at SciVerse ScienceDirect

Neurobiology of Aging xxx (2012) 1e13

Contents lists available

Neurobiology of Aging

journal homepage: www.elsevier .com/locate/neuaging

Effects of different amyloid b-protein analogues on synaptic function

Cristian Ripoli a,1, Roberto Piacentini a,1, Elisa Riccardi a, Lucia Leone a, Domenica D. Li Puma a,Gal Bitan b,c,d, Claudio Grassi a,*a Institute of Human Physiology, Università Cattolica, Rome, ItalybDepartment of Neurology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USAcBrain Research Institute, University of California at Los Angeles, Los Angeles, CA, USAdMolecular Biology Institute, University of California at Los Angeles, Los Angeles, CA, USA

a r t i c l e i n f o

Article history:Received 2 August 2011Received in revised form 31 May 2012Accepted 3 June 2012

Keywords:Amyloid-b proteinLong-term potentiationSynaptic transmissionDentritic spinesMethionine 35SynaptophysinPSD-95mEPCSAlzheimer’s disease

* Corresponding author at: Institute of Human PhyLargo Francesco Vito 1, 00168 Rome, Italy. Tel.: þ393015 4665.

E-mail address: grassi@rm.unicatt.it (C. Grassi).1 C.R. and R.P. contributed equally to this work.

0197-4580/$ e see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.neurobiolaging.2012.06.027

Please cite this article in press as: Ripoli, C., e(2012), http://dx.doi.org/10.1016/j.neurobiol

a b s t r a c t

Perisynaptic accumulations of amyloid b-protein (Ab) play a critical role in the synaptic dysfunctionunderlying the cognitive impairment observed in Alzheimer’s disease. The methionine residue at posi-tion 35 (Met35) in Ab is highly subject to oxidation in Alzheimer’s disease brains. In hippocampal brainslices we found that long-term potentiation at CA3eCA1 synapses was significantly inhibited by wildtype Ab42 in which Met35 is reduced, but not by Ab42 harboring Met35 sulfoxide. Similar differenceswere observed when basal synaptic transmission was investigated in autaptic hippocampal neurons. Thesignificant decreases in excitatory postsynaptic current amplitude, vesicle release probability andminiature excitatory postsynaptic current frequency caused by 20-minute exposure to wild type Ab42were not observed after exposure to Ab42 harboring Met35 sulfoxide. With longer (24-hour) Ab treat-ments, this early impairment of the presynaptic terminal function extended to involve the postsynapticside as well. The Met35 oxidation also affected Ab42 negative impact on dendritic spine density andexpression of pre- and postsynaptic proteins (synaptophysin and postsynaptic density protein-95). Ourfindings suggest that oxidation of Met35 is critical for molecular, structural, and functional determinantsof Ab42 synaptotoxicity.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Synaptic dysfunction is likely to play amajor role in the cognitivedeficits associated with Alzheimer’s disease (AD), particularly thoseseen before neuronal death occurs (Hsia et al., 1999; Selkoe, 2002).A key initiating pathogenic event in AD is amyloid b-protein (Ab)accumulation in the hippocampus and brain cortex (LaFerla andOddo, 2005). Ab is generated by b- and g-secretase-mediatedcleavage of the amyloid-b precursor protein (APP) and released intothe extracellular environment and lumens of intracellular organ-elles. Low Ab concentrations play critical physiological roles insynaptic plasticity (Puzzo et al., 2008, 2011) and activity-dependentregulation of synaptic vesicle release (Abramov et al., 2009), butabnormal accumulations lead to the self-assembly of neurotoxic Aboligomers, which interfere with synaptic function and cause neu-rodegeneration (see references in Querfurth and LaFerla, 2010).

siology, Università Cattolica,06 3015 4966; fax: þ39 06

ll rights reserved.

t al., Effects of different amyaging.2012.06.027

The Ab forms that are most abundant in AD brains comprise 40(Ab40) or 42 (Ab42) amino acid residues (Kuo et al., 2001; Näslundet al., 1994). The hydrophobic C-terminal region plays crucial rolesin the peptide’s assembly and neurotoxicity (Bitan et al., 2003a; Yanand Wang, 2006). It contains a single methionine residue at posi-tion 35 (Met35), whose sulfur atom is highly subject to oxidation,an event that markedly alters C-terminal hydropathy and thusaffects Ab assembly (Bitan et al., 2003b). Ab40 and Ab42 harboringMet35-sulfoxide have been demonstrated in the brains of ADpatients and of transgenic mouse models of AD (Boutte et al., 2006;Dong et al., 2003; Kuo et al., 2001; Näslund et al., 1994), which is notsurprising considering the extensive oxidative stress documentedin AD brains (see references in Querfurth and LaFerla, 2010). Wehave shown that Met35 oxidation critically affects Ab neurotoxicity.Indeed, compared with Ab42 containing reduced Met35 (Ab42WT),the form harboring Met35-sulfoxide (Ab42MO) has a considerablymilder proapoptotic action (Clementi et al., 2006; Maiti et al., 2011;Piacentini et al., 2008b).

Some investigators have attributed Ab’s toxicity to its interactionwith cellular membranes (Barnham et al., 2003; Ciccotosto et al.,2011). Ab oligomers show high affinity for synaptic membranes,especially at the level of dendritic spines (Di Paolo and Kim, 2011),

loid b-protein analogues on synaptic function, Neurobiology of Aging

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e132

and this might explain the efficacy of Ab42 oligomers in alteringsynaptic function. Links between Ab aggregation and its toxicity arealso being investigated. For example, Ab42WT and Ab42MO havedistinct oligomerization patterns (Bitan et al., 2003b), whichcorrelate with their differential neurotoxicity (Maiti et al., 2011). Incontrast, Ab42 in which Met35 has undergone further oxidation tosulfone (Ab42MO2) has an oligomer-size distribution indistin-guishable from that of Ab42MO (Bitan et al., 2003b), and yet it is justas neurotoxic as Ab42WT (Maiti et al., 2011).

This study was conducted to investigate the effects of reducedand oxidized Ab42 at the synaptic level. We compared the effects ofAb42WT, Ab42MO, and Ab42MO2 on basal synaptic transmission,synaptic plasticity, expression of synaptic proteins, and dendriticspine density. Ab42MO proved to be markedly less synaptotoxicthan Ab42WT, suggesting that Met35 oxidation might be a seren-dipitously protective reaction that limits the functional synapticdamage provoked by Ab.

2. Methods

2.1. Preparation of Ab42 solutions

Freeze-dried purified Ab42 analogues (Ab42WT, Ab42MO, andAb42MO2) were purchased from Anaspec (Freemont, CA, USA).Peptideswere diluted to 1mM in 1,1,1,3,3,3,-hexafluoro-2-propanol,as previously described (Rahimi et al., 2009), to disassemble pre-formed aggregates and stored as dry films at �20 �C before use.

Protein solutions were prepared according to Berman et al.(2008) by dissolving the films at 1 mM in dimethyl sulfoxide(DMSO), sonicated for 10 minutes, and then diluted to the finalworking concentration in culture media or artificial cerebrospinalfluid (aCSF). In some experiments, we used these Ab solutionsimmediately after their preparation. In others, we used “aged” Absolutions. In these cases, before the final dilution in culturemedium,Tyrode’s solution or aCSF, the 1 mM protein solution in DMSO wasdiluted to 100 mM in cold phosphate-buffered saline (PBS) andincubated for 12 hours at 4 �C to promote protein oligomerization.

In a subset of experiments, Ab preparations were subjected tocentrifugation (18,000g for 10 minutes at 4 �C) to remove largeaggregates and fibrils (Resende et al., 2008; Sondag et al., 2009)possibly formed during the “aging” process.

To study the interaction of our Ab preparations with neurons,each Ab analogue was labeled with the IRIS 5-NHS active ester dye(IRIS 5; lex: 633 nm; lem:650e700 nm; Cyanine Technology, Turin,Italy) according to manufacturer’s protocol. Briefly, Ab solutions(100 mM in PBS) were mixed with 6 mM IRIS 5 in DMSO for 4 hoursin the dark under mild shaking conditions. After this time, labeledAbs were purified with Vivacon 500 ultrafiltration spin columns(Sartorius Stedim Biotech GmbH, Goettingen, Germany) and thenresuspended in PBS at a concentration of 100 mM before finaldilution in the culture medium.

2.2. Hippocampal slice preparation

All experiments were performedwith 2-month-old male C57BL/6 mice. The protocol was approved by the Ethics Committee of theCatholic University. Animals were housed and handled in accor-dance with European Community guidelines (Council Directive 86/609/EEC of 24/11/1986). Unless otherwise specified, all commercialproducts were used according to manufacturers’ instructions.

