comparing OA treated to untreated cells with fainter signalsin the SH-SY5Y cells compared to the SH−LMO4 clone, as ex-pected from the Western blots. Thus, PSD-95 expression levelremained unchanged in both SH−LMO4 and wild type SH-SY5Ycells treated with OA mimicking both AD-type phosphorylation ofTau and neurofilament (NF-H and -M), and neuritic retraction.

Discussion

Human entorhinal cortex

Our main result in the present study relates to a differentialquantitative staining of defined synaptic markers in the entorhinalcortex (EC) of AD patients, showing mainly a significant decrease ofpresynaptic synaptophysin and a significant increase in postsynapticPSD-95. The decrease in synaptophysin, whose expression is relatedto axonal retraction, corroborated the data obtained by others indifferent brain regions and with different methods (Davidsson andBlennow, 1998; Honer, 2003; Reddy et al., 2005; Love et al., 2006),taking into account that this marker doesn't seem to depend onpostmortem delay (Lassmann et al., 1992). A decrease in mRNAexpression was also reported in AD cases, indicating that synapto-physin transcription diminished in NFT bearing neurons (Ginsberget al., 2000; Callahan et al., 2002). α-synuclein, another pre-synapticprotein (Galvin et al., 2001), remained unchanged in our data butappeared to decrease in another AD study (Western blots), correlatingwith mild functional deterioration (Wang et al., 2004). However, therole of α-synuclein in synucleinopathies is recognized (Galvin et al.,2001; Bennett, 2005) and positive α-synuclein brain structures seemto increase with age and with the number of Aβ senile plaques (Dinget al., 2006), but we did not observe Lewy bodies or neurites in ourcases. Yet the most striking observation was a significant increasedimmunoreactivity in the postsynaptic density protein PSD-95, whichexhibited a mostly punctate but also filamentous distribution(Fig. 2A), particularly marked in the superficial and deep EC layers.Vertical fibers were exceptionally well marked in the deep layers, andmore so in AD compared to old controls or to 2 young controls. Notethat in the near subiculum and hippocampal region, an increasedstaining of PSD-95 was equally observed, particularly in the CA1subdivision. No fresh tissue was available forWestern blots. The anti-PSD-95 monoclonal antibody from Upstate Biotechnology was usedfor its specificity as shown in previous immunohistochemical and-cytochemical studies (Jones et al., 2002; Almeida et al., 2005; Roselliet al., 2005), including one in human cortex in schizophrenia(Kristiansen et al., 2006). Further, we obtained comparable data withanother monoclonal anti-PSD-95 antibody (ABR/Affinity BioRea-gents; data not shown). However, in other papers, ELISA measure-ments indicated a decreased expression of PSD-95 either insynaptosome fraction from AD association cortex (Gylys et al.,2004) or in the superior temporal gyrus of AD patients, with a greaterdecrease for ApoE4 genotyped patients (Love et al., 2006). However,

no significant decrease of gene expression could be shown by real-time PCR in the prefrontal and occipital AD cortex (Dracheva et al.,2001) and more recently no significant change in PSD-95 wasrevealed in a Western blot study of the human frontal cortex in oldestold humans (Head et al., 2007). Thus, in contrast to data obtainedwithother brain samples (e.g. synaptosome and temporal cortex) andmethods (e.g. ELISA and Western blots), our immunohistochemicalanalysis demonstrates rather an increase in PSD-95 expression and achange in its distribution in AD. Furthermore, the postsynapticspinophilin remained stable in our data. Thismight indicate a differentfate of postsynaptic proteins compared to presynaptic ones in thecourse of neurodegeneration (see further).

An APP mutant model showed diminished levels of PSD-95protein in primary neuronal culture (Almeida et al., 2005) and Aβoligomers seem to induce a NMDA-dependent PSD-95 degrada-tion in an in vitro model (Roselli et al., 2005). Taking into accountthat APP mice models exhibit spine abnormalities and cognitiveimpairments (Janus et al., 2000; Spires et al., 2005; Spires andHyman, 2005), this would suggest a positive correlation betweencognitive performance and PSD-95 expression, fitting with otherdata showing an increased PSD-95 transcription in the cortex ofmice undergoing enriched environment (Rampon et al., 2000).However, in another study, PSD-95 protein expression wasselectively increased in the hippocampus of aged learning-impairedrats (Nyffeler et al., 2007), suggesting rather an inverse correlationbetween cognitive performance and PSD-95 expression in aging.Finally, PSD-95 mutant mice show altered behavioral plasticity,together with an increase in hippocampal neuron's spine density(Vickers et al., 2006). But the overexpression of mutated APP inmice models does not necessarily recapitulate the whole extent ofpathological changes occurring in AD, the latter representing aslower and more complex phenomenon, including widespreadneurofibrillary pathology and neuronal loss. Thus, these data inmice do not necessarily contradict our study in the human EC, thatsuggests some compensatory phenomena possibly related to denovo PSD-95 synthesis, but also to some neuritic alterations asinvestigated in our in vitro model.