Mice were anesthetized with isoflurane and decapitated. Thebrain was rapidly removed and put in ice-cold cutting solutioncontaining (in mM): 124 NaCl, 3.2 KCl, 1 NaH2PO4, 26 NaHCO3, 2MgCl2, 1 CaCl2, 10 glucose, 2 Na-pyruvate, and 0.6 ascorbic acid (pH7.4, 95% O2/5% CO2). Hippocampal slices (400 mm thick) were cut

with a vibratome (VT1000S, Leica Microsystems, GmbH, Wetzlar,Germany), incubated in cutting solution at 30 �Ce32 �C for at least60 minutes, and stored until use in the same solution at roomtemperature (RT; 21 �Ce23 �C) as previously described (Cuccurazzuet al., 2010; Podda et al., 2008).

For electrophysiological recordings, slices were transferred intoa submerged recording chamber and continuously perfused withaCSF containing (in mM): 124 NaCl, 3.2 KCl, 1 NaH2PO4, 1 MgCl2, 2CaCl2, 26 NaHCO3, and 10 glucose (pH 7.4, 95% O2/5% CO2). The flowratewas kept at 1.5mL perminutewith a peristaltic pump (Minipuls3; Gilson, Villiers, France), and bath temperature was maintained at30 �Ce32 �C with an in-line solution heater and temperaturecontroller (TC-344B; Warner Instruments, Hamden, CT, USA).

2.3. Long-term potentiation recordings

For extracellular recordings of field potentials, the Schaffercollateral fibers were stimulated with a bipolar tungsten electrode(Warner Instruments). Recordings were made in the CA1 stratumradiatum using a glass electrode filled with aCSF (tip resistance 2e5MU) and connected to a MultiClamp 700A amplifier (MolecularDevices, Sunnyvale, CA, USA). A Digidata 1440 Series interface andpClamp 10 software (Molecular Devices) were used for dataacquisition and stimulation protocols. Data were filtered at 1 kHz,digitized at 10 kHz, and analyzed on- and off-line.

Hippocampal subfields and electrode positions were identifiedwith the aid of 4� and 40� water immersion objectives on anupright microscope equipped with differential interferencecontrast optics under infrared illumination (BX5IWI; Olympus,Tokyo, Japan) and video observation (C3077-71 CCD camera;Hamamatsu Photonics, Japan).

To study basal synaptic transmission, we measured the ampli-tudes of field excitatory postsynaptic potentials (fEPSPs) elicited bystimuli of increasing amplitudes, before and after slice exposure toaCSF containing either 200 nM Ab42WT or the same amount ofDMSO contained in Ab42 solutions (vehicle), and plotted input/output curves. The stimulation intensity that elicited one-third ofthe maximal response was used for delivering test pulses every 20seconds. To assess the effects of Ab42WT, Ab42MO, and Ab42MO2 onsynaptic plasticity, we diluted the oligomer preparations in aCSF toa concentration of 200 nM and applied them to brain slices for the20 minutes before tetanic stimulation. Long-term potentiation(LTP) was induced with a standard stimulation paradigm consistingof 4 trains of 50 stimuli at 100 Hz (500 ms each) repeated every 20seconds (Fusco et al., 2012). Responses to test pulse were recordedevery 20 seconds for 60 minutes to assess LTP. The amplitude andslope of fEPSPs at 60 minutes were averaged from values obtainedduring the last 5 minutes of posttetanus recordings (from minute55 to minute 60). LTP magnitude was expressed as the percentagechange in the mean fEPSP peak amplitude and slope normalized tobaseline values (i.e., mean values for the last 10 minutes ofrecording before tetanus). The fEPSP amplitude was measured frombaseline to peak, and the slope was calculated between 20% and80% of the maximal amplitude. Data included in the study refer tobrain slices exhibiting stable fEPSP amplitude and slope increases ofat least 15% at the end of the LTP recording. In a subset of experi-ments the effects of Ab42MO2 on LTP were studied after 2-hourtreatment of hippocampal brain slices with the pan-caspaseinhibitor Z-VAD-FMK (50 mM; Tocris Bioscience, Bristol, UK).

2.4. Primary cortical neuron cultures

Primary cultures of cortical neurons were obtained from E18Wistar rat embryos as previously described (Piacentini et al., 2011).In brief, dissected cortices were incubated for 10 minutes at 37 �C in

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e13 3

PBS containing trypsin-ethylenediaminetetraacetic acid (0.025%/0.01% wt/vol; Biochrom AG, Berlin, Germany), and the tissue wasmechanically dissociated at RT with a fire-polished Pasteur pipette.The cell suspension was harvested and centrifuged at 235g for 8minutes. The pellet was suspended in 88.8% Minimum EssentialMedium (Biochrom), 5% fetal bovine serum, 5% horse serum, 1%glutamine (2 mM), 1% penicillin-streptomycin-neomycin antibioticmixture (Invitrogen, Carlsbad, CA, USA), and glucose (25 mM). Cellswere plated at a density of 105 cells on 20-mm coverslips precoatedwith poly-L-lysine (0.1 mg/mL; Sigma, St. Louis, MO, USA). Twenty-four hours later, the culture mediumwas replaced with amixture of96.5% Neurobasal medium (Invitrogen), 2% B-27 (Invitrogen), 0.5%glutamine (2 mM), and 1% penicillin-streptomycin-neomycin anti-biotic mixture. After 72 hours, this medium was replaced witha glutamine-free version of the same medium, and the cells weregrown for 10 more days before experiments.

2.5. Autaptic neuron culture preparation and synaptic transmissionstudies

Autaptic neuron microcultures were prepared according to pub-lishedprocedures (Bekkers andStevens,1991; Pyott andRosenmund,2002) with minor modifications. Glass coverslips coated withagarose, which does not allow cell growth, were sprayed witha mixture of poly-D-lysine and collagen (both from Sigma) to createmicroislands in which glial cells could be grown. Cortical astrocytesfrom the brains of postnatal day 0e2 Wistar rats were culturedaccording to standard procedure (Galea et al.,1992). Dissociated cells(suspended inDulbecco’sminimun essentialmedium supplementedwith 10% fetal bovine serum and antibiotics) were plated onto thecoverslips. After 4e6 days, when microislands of glial cells hadformed, half of the medium volume was replaced with neuronalmedium (consisting of Neurobasalmedium, 2% B-27, 0.5% glutamine,and 1% penicillin-streptomycin-neomycin antibiotic mixture). Thisallowed the medium to be conditioned by the astrocytes beforeneuron plating. Hippocampal neurons from postnatal day 0e2 Wis-tar rat brains were prepared according to standard procedure(Piacentini et al., 2008a) and suspended in neuronal medium. Later,neurons were plated onto glial microislands at low density (25,000/cm2) to obtain a ratio of one neuron per island. Two weeks later,autapses had formed and were ready to be studied. Every 4 dayshalf the neuronal mediumvolumewas replaced with fresh neuronalmedium supplemented with 2 mM cytosine arabinoside.

We studied synaptic transmission in autaptic neurons asdescribed in Gerber et al. (2008), using the patch-clamp technique inthe whole-cell configuration. An Axopatch 200B amplifier (Molec-ular Devices) was used, and stimulation and data acquisition wereperformed with the Digidata 1200 series interface and pCLAMP 10software (Molecular Devices). Recording electrodes were fabricatedfrom borosilicate glass capillaries with the aid of a micropipettepuller (P-97; Sutter Instruments, Novato, CA, USA). The extracellularsolution (pH 7.4) contained (in mM): 140 NaCl; 2 KCl; 10 hydroxy-ethyl piperazineethanesulfonic acid (HEPES); 10 glucose; 4 MgCl2;and 4 CaCl2. The internal solution (pH 7.4) contained (in mM): 136KCl, 17.8 HEPES, 1 ethylenediaminetetraacetic acid (EGTA), 0.6 MgCl2,4 adenosine 5’-triphosphate, 0.3 guanosine 5’-triphosphate, 12creatine phosphate, and 50 U/mL phosphocreatine kinase.

Neurons were voltage-clamped at a membrane potential of �70mV, and stimuli mimicking action potentials (2 ms at 0 mV) weredelivered to evoke excitatory postsynaptic currents (EPSCs), whichwere recorded every 6 or 20 seconds. Input resistance, access resis-tance, andmembrane capacityweremonitored before and at the endof the experiments to ensure recording stability and the health ofstudied cells. The amplitudes and frequency of spontaneous minia-ture excitatory postsynaptic currents (mEPSCs) were evaluated in

60-second recordings. The detection threshold was set to 3.5 timesthe baseline standard deviation. The size of the readily releasablepool (RRP) of synaptic vesicles was estimated as described elsewhere(Gerber et al., 2008). In brief, we measured the charge induced bya 4-second extracellular application of hypertonic (0.5 M) sucrose,which triggers vesicle exocytosis by increasing the transmembraneosmotic pressure in the presynaptic terminal. The total RRP chargewas then estimated as the integral of the fast, transient, inwardcurrent component, after subtraction of the steady state component.The probability of vesicular release (Pvr), estimated from the samecell, was expressed as the EPSC charge/RRP charge ratio. RRP refillingwas investigated in paired-pulse experiments, in which 0.5 Msucrose solution was applied for 4 seconds at 4-second interpulseintervals. The RRP recovery ratewas expressed as the peak amplitudeof the second response normalized to that of the first response. Allparameters were measured before and after 20-minute and 24-hourexposures to 200 nM Ab42WT, oxidized Ab42 analogues, or vehiclealone. All experiments were performed at RT.

2.6. Immunocytochemistry

Cortical neurons cultured for 7e15 days in vitro (DIV) andtreated with Ab42 analogues were fixed with 4% paraformaldehyde(Sigma) in PBS for 15 minutes at RT. After being permeabilized (15-minute incubation with 0.3% Triton X-100 [Sigma] in PBS), the cellswere incubated for 20 minutes with 0.3% bovine serum albumin inPBS to block nonspecific binding sites and then overnight at 4 �Cwith different pairs of the following antibodies: rabbit monoclonalanti-synaptophysin (1:250; Abcam, Cambridge, UK); mouse anti-postsynaptic density protein-95 (PSD-95; 1:250, Abcam); mouseanti-microtubule associated protein-2 (MAP-2; 1:300; Sigma). Thenext day, cells were incubated for 90 minutes at RT with a mixtureof the following secondary antibodies: Alexa Fluor 488 donkey anti-rabbit (1:1000; Invitrogen); Alexa Fluor 546 donkey anti-mouse(1:1000; Invitrogen); or rhodamine-conjugated goat anti-mouse(1:300; Millipore, Billerica, MA, USA). Finally, nuclei were coun-terstainedwith 4’,6-diamidino-2-phenylindole (DAPI; 0.5 mg/mL for10 minutes; Invitrogen), and the cells were coverslipped withProLong Gold anti-fade reagent (Invitrogen).

For the experiments in which neuronal cultures were treatedwith IRIS 5-labeled Ab analogues, cells were immunoprocessed forMAP-2 only.

Images (1024�1024 pixels) were acquired at 63�magnificationwith a confocal laser scanning system (TCS-SP2; Leica) and anoil-immersion objective (numerical aperture 1.4; physical pixel size233 nm). 4’,6-diamidino-2-phenylindole staining was imagedafter 2-photon excitation with an ultrafast, tunable, mode-lockedtitanium:sapphire laser (Chamaleon; Coherent Inc., Santa Clara,CA, USA). All experiments were repeated at least 3 times, and atleast 20 randomly chosenmicroscopic fields were analyzed for eachcondition. Immunofluorescence for synaptophysin and PSD-95 wasquantified as the sum of fluorescence intensities (8-bit depth)measured for every pixel in the recorded field. In some fields, wecalculated the “synaptophysin density,” that is, the total fluores-cence intensity of synaptophysin labeling divided by the total areain the field that was occupied by neurons (identified by MAP-2immunoreactivity and calculated by ImageJ software [available athttp://rsbweb.nih.gov/ij/]). The operator was blinded to the studyconditions.

2.7. Western immunoblot analyses

2.7.1. Western blot analysis of Ab42 preparationsFresh and aged Ab42 solutions, prepared as described in Section

2.1., were diluted in aCSF and then incubated for 20 minutes at

Fig. 1. Effects of amyloid b-protein (Ab)42 analogues on long-term potentiation (LTP) at CA3eCA1 synapses. (A) Representative traces of field excitatory postsynaptic potentials(fEPSPs) recorded before (dotted lines) and after (solid lines) tetanic stimulation in hippocampal slices exposed to different Ab42 forms. Time course of fEPSP amplitudes before andafter tetanic stimulation (indicated by arrow) in slices treated for 20 minutes with 200 nM Ab42 containing reduced methionine residue at position 35 (Met35) (Ab42WT) (B), theAb42 form harboring Met35-sulfoxide (Ab42MO) (D), and Ab42MO2 (F) versus vehicle alone. Results are expressed as percentages of baseline fEPSP amplitude (¼ 100%). (C), (E), and(G) Time course of fEPSP slope recorded from slices treated as described for (B), (D), and (F). (H) LTP amplitudes measured during the last 5 minutes of recording under theconditions described for (B), (D), and (F). (I) LTP slopes measured during the last 5 minutes of recording under the conditions described for (C), (E), and (G). Representative Westernblots of Ab42 analogues (200 nM) incubated for 12 hours at 4 �C (J) and for 20 minutes at 32 �C (K). Oligomeric species were separated by Tricine precast gels and probed with 6E10antibody. ** p < 0.001.

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e134

32 �C. After this step, NuPAGE LDS sample buffer 4� was addedand the samples (at the final Ab concentration of 200 nM) wereseparated on 10%e20% Novex Tricine precast gels (Invitrogen)according to the manufacturer’s protocol. Gels were transferred to0.2 mm nitrocellulose membranes (Amersham Biosciences, Buck-inghamshire, UK). Membranes were blocked for 1 hour, at RT, ina solution of 5% nonfat dry milk in Tris-buffered saline containing0.1% Tween-20 before incubation overnight at 4 �C with the mousemonoclonal antibody 6E10 (1:1000; Covance, Princeton, NJ, USA).Membranes were washed 3 times with Tris-buffered saline con-taining 0.1% Tween-20 and then incubated with the horseradishperoxidase-conjugated Ig anti-mouse antibody (1:2000; CellSignaling Technology Inc., Danvers, MA, USA) at RT for 1 hour.Development was done after 5-minute incubation with enhancedchemiluminescence reagents (Amersham Biosciences) and exposedto Kodak X-OMAT films. Images of the blots were acquired using

a high-resolution scanner. Molecular weights were estimated usingRainbow Molecular Weight Markers (Amersham Biosciences).

2.7.2. Western blot analysis of synaptic proteinsLysates were prepared from primary cortical neurons (15 DIV)

with modified radio-immuno-protein assay (RIPA) buffer (ThermoScientific, Rockford, IL, USA) containing 25 mM Tris-HCl (pH 7.4), 1%Nonidet P-40, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1%sodium dodecyl sulfate, 1mM EGTA, and 1% protease inhibitorcocktail (Sigma). In brief, neurons were treated for 72 hours withvehicle or with 500 nM of one of the Ab42 analogues, scraped intoPBS, and pelleted. These samples were homogenized in ice-coldlysis buffer, and the protein concentration was measured withthe microBCA protein assay (Thermo Scientific). Equal amountof proteins (30 mg) were then loaded onto 10% Tris-glycine poly-acrylamide gels for electrophoretic separation. Colorburst

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e13 5

electrophoresis markers (Sigma) were used as molecular massstandards. Proteins were then transferred onto nitrocellulosemembranes (90 minutes in transfer buffer containing 25 mM Tris,192 mM glycine, 0.1% sodium dodecyl sulfate, and 20% methanol).The membranes were stained with Ponceau S (Sigma), incubatedfor 1 hour with blocking buffer (5% skimmilk in Tris-buffered salinecontaining 0.5% Tween 20), and then incubated overnight at 4 �Cwith primary antibodies directed against PSD-95 (1:1,000; CellSignaling); synaptophysin (1:10,000; Abcam); or tubulin (1:2500;Sigma). After three 10-minute rinses in Tris-buffered saline withTween, membranes were incubated for 1 hour at RT with horse-radish peroxidase-conjugated anti-rabbit or anti-mouse antibodies(1:1000; Cell Signaling). Themembranes were thenwashed and thebands visualized with an enhanced chemiluminescence detectionkit (Thermo Scientific). Protein expression was quantified bydensitometric analysis performed with TINA software version2.09f; (Raytest, Strauberhardt, Germany).

All Western blot experiments were repeated at least 3 times.

2.8. Dendritic spine staining and quantification

We assessed the density and shape of dendritic spines inprimary cortical neurons at 15 DIV. Neurons were stained withMAP-2 (see immunocytochemistry Section 2.6.) to identifydendrites and then relabeled for 30 minutes with rhodamine-conjugated phalloidin (Invitrogen, 1:500 in PBS) to reveal fila-mentous actin, which is abundant in dendritic spines (Halpain et al.,1998). Spines were imaged with a Leica TCS-SP2 confocal scanningsystem and a 100� magnification objective (numerical aperture1.47) plus additional magnification of 9�. Images were taken at512 � 512 pixel resolution (physical pixel size: 33 nm). Spinedensity was analyzed under blinded conditions in randomly chosensegments (length: 38e62 mm) of secondary dendrites from apicalbranches and expressed as the number of spines per 100 mmdendrites. A total length of at least 1 mm was analyzed for eachexperimental condition.