SH-SY5Y neuroblastoma cells

In order to investigate how PSD-95 changes could be related toAD-type neuritic alterations, we used a cell model commonly em-ployed to induce Tau phosphorylation as an early stage of NFTformation (Caillet-Boudin and Delacourte, 1996; Shea and Fischer,1996). SH-SY5Y neuroblastoma cells were treated with okadaic acid,an inhibitor of phosphatases PP1 and PP2A (Goedert et al., 1992;Drewes et al., 1993; Harris et al., 1993; Tanaka et al., 1998)mimicking the decrease of Tau phosphatases activity observed in ADbrains (Gong et al., 1995). We observed neuritic retraction withincreased OA concentration and both AD2 immunoreactive signal—corresponding to phosphorylated Tau protein— and SMI-31 signal—corresponding to phosphorylated neurofilaments NF-H and NF-Mincreased. But in spite of neuritic retraction, PSD-95 expressionremained unchanged (Figs. 4B, 5A), while synaptophysin immunor-eactive signal was not detectable (not shown). To clarify the linkbetween PSD-95 expression level and neuritic changes, we used oneof our SH-SY5Y clones with decreased LMO4 expression andenhanced neuritic outgrowth (clone SH−LMO4; Fig. 3) (Vu et al.,2003). Its phenotype is genuine as reproduced by independent clonesand different from clones with the transfected vector without insertthat maintain the wild type SH-SY5Y phenotype. Furthermore, this

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clone grows neurites as long as SH-SY5Y cells differentiated withretinoic acid (Vu et al., 2003). The increase in PSD-95 immunor-eactivity on Western blots (together with Tau-1) indicates a linkbetween PSD-95 expression and neuritic expansion. A similar rela-tionship has been shown in primary hippocampal neurons (Rao et al.,1998), supporting the validity of our model and suggesting that theincreased PSD-95 immunostaining in Alzheimer EC is, at least in part,related to compensatory dendritic outgrowth. Yet, a link between neu-ritic outgrowth and LMO4 expression has not been demonstrated inbrain tissue, despite the fact that LMO4 in the somatosensory cortex isinvolved in the formation of proper neuronal connections with afferentthalamocortical fibers (Kashani et al., 2006). When clone SH−LMO4was treated with OA, neurites retracted as in OA-treated SH-SY5Ycells, without significant decrease in PSD-95. In contrast, the study ofclone SH+LMO4with increased LMO4 expression leading to neuriticretraction compared to SH-SY5Y cells (Vu et al., 2003), indicateddecreased PSD-95 and Tau-1 immunoreactivity. But in this case, AD2signal did not increase as inOA-treated SH-SY5Y cells, demonstratingthat neuritic retraction relies on a different process than in AD. Theabsence of Tau-1 immunoreactivity is not at odds with the absence ofAD2 signals as the corresponding antibodies recognize different Tauphosphorylation sites. Thus, there is no direct causal relation betweenLMO4 expression and altered AD-like Tau-phosphorylation, assuggested in Leuba et al. (2004). The SH+LMO4 clone is indeed abetter model for neuritic retraction than for AD-like degeneration.

Neuritic degeneration and PSD-95

On the whole, these observations demonstrate that PSD-95changes are related to the length of neurites in SH-SY5Y clones, butthat an AD-like phosphorylated Tau linked retraction (OA treatment)does not appear to induce any PSD-95 change in expression level,providing a plausible explanation for our observations in theAlzheimer EC. An increased PSD-95 immunoreactivity might resultpartially from a compensatory dendritic outgrowth (similarly to theobservation in the SH−LMO4 clone), and not from increased Tauphosphorylation as suggested by the OA-treated cells. However, wecannot exclude with the present cell models that impaired PSD-95degradation might also contribute to the increased PSD-95 immuno-positive signals in AD. Indeed, neurodegenerative events and par-ticularly paired helical filaments are known to impair mechanisms ofprotein transport and degradation (Keck et al., 2003). As a con-sequence, PSD-95 and other spine proteins could accumulate in thedendritic shafts showing a pattern of abnormal protein distribution andincreased density. PSD-95 might be particularly concerned as it canact as a kinesin “motor receptor” (Kim and Sheng, 2004). Theimportance of dendritic shafts in AD process is underlined by theobservation that the presence of Tau protein is necessary for Aβneurotoxicity (Rapoport et al., 2002; Robertson and Scott, 2007). Inour AD brains, the correlated increase in PSD-95, β amyloid andphosphorylated Tau (AD2) suggests a strong interrelated evolution ofPSD-95with pathological events in EC. A beneficial effect of PSD-95to protect synapses or enhance synaptic formation (El-Husseini et al.,2000), appears likely limited in time. Rather, PSD-95 accumulationmight rapidly become a marker of learning impairment (Nyffeleret al., 2007) and later of pathological events.

In conclusion, while presynaptic degeneration with decreasedmarkers such as synaptophysin seems generally admitted in AD,postsynaptic changes could be more complex events, as shown bystable spinophilin and increased PSD-95 immunoreactivity. Thesechanges could be due, at least partly, to compensatory mechanisms,

as shown in the present publication. The altered distribution of PSD-95 could thus be considered as a pathological marker in AD. On theother hand, the plasticity of postsynaptic sites including PSD-95distribution might also represent an early therapeutic target, par-ticularly with regards to NMDA receptor-mediated excitotoxicity(Wen et al., 2006).

Acknowledgments

We thank Prof. P. Magistretti and P. Giannakopoulos fordiscussion and support, E. Bernardi for technical help, Drs J.E.Visvader and E.Y. Sum, both from theWalter and Eliza Hall Instituteof Medical Research, Melbourne, Australia, for the rat anti-LMO4antibody.

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