2.9. Statistical analysis

All data are shown as mean � standard error of the mean.Statistical analyses (Student paired and unpaired t tests) wereperformed with SYSTAT 10.2 software (Statcom, Inc., Richmond, CA,USA). The level of significance was set at 0.05.

3. Results

3.1. Effects of the Ab42 analogues on synaptic plasticity

To determine whether Met35 oxidation affects Ab42 synapto-toxicity, we first investigated LTP at Schaffer collateraleCA1synapses after exposure to Ab42WT and its oxidized analoguesAb42MO and Ab42MO2. When hippocampal slices were perfusedwith vehicle alone for 20 minutes before tetanization (controlconditions), the fEPSP amplitude and slope recorded 60 minutesafter tetanization displayed increases of 138.3 � 18.2% and 132.3 �14.8%, respectively (n ¼ 10; Fig. 1AeC). In agreement with previousreports (Jo et al., 2011; Knobloch et al., 2007; Li et al., 2011; Oliveiraet al., 2010), when slices were perfused with 200 nM aged Ab42WT

(prepared 12 hours before use), the LTP was weaker (amplitude andslope increases of 76.1 � 10.1% and 68.0 � 9.9%, respectively, overbaseline values; n¼ 9; p< 0.01; Fig.1B and C). This treatment had noeffect on basal synaptic transmission (as reflected by the stability ofpretetanus fEPSP recordings and the absence of significant differ-ences between input/output curves obtained before and afterAb42WT application). However, as noted by others (Cerpa et al.,

2010; Rönicke et al., 2011), higher concentrations of Ab42WT (500nM) inhibited basal synaptic transmission in our brain slice prepa-rations, as reflected by reduced fEPSP amplitudes (data not shown).As for the oxidized Ab analogues, 200 nM aged Ab42MO had nosignificant effect on LTP (130.1 � 14.8% of baseline amplitude and134.0 � 21.3% of baseline slope; n ¼ 10; Fig. 1D and E), but sliceexposure to 200nMAb42MO2 produced LTP inhibition similar to thatobserved with Ab42WT (62.2 � 8.7% of baseline amplitude and 69.1� 14.4% of baseline slope; n¼ 10; p< 0.01 vs. vehicle; Fig. 1F and G).

To check whether large size Ab aggregates and fibrils possiblyproduced during the peptide aging contributed to the observedeffects, LTP experiments were repeated with Ab preparations thatwere subjected to centrifugation (at 18,000g for 10 minutes at 4 �C)before application to brain slices. The centrifuged Ab42WT inducedincreases in fEPSP amplitude that were not significantly differentfrom those caused by the noncentrifuged Ab preparations (44.2 �8.3% [n ¼ 5] and 50.3 � 4.2% [n ¼ 13], respectively). These dataindicate that, if present, large size Ab aggregates and fibrils did notcontribute to the LTP inhibition we observed.

To determine whether the different effects of the tested Ab42variants on LTP were somehow related to differences in their olig-omerization occurring during the 12-hour interval preceding ourrecordings, we performed LTP experiments with freshly preparedAb42WT, Ab42MO, and Ab42MO2 solutions (200 nM). Fresh Ab42WT

produced robust LTP inhibition, which was not significantlydifferent from that caused by its 12-hour-old counterpart (ampli-tude: 76.5� 7.4% vs. 135.0� 7.0% of control slices; slope: 82.5� 7.5%vs. 160.0 � 8.9% of controls; n ¼ 10 and 9, respectively for Ab- andvehicle-treated slices; p < 0.01). Freshly prepared Ab42MO2 alsoinhibited LTP (58.4� 9.9% of baseline amplitude and 81.8� 16.4% ofbaseline slope; n ¼ 9; p < 0.01 compared with vehicle), but thechanges observed in slices perfused with fresh Ab42MO were smalland nonsignificant (110.2 � 10.6% and 148.2 � 8.4% for baselineamplitude and slope, respectively; n ¼ 10). These data demonstratethat Met35 oxidation to Ab42MO abolishes the protein’s capacity toinhibit LTP, and suggest that differences between the effects of thetested Ab analogues are independent of their aggregation.

To check more specifically whether the different synaptotoxicityof Ab42MO and Ab42MO2 correlated with their different aggregationproperties, we characterized the oligomer distribution patterns ofall our Ab42 preparations by Western blot focusing on the small Aboligomers whose synaptotoxic action is well documented (Li et al.,2011; Rönicke et al., 2011; Selkoe, 2008). As shown in Fig. 1J, 6E10antibody only revealed monomers and dimers in 12-hour agedAb42MO and Ab42MO2 preparations (200 nM) whereas Ab42WT

contained small oligomers ranging frommonomers to tetramers. Toreproduce the experimental conditions of our LTP experiments,Western blot analysis was also performed on 200 nM Ab42WT

solutions prepared 20 minutes earlier. In agreement with previousliterature reports (Berman et al., 2008; Rahimi et al., 2009; Stineet al., 2003) the “fresh” Ab42WT solution also contained small Abassembly forms (especially dimers along with some trimers andtetramers; Fig. 1K) that were probably produced as soon as thepeptide was dissolved in the aqueous buffer. More importantly, thefreshly-prepared Ab42MO and Ab42MO2 exhibited a very similaroligomer distribution pattern consisting of monomers and dimersonly.

These findings clearly indicate that the different effects exertedby our Ab analogues on LTP do not correlate with their oligomerdistribution patterns. In particular, despite the dramatic differencesin LTP inhibitions induced by Ab42MO and Ab42MO2 these two Abpreparations exhibited virtually identical contents in monomersand dimers.

Because Jo and coworkers (2011) recently demonstrated theinvolvement of caspase-3 in LTP impairment induced by Ab42WT

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e136

we checked whether caspase activation also underlies the syn-aptotoxic effects of Ab42MO2. To address this issue we repeated LTPexperiments in hippocampal brain slices treated with the pan-caspase inhibitor Z-VAD-FMK (50 mM) for 2 hours before expo-sure to aged Ab42MO2. We found that Z-VAD-FMK pretreatmentsignificantly counteracted the LTP inhibition caused by 20-minuteexposure to Ab42MO2 (200 nM). Indeed, LTP was 51.3 � 11.2% ofbaseline fEPSP amplitude (n¼ 5) in brain slices exposed to Ab42MO2

alone and 95.5 � 8.2% (n ¼ 10) in slices treated with Z-VAD-FMKbefore exposure to Ab42MO2 (p < 0.005; data not shown).

3.2. Effects of Ab42 analogues on basal synaptic transmission

Whole-cell patch-clamp recordings were made in autapticmicrocultures of hippocampal neurons to investigate the Ab42analogues’ effects on basal synaptic transmission. EPSCs evoked bystimuli mimicking action potentials were recorded every 20seconds during the 20-minute period after Ab application. Experi-ments were confined to autapses exhibiting stable EPSC amplitudesduring the 5-minute period preceding Ab application (T0). After 20minutes of exposure (T20) to vehicle alone, EPSC amplitudes werealmost identical to those measured at T0 (6.53 � 0.55 nA vs. 6.72 �0.50 nA; n ¼ 20; Fig. 2AeC), but those measured after neurons hadbeen exposed for 20 minutes to 200 nM Ab42WT were significantlysmaller (�32.1�3.9%; n¼ 20; p< 0.05 vs. T0; Fig. 2AeC), indicatingthat Ab42WT markedly depresses hippocampal excitatory neuro-transmission. Significant reductions were also seen after treatmentwith Ab42MO2 (�24.1 � 6.8%; n ¼ 11; p < 0.05), but no significanteffects were produced by 200 nM Ab42MO (6.02� 1.16 nA vs. 6.46�1.29 nA at T20 and T0, respectively; n ¼ 9; Fig. 2AeC).

Next, we analyzed the amplitude and frequency of mEPSCs todetermine whether presynaptic and/or postsynaptic mechanismswere responsible for the effects of Ab42WT and Ab42MO2. Therewere no significant differences in either parameter before and after20-minute exposure to vehicle (amplitude: 18.6 � 1.5 pA vs. 20.0 �1.8 pA; frequency: 7.9 � 1.0 Hz vs. 9.4 � 1.3 Hz, respectively; n ¼ 17;Fig. 2D1e3). Perfusion with 200 nM Ab42WT significantly reducedmEPSC frequency (�34.1�9.4% vs. T0; n¼ 16; p< 0.005; Fig. 2E1e2)but had no effect on mEPSC amplitude (19.8 � 2.3 pA vs. 18.2 � 1.2pA; Fig. 2E1 and 3). In accordance with other electrophysiologicalmeasurements, Ab42 harboring Met35-sulfoxide did not signifi-cantly modify either the amplitude (14.3 � 2.3 pA vs. 14.4 � 3.7 pA,at T20 and T0, respectively; n ¼ 9; Fig. 2F1 and 3) or frequency (7.9 �1.3 Hz vs. 7.5�1.1 Hz; Fig. 2F1 and 2) of mEPSCs. When neurons wereexposed to 200 nM Ab42MO2, mEPSC frequency dropped signifi-cantly (�30.6� 8.0%; n¼ 11; p< 0.005; Fig. 2G1 and 2), although theamplitude did not change (17.5 � 2.3 pA vs. 17.0 � 2.7 pA; Fig. 2G1

and 3). The fact that Ab42WT and Ab42MO2 decreased only thefrequency of mEPSCs suggests that postsynaptic receptors are notaffected by brief exposure to Ab. Therefore, in all probability, thesynaptic transmission alterations observed after 20-minute expo-sure to Ab42WT or Ab42MO2, which clearly depend on the oxidationstate of Met35, are caused entirely by impaired presynapticfunction.

The next set of experiments explored the functional alterationsinduced by the Ab analogues at presynaptic terminals. To determinewhether the diminished release of excitatory neurotransmitter inAb42WT- and Ab42MO2-treated hippocampal neurons was becauseof a decrease in RRP size, we challenged neurons with hypertonicsucrose solution at T0 and again 20 minutes after each Ab exposure.The size of the RRP was estimated as the integrated charge transfer(see also 2. Methods). RRPT20/RRPT0 ratios for the Ab42WT-,Ab42MO2-, and vehicle-treated groups of neurons were not signifi-cantly different (Fig. 3A and B), which suggests that the number ofdocked vesicles was unaffected by treatments. As for postrelease

RRP recovery, the refilling rate after 20 minutes of perfusion withvehicle was 80.4 � 7.0%, and rates were similar in neurons exposedto 200 nM Ab42WT or Ab42MO2 (82.0 � 5.2% and 82.0 � 7.0%,respectively; Fig. 3C and D). At T20 the estimated Pvr was signifi-cantly reduced in cells treatedwith Ab42WT (�40.7�6.8% of T0; n¼10; p < 0.005) or Ab42MO2 (�38.9 � 7.7%; n ¼ 7; p < 0.005; Fig. 3E).These data indicate that Ab42 rapidly impairs the presynapticglutamate-release machinery when its Met35 is reduced oroxidized to sulfone, but this ability is lost when the sulfur atom isoxidized to sulfoxide.

Ab42 treatments lasting longer than 20 minutes produced morestriking synaptic depression. As shown in Fig. 4A, mean EPSCamplitudes in autaptic neurons exposed for 24 hours to Ab42WT orAb42MO2 were 3.59 � 0.33 nA (n ¼ 26) and 3.98 � 0.59 nA (n ¼ 24),respectively, versus 7.45 � 0.50 nA (n ¼ 38; p < 0.05) in controlcultures. Interestingly, 24-hour exposure to Ab42MO (which hadproduced no significant effects when applied for 20 minutes)significantly reduced EPSC amplitudes (5.69 � 0.42 nA; n ¼ 31; p <

0.05) although its effects were still appreciably weaker than thoseof Ab42WT and Ab42MO2 (�23.6% vs. �51.8% and �46.5%, respec-tively; p < 0.005). Twenty-four-hour treatments had similar effectson mEPSC amplitude (Ab42WT: �34.5% of controls [p < 0.005];Ab42MO2: �31.8% [p < 0.005 vs. control]; Ab42MO: �18.7% [p < 0.05vs. controls]; Fig. 4B) and mEPSC frequency (reductions of 41.6%,44.0%, and 28.2% in neurons cultured with Ab42WT, Ab42MO2, andAb42MO, respectively; p < 0.005 for Ab42WT and Ab42MO2 vs.control; p < 0.05 for Ab42MO vs. control; Fig. 4C). The amplitudedecreases observed in both evoked and spontaneous neurotrans-mission suggest that the number and/or responsiveness of post-synaptic receptors, which were unaffected by short (20-minute)exposures to Ab, were indeed diminished by 24-hour treatment,and the magnitude of this effect was clearly related to the oxidationstate of Met35.

Fig. 4D and E show the RRP charge and Pvr for autaptic culturestreated for 24 hours with the different Ab42 analogues. The formerparameter was significantly reduced, relative to vehicle-treatedcultures, by all three Ab42 analogues (reductions of 70.0%, 51.7%,and 45.1% with 200 nM Ab42WT, Ab42MO2, and Ab42MO, respec-tively; p < 0.005; Fig. 4D). Interestingly, the Pvr (0.23 � 0.03 incontrol neurons, n ¼ 30) was not significantly different after 24-hour treatment with Ab42WT (0.29 � 0.06, n ¼ 23), Ab42MO (0.25� 0.03, n ¼ 26), or Ab42MO2 (0.29 � 0.05, n ¼ 21), suggesting thatthe preserved synapses efficiently released glutamate.

In light of these data, Ab-induced synaptic depression seems toconsist of early presynaptic impairment followed by combined pre-and postsynaptic compromise, which is more pronounced inneurons treated with Ab42WT or Ab42MO2 than in those exposed toAb42MO.

3.3. Effects of Ab42 analogues on synaptic protein expression

Next, we investigated the possibility that the altered synaptictransmission observed after Ab treatments was associated withchanges in the expression of specific pre- and/or postsynapticproteins. To this end, we incubated DIV-15 cortical neurons witheach of the Ab42 variants for 24e72 h and evaluated postexposureexpression of the presynaptic protein, synaptophysin, and PSD-95with immunofluorescence and Western blot analyses.

Synaptophysin and PSD-95 immunolabeling resulted in abun-dant punctate staining of axons and dendrites (Fig. 5A and B), whichis consistent with the expression patterns reported for theseproteins (Fletcher et al., 1991; Lacor et al., 2004). Exposure ofneurons to Ab induced time- and dose-dependent reductions insynaptophysin labeling. The total fluorescence intensity was notsignificantly modified by a 24-hour incubation with 200 nM

Fig. 2. Effects of amyloid b-protein (Ab)42 analogues on evoked and spontaneous synaptic neurotransmission in autaptic hippocampal neurons. (A) Time course of excitatorypostsynaptic potential (EPSC) amplitudes recorded under basal conditions before and during 20-minute perfusion with vehicle (open circles), or solutions containing 200 nM Ab42containing reduced methionine residue at position 35 (Met35) (Ab42WT) (red circles), the Ab42 form harboring Met35-sulfoxide (Ab42MO) (green circles), or Ab42MO2 (blue circles).(B) Bar graph showing mean EPSC amplitudes recorded in neurons treated with vehicle, Ab42WT, Ab42MO, or Ab42MO2. Amplitudes (mean � standard error of the mean) arenormalized to those recorded under basal conditions. (C) Representative traces showing EPSCs recorded during the 5-minute period preceding application (T0) and after 20 minutesof exposure (T20) in neurons subjected to each treatment. Representative traces of mEPSCs recorded at T0 and T20 of perfusion with (D1) vehicle, (E1) Ab42WT, (F1) Ab42MO, and (G1)Ab42MO2. Mean frequency of miniature EPSC (mEPSC) after 20 minutes of perfusion with (D2) vehicle, (E2) Ab42WT, (F2) Ab42MO, and (G2) Ab42MO2. Mean mEPSC amplitude after20 minutes of perfusion with (D3) vehicle, (E3) Ab42WT, (F3) Ab42MO, and (G3) Ab42MO2. * p < 0.05.

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e13 7

Fig. 3. Effects of 20-minute exposure to amyloid b-protein (Ab)42 containing reducedmethionine residue at position 35 (Met35) (Ab42WT) or the Ab42 form harboringMet35-sulfoxide-2 (Ab42MO) on glutamate release from hippocampal neurons. (A)Representative traces of currents induced by 4-second applications of 0.5 M sucroseduring the 5-minute period preceding application (T0) and after 20 minutes of expo-sure (T20) (used to assess the readily releasable pool [RRP] size). (B) Ratios of RRPcharge at T20 to that observed at T0 in Ab- and vehicle-treated neurons. (C) Repre-sentative traces of currents obtained during paired sucrose applications (used to studypostrelease RRP refilling rates, i.e., RRP recovery). Bar graphs showing RRP recoveryrates (D) and mean probability of vesicular release (Pvr) ratios (E) after 20-minexposure to vehicle, Ab42WT, or Ab42MO2. * p < 0.05.

Fig. 4. Effects of 24-hour exposure to amyloid b-protein (Ab)42 analogues on evokedand spontaneous synaptic neurotransmission in autaptic hippocampal neurons. Bargraphs showing the mean excitatory postsynaptic current (EPSC) amplitude (A);miniature EPSC (mEPSC) amplitude (B); and mEPSC frequency (C), recorded after 24hours of treatment with vehicle, 200 nM Ab42 containing reduced methionine residueat position 35 (Met35) (Ab42WT), the Ab42 form harboring Met35-sulfoxide (Ab42MO),or Ab42MO2. (D) The readily releasable pool (RRP) charge measured after 0.5 M sucroseapplication under the same conditions described in (AeC). (E) Mean probability ofvesicular release (Pvr) value recorded after each treatment. * p < 0.05; ** p < 0.001.

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e138

Ab42WT, but appreciable reductions (�20% comparedwith controls;n ¼ 20 microscopic fields for each condition; p < 0.001) were seenafter 72 hours of exposure. At higher concentrations (500 nM),24 hours of exposure also produced significant effects (�26%; n ¼20; p < 0.001), and greater reductionwas found at 72 hours (�35%;n ¼ 20; p < 0.001; Fig. 5C, E, and F).

To determine whether these reductions reflected down-regulated protein expression and/or decreased numbers of immu-nofluorescent puncta secondary to neurite loss, we evaluated thesynaptophysin density, expressed as the ratio of total synaptophy-sin fluorescence intensity to the total area occupied by neurons(quantified byMAP-2 immunolabeling). Synaptophysin densities inAb42WT-treated cells were significantly lower than those of controls(�16% after 72-hour exposure; n ¼ 20; p < 0.05) although thereduction was smaller than that estimated on the basis of fluores-cence intensity of synaptophysin labeling alone. The diminishedsynaptophysin immunolabeling observed after protracted exposureto Ab42WT thus appears to reflect downregulated protein expres-sion as well as dendrite loss, and this conclusion was confirmed bypostexposure size reductions in the MAP-2-labeled area in theimaged fields (�27%, n ¼ 20; p < 0.001).

Analysis of mean values of total fluorescence intensity for syn-aptophysin labeling revealed no significant decrease in proteinexpression after 24 hours of treatment with 500 nM Ab42MO, buta small reduction (�14%; p < 0.01; n ¼ 20; Fig. 5C and G) was

observed after 72 hours of exposure. As for Ab42MO2, its effectswere similar to those observed with Ab42WT. Exposures of 24 and72 hours reduced synaptophysin immunolabeling by 17% (p < 0.05)and 48% (p< 0.001), respectively, relative to controls (Fig. 5C, E, andH).

A similar trend was observed for PSD-95 expression. Reductionsof 19% and 17%were observed after 24 hours of incubationwith 500nM Ab42WT or Ab42MO2, respectively (p < 0.01; Fig. 5D), and largerreductions (�23% and �25%, respectively) were seen after 72 hoursof exposure (Fig. 5I, J, and L). In contrast, treatment with Ab42MO

had no significant effect on PSD-95 immunolabeling, regardless ofthe duration of exposure (Fig. 5D and K).

To further investigate the effects of Met35 oxidation on syn-aptophysin and PSD-95 expression, weusedWestern blot to analyzelysates of DIV-15 cortical neurons treated for 72 hours with 500 nMof the Ab42 analogues. Once again, cells treated with Ab42WT orAb42MO2displayed significantly reducedexpressionof both synapticproteins, whereas the changes observed in Ab42MO-treated cellswere modest and nonsignificant (Fig. 6). Densitometric analysisrevealed that Ab42WT and Ab42MO2 treatments reduced synapto-physin expression by 25.7 � 1.3% and 14.9 � 3.0%, respectively (p <

0.01) and PSD-95 expression by 28.5 � 2.0% and 15.1 � 1.4%,respectively (p < 0.01). In contrast, the decreases produced byAb42MO were considerably smaller (�8.7 � 2.4 [p < 0.05] forsynaptophysin,�2.3�0.7% [p¼not significant] for PSD-95) (Fig. 6B).

Fig. 5. Effects of amyloid b-protein (Ab)42 analogues on neuronal immunoreactivity for synaptophysin and postsynaptic density protein-95 (PSD-95). (A) Double staining forsynaptophysin and microtubule associated protein-2 (MAP-2) in 15 days in vitro (DIV) cortical neurons. Blue staining (4’,6-diamidino-2-phenylindole; DAPI) identifies nuclei. (B)PSD-95 immunolabeling. (C) and (D) Bar graphs showing mean fluorescent intensities of synaptophysin and PSD-95, respectively, in neurons exposed to Ab42 analogues for 24 hours(open bars) and 72 hours (hatched bars). Representative images showing changes in synaptophysin immunoreactivity after 72-hour treatments with vehicle (E); Ab42 containingreduced methionine residue at position 35 (Met35) (Ab42WT) (F); the Ab42 form harboring Met35-sulfoxide (Ab42MO) (G); or Ab42MO2 (H). Neurons are labeled with MAP-2. (IeL)Representative differential interference contrast (DIC) images showing changes in PSD-95 immunoreactivity under the same experimental conditions described for panels (EeH).Representative examples of neurons treated for 24 hours with 500 nM IRIS-labeled Ab42WT (M), Ab42MO (N), and Ab42MO2 (O). Red staining indicated MAP-2 immunoreactiveneurons. Bottom boxes represent XZ cross-sections from the Z-stack acquisitions showing the different neuronal accumulation of Ab42 analogues after 24-hour treatments. Scalebars: 20 mm for A, B, and IeO; 40 mm for EeH. * p < 0.05; ** p < 0.001.

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e13 9

3.4. Ab42 analogues interact differently with neuronal membranes

The different synaptotoxicity of the Ab42 analogues we studiedmight depend on differences in their interactions with neuronal

membranes and cell internalization. To test this hypothesis welabeled Ab42WT, Ab42MO, and Ab42MO2 with the fluorescent dyeIRIS-5-NHS and studied their localization by high-resolutionconfocal microscopy. After 24-hour treatments, both Ab42WT and

Fig. 6. Effects of amyloid b-protein (Ab)42 analogues (Ab42 containing reducedmethionine residue at position 35 [Met35]: Ab42WT; the Ab42 form harboring Met35-sulfoxide: Ab42MO; and Ab42MO2) on synaptophysin and postsynaptic density protein-95 (PSD-95) protein expression. (A) Representative Western blots showing the effectsof 72-hour exposure to Ab42 analogues on PSD-95 and synaptophysin expression in 15days in vitro (DIV) cortical neurons. (B) Densitometric analysis of the bands shown in(A). Bars indicate mean � standard error of the mean protein levels normalized totubulin and expressed as percentages of control (ctrl) values. * p < 0.05; ** p < 0.001.

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e1310

Ab42MO2 were abundantly taken up by neurons and were found inboth cell bodies and neuronal processes (Fig. 5M and O). Instead,cells treated with Ab42MO only exhibited very weak labeling ofsomata and occasional staining of neuronal processes. Most of thelabeled Ab42MO was confined outside the cells (Fig. 5N), as alsoshown by the XZ cross-sections from the Z-stack acquisitions (boxesin Fig. 5M and O). These findings support the view that the differentsynaptotoxicity of Ab42 analogues we studied depends on theirdifferent ability to interact with and cross neuronal membranes.

3.5. Effects of Ab analogues on dendritic spine density andmorphology

Dendritic spines are small protrusions extending from dendritesthat serve as the postsynaptic component for the vast majority ofexcitatory synapses in the central nervous system (Calabrese et al.,2006). Their number is highly variable, and morphologic differ-ences between spines reflect functional differences (Benavides-Piccione et al., 2002). Changes in the actin cytoskeleton ofdendritic spines underlie certain forms of synaptic plasticity (Schelland Irvine, 2006). Spine number, shape, and size are dynamicallyregulated in response to a large number of physiological stimuli,and they are also affected by pathologic conditions, including ADand other neurodegenerative diseases (Penzes et al., 2011; Weiet al., 2009).

Our final set of experiments explored dendritic spines of corticalneurons treated with Ab analogues at concentrations exertingmaximum effects on synaptic protein expression. At 15 DIV, themean spine density (number of spines per 100 mm dendrite length)in control neurons was 78 � 3 (n ¼ 50 dendrite segments). When

neurons were incubated for 3 hours with 500 nM Ab42WT, spinedensity dropped to 55 � 5 (n ¼ 10; p < 0.05). Longer exposures(24e72 hours) further reduced the number of dendritic spines to 41� 3 (n¼ 14) and 39� 5 (n¼ 17) spines per 100 mm, respectively (p<

0.001 vs. control cells). Morphologic changes were also frequentlyobserved, i.e., reduced presence of mushroom-shaped spines,prevalence of long, thin spines resembling filopodia, and abundantdendritic beading (Fig. 7). Similar results were obtained inAb42MO2-treated neurons, which had 47 � 3 (n ¼ 10), 41 � 3 (n ¼18), and 30� 2 (n¼ 37) spines per 100 mmafter 3-, 24-, and 72-hourtreatments, respectively (Fig. 7D and E). When neurons weretreatedwith Ab42MO for 3 hours, no significant changes in dendriticspine density was observed (81 � 2 spines per 100 mm). The spineloss caused by 24-hour treatment with Ab42MO was significantlysmaller (65 � 3 spines per 100 mm; �18% of controls; n ¼ 24) thanthat induced by Ab42WT (�37%; p < 0.001). In agreement with theresults of our electrophysiological studies (reported above), 72-hour treatments with Ab42MO caused larger effects (49.5 � 3.7spines per 100 mm; a 37% decrease in spine density; n ¼ 25; p <

0.001; Fig. 7C and E).

4. Discussion

A principal goal of AD research is to identify the mechanismsunderlying Ab synaptotoxicity and their correlation with thecognitive decline observed in patients with AD. Although Ab42-induced perturbations of synaptic transmission and plasticityhave been widely investigated, these phenomena are by no meansfully understood.

Our findings provide novel evidence that the oxidative state ofthe Ab42 Met35 residue strongly affects the protein’s capacity toinhibit LTP, depress synaptic transmission, downregulate synapticprotein expression, and reduce dendritic spine density. Theseresults extend our previous observations (Clementi et al., 2006;Maiti et al., 2011; Piacentini et al., 2008b), which showed that: (1)the oxidation state of Met35 is a critical determinant of Ab42-induced neurotoxicity; (2) Ab42MO induces low neuronal deathrates because it does not activate calcium-dependent proapoptoticpathways; and (3) Ab42MO2 is as toxic as Ab42WT although itsoligomer size distribution is indistinguishable from those ofAb42MO and Ab40 (Bitan et al., 2003b).

These findings prompted us to investigate the effect of Met35oxidation on Ab42-induced synaptotoxicity, as reflected by func-tional, morphologic, and molecular parameters that are closelycorrelated with AD-associated memory loss. Ab-induced changes insynaptic function have been attributed to their effects on post-synaptic processes (Lacor et al., 2004), presynaptic functions (Kellyet al., 2005; Parodi et al., 2010; Zhang et al., 2009), and both (Tinget al., 2006). The heterogeneous conclusions reached thus farreflect the use of different experimental approaches, various animalmodels, a broad range of Ab concentrations, different protocols forpreparing Ab oligomers, and different time windows for evaluatingsynaptic effects. Consequently, some groups have found that Ab hasno effect on basal synaptic transmission (Chapman et al., 1999;Malinow et al., 2008; Stern et al., 2004; Wang et al., 2004) andothers have observed marked synaptic depression, with (Kesselset al., 2010) or without (Hsia et al., 1999) LTP deficits.

Transgenic mouse models of AD are useful for studying Ab-induced synaptotoxicity, but the constant production of Ab in theseanimals makes it difficult to control for effects related to intracel-lular versus extracellular Ab accumulation, type of Ab assembly,synapse exposure time and duration, and specific Ab alloforms,including the oxidized peptides found in the brains of AD patientsand transgenicmice (Boutte et al., 2006; Dong et al., 2003; Kuo et al.,2001; Näslund et al.,1994). The in vitro and ex vivo neuronalmodels

Fig. 7. Effects of amyloid b-protein (Ab)42 analogues (Ab42 containing reducedmethionine residue at position 35 [Met35]: Ab42WT; the Ab42 form harboring Met35-sulfoxide: Ab42MO; and Ab42MO2) on dendritic spine density and morphology. Ratcortical neurons (15 days in vitro [DIV]) exposed to Ab42 analogues for 3 hours, 24hours, or 72 hours were immunolabeled for the neuronal marker microtubule asso-ciated protein-2 (MAP-2) (red) and phalloidin (green), which stains filamentous (F)-actin. Representative confocal images of dendrites after 72-hour exposure to vehicle(A); Ab42WT (B); Ab42MO (C); or Ab42MO2 (D). (E) Bar graphs showing spine densities inneurons exposed to Ab42 analogues for 3 hours (gray bars), 24 hours (open bars), and72 hours (hatched bars). Scale bar: 3 mm. ** p < 0.001 versus vehicle; # p < 0.05 versus3-hour Ab treatment.

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e13 11

we used allowed us to investigate the effect of Ab42 Met35 oxida-tion with exposure times ranging from 20 minutes to 72 hours andwith control of many of the conditions listed above.

Oxidation of Met35 to sulfoxide rendered Ab42 incapable ofinhibiting LTP, but additional oxidation reversed this effect. Indeed,Ab42MO2 reduced hippocampal synaptic plasticity as much as didAb42WT. Although Ab42MO2 and Ab42MO differ markedly in terms ofassembly kinetics (Maiti et al., 2011), they have very similar olig-omer distributions (Bitan et al., 2003b; Fig. 1), so their markedlydifferent effects on LTP cannot be attributed to differences in theamounts of those small Ab assembly forms that are consideredprimarily responsible for the peptide synaptotoxicity (Li et al., 2011;Rönicke et al., 2011; Selkoe, 2008). This conclusion is also consistentwith our finding that the synaptotoxicity of each tested Ab variant

was not significantly affected by whether the protein solutionswere allowed to oligomerize for 12 hours before use. Indeed, theresults obtained with “fresh” and “aged” Ab solutions were almostidentical for each of the Ab variants tested.

Recent in vivo work has also highlighted the importance ofMet35 in the neurotoxicity and oxidative stress induced by Ab(Butterfield et al., 2010). These authors used PDAPP transgenic micein which Met35 of Ab (corresponding to Met631 in APP) wassubstituted with leucine that exhibits similar hydrophobicity asmethionine. Met35 substitution prevented the increased oxidativestress but it failed to rescue the deficits in learning and memoryobserved in PDAPPmice. The authors attributed the lack of recoveryfrom memory impairment in their PDAPPM631L mice to microglialactivation and the formation of non-Ab peptides like sAPPa, Jcasp,and C31 (Butterfield and Sultana, 2011).

Previous reports have documented changes in basal synaptictransmission because of Ab42 treatment or deposition, althoughthe underlying mechanisms have not been fully elucidated (seereferences in Malinow et al., 2008). In our hippocampal brain slices,20-minute exposure to 200 nM Ab42WT strongly inhibited LTPwithout affecting the basal transmission recorded before tetanicstimulation, but higher Ab concentrations (�500 nM) also rapidlydiminished pretetanus fEPSPs. However, in autaptic micro-islandsda more specific model for studying basal synaptic trans-missiondneurotransmission was inhibited within the same timeframe with an Ab42WT concentration of only 200 nM, probablybecause the target synapses in this model are more accessible toexogenous Ab. In autaptic hippocampal microcultures, we foundearly defects in presynaptic neurotransmitter release, which mightrepresent the first step in a cascade that leads to synapse losswithin24 hours. In fact, 20-minute exposure to Ab42WT significantlyreduced both EPSC amplitude and mEPSC frequency withoutaffecting mEPSC amplitude, but after 24 hours of exposure, all threeparameters were significantly reduced, suggesting that the earlypresynaptic damage is followed by injury involving both sides ofthe synapse. Similarly, RRP charges were also essentially unchangedafter 20minutes of Ab42WTexposure but dropped significantly after24 hours of treatment. It is possible that Ab42 reduces the fusionefficiency of synaptic vesicles before provoking complete synapticloss, a hypothesis supported by the decreased Pvr we observed after20-minute exposure to Ab42. These early deficits might reflectAb-mediated membrane changes that hinder vesicle fusion ormitochondrial dysfunction resulting in energy production that isinadequate to drive fusion. Our results are consistent with previousobservations which attribute defective presynaptic neurotrans-mitter release as a possible mechanism underlying AD-relatedneurodegeneration (Zhang et al., 2009).

The markedly decreased RRP charge observed after 24-hourAb42 treatments correlates with the significant reductions in syn-aptophysin and PSD-95 expression and dendritic spine density weobserved after 24- and 72-hour treatments. These results are in linewith those of Priller et al. (2007, 2009). In transgenic miceexpressing human presenilin 1 harboring an A246E mutation, theseauthors found depressed synaptic transmission caused by physicalloss of synapses, which was documented by a reduction in the RRPcharge. In other studies, Ab40-treated neurons also displayed earlypresynaptic damage that later extended to postsynaptic structuresas well (Parodi et al., 2010).

Comparative analysis of the early and late effects of the Ab42analogues shows that Ab42 harboring Met35-sulfone and Ab42WT

cause similar impairment of synaptic function, whereas Ab42MO

had no effects after 20-minute exposure, and 24-hour exposureproduced only moderate synaptotoxicity that was far less intensethan that caused by Ab42WT and Ab42MO2. Our data also showedthat reduction in dendritic spine density was evident after 3-hour

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e1312

exposure to Ab42WT and Ab42MO2, but not with Ab42MO. Themildersynaptotoxicity observed after 24- and 72-hour exposure toAb42MO might reflect its conversion to Ab42WT via methioninesulfoxide reductase-mediated reduction of Met-sulfoxide to Met.

Several reports have demonstrated high-affinity bindingbetween Ab and Cu2þ, Zn2þ, and Fe3þ, metals found at high levels inAD amyloid deposits. The neurotoxicity of Ab has been linked to itsability to form complexes with redox-susceptible ions in thesynaptic cleft, particularly Cu2þ (Barnham et al., 2003). The Ab:Cucomplexes generate reactive oxygen species by Fenton chemistry,thereby producing oxidative stress, but Fenton chemistry is prob-ably not involved in the Met35-dependent synaptotoxicity of Ab,because Ab42MO2, which does not participate in the Fenton reaction(Butterfield and Boyd-Kimball, 2005), is as synaptotoxic as Ab42WT.

The differential synaptotoxicity of oxidized and reduced Ab42analogues probably reflects their relative capacities for bindingto or interact with synaptic membranes. This hypothesis is consis-tent with Barnham and colleagues’ (2003) demonstration thatAb42MOdunlike Ab42WTdassumes a peculiar secondary structure(i.e., random coil) in the proximity of the lipid bilayer that renders itincapable of penetrating themembrane. Ab42WT and Ab42MO2 formsimilar secondary structures with similar kinetics (Maiti et al.,2011), so they can reasonably be expected to insert themselves inthe lipid bilayer in the same manner and cause similar effects.Ciccotosto et al. (2011) also showed that the D-enantiomer of Ab42is not neurotoxic. It resembles L-Ab42 in several respects (aggre-gation profiles, other biophysical and biochemical properties), butthe two enantiomers bind phosphatidylserine in different ways,suggesting that the mechanisms by which Ab42 interacts withneuronal membranes are indeed key determinants of its toxicity.Here we report that Ab42WT and Ab42MO2 cross the cell membranesand enter the cells. After 24-hour treatments they are abundantlyfound inside cell bodies and neuronal processes whereas Ab42MO

remains confined outside the neurons. We also found that 24-hourtreatments with 1 mM Ab42WT alters membrane phospholipidorganization in cortical neurons and astrocytes in vitro (data notshown), as revealed by Laurdan 2-photon microscopy (reviewed inGaus et al., 2006), but similar exposure to Ab42MO produced onlyweak, insignificant effects compared with vehicle alone. Thesefindings support the view that Ab42-mediated disruption of thestructural integrity of synaptic membranes might underlie theprotein’s synaptotoxicity.

From amolecular point of view, the different effects of the testedAb42 analogues on the synaptic function might also depend ontheir different ability to activate BAX and caspase-3. The critical roleplayed by BAX in the Ab-mediated inhibition of LTP was recentlydemonstrated by Olsen and Sheng (2012) who reported that LTP isunaffected by Ab in hippocampal slices from BAX knockout mice.Recent reports have also demonstrated the involvement of caspase-3 activation in the alteration of synaptic function. In particular, theintensity and duration of BAX-dependent caspase-3 activationdetermines whether the latter leads to apoptotic cell death orsynaptic depression (Jiao and Li, 2011; Li et al., 2010). The non-apoptotic effect of caspase-3 activation was also responsible for theearly synaptic dysfunction observed in in vivo and ex vivo micemodels of AD (D’Amelio et al., 2010; Jo et al., 2011). We previouslydemonstrated that Ab42MO has significantly milder effects oncaspase-3 activity than Ab42WT (Clementi et al., 2006). In this study,we found that pretreatment of hippocampal brain slices with thepan-caspase inhibitor Z-VADeFMK prevented the LTP impairmentinduced by Ab42MO2. These findings support the hypothesis thatthe different synaptotoxicity of Ab analogues we tested reflectstheir ability to affect caspase-3 activity.

Collectively, data reported in the present report shed light onthe time course and mechanisms underlying the Ab-induced

synaptic failure. Although oxidative stress per se contributessignificantly to AD pathogenesis (see references in Querfurth andLaFerla, 2010) our findings on the remarkably reduced synapto-toxicity of Ab42MOdrelative to Ab42WTdsuggest that Met35oxidation to sulfoxide is a serendipitously protective effect thatlimits the profound synaptic dysfunction provoked by Ab42.

Disclosure statement

None of the authors has any actual or potential conflicts ofinterest to report.

All animal procedures were approved by the Ethics Committeeof the Catholic University and complied with Italian Ministry ofHealth guidelines and with national laws (Legislative decree 116/1992) and European Union guidelines on animal research (No. 86/609/EEC).

Acknowledgements

This workwas supported by grants from Università Cattolica (D1funds) and Italian Ministry of University and research (PRIN 2009grant) to C.G. and from Alzheimer’s Association (IIRG-07-58334) toG.B. The authors thank Mr. Daniele Mezzogori for technical assis-tance, and Dr Panchanan Maiti for preparing dry films of dis-assembled Ab42 analogues.

References

Abramov, E., Dolev, I., Fogel, H., Ciccotosto, G.D., Ruff, E., Slutsky, I., 2009. Amyloid-beta as a positive endogenous regulator of release probability at hippocampalsynapses. Nat. Neurosci. 12, 1567e1576.

Barnham, K.J., Ciccotosto, G.D., Tickler, A.K., Ali, F.E., Smith, D.G., Williamson, N.E.,Lam, Y.H., Carrington, D., Tew, D., Kocak, G., Volitakis, I., Separovic, F., Barrow, C.J.,Wade, J.D., Masters, C.L., Cherny, R.A., Curtain, C.C., Bush, A.I., Cappai, R., 2003.Neurotoxic, redox-competent Alzheimer’s beta-amyloid is released from lipidmembrane by methionine oxidation. J. Biol. Chem. 278, 42959e42965.

Bekkers, J.M., Stevens, C.F., 1991. Excitatory and inhibitory autaptic currents in iso-lated hippocampal neurons maintained in cell culture. Proc. Natl. Acad. Sci.U. S. A. 88, 7834e7838.

Benavides-Piccione, R., Ballesteros-Yáñez, I., DeFelipe, J., Yuste, R., 2002. Corticalarea and species differences in dendritic spine morphology. J. Neurocytol. 31,337e346.

Berman, D.E., Dall’Armi, C., Voronov, S.V., McIntire, L.B., Zhang, H., Moore, A.Z.,Staniszewski, A., Arancio, O., Kim, T.W., Di Paolo, G., 2008. Oligomeric amyloid-beta peptide disrupts phosphatidylinositol-4,5-bisphosphate metabolism. Nat.Neurosci. 11, 547e554.

Bitan, G., Kirkitadze, M.D., Lomakin, A., Vollers, S.S., Benedek, G.B., Teplow, D.B.,2003a. Amyloid b -protein (Ab) assembly: Ab40 and Ab42 oligomerize throughdistinct pathways. Proc. Natl. Acad. Sci. U. S. A. 100, 330e335.

Bitan, G., Tarus, B., Vollers, S.S., Lashuel, H.A., Condron, M.M., Straub, J.E.,Teplow, D.B., 2003b. A molecular switch in amyloid assembly: Met35 andamyloid beta-protein oligomerization. J. Am. Chem. Soc. 125, 15359e15365.

Boutte, A.M., Woltjer, R.L., Zimmerman, L.J., Stamer, S.L., Montine, K.S., Manno, M.V.,Cimino, P.J., Liebler, D.C., Montine, T.J., 2006. Selectively increased oxidativemodifications mapped to detergent-insoluble forms of Ab and b-III tubulin inAlzheimer’s disease. FASEB J. 20, 1473e1483.

Butterfield, D.A., Boyd-Kimball, D., 2005. The critical role of methionine 35 in Alz-heimer’s amyloid b-peptide (1-42)-induced oxidative stress and neurotoxicity.Biochim. Biophys. Acta. 1703, 149e156.

Butterfield, D.A., Galvan, V., Lange, M.B., Tang, H., Sowell, R.A., Spilman, P.,Fombonne, J., Gorostiza, O., Zhang, J., Sultana, R., Bredesen, D.E., 2010. In vivooxidative stress in brain of Alzheimer disease transgenic mice: Requirement formethionine 35 in amyloid b-peptide of APP. Free Radic. Biol. Med. 48, 136e144.

Butterfield, D.A., Sultana, R., 2011. Methionine-35 of Ab(1-42): importance foroxidative stress in Alzheimer disease. J. Amino Acids 2011, 198430.

Calabrese, B., Wilson, M.S., Halpain, S., 2006. Development and regulation ofdendritic spine synapses. Physiology (Bethesda) 21, 38e47.

Cerpa, W., Farías, G.G., Godoy, J.A., Fuenzalida, M., Bonansco, C., Inestrosa, N.C., 2010.Wnt-5a occludes Abeta oligomer-induced depression of glutamatergic trans-mission in hippocampal neurons. Mol. Neurodegener. 5, 3.

Chapman, P.F., White, G.L., Jones, M.W., Cooper-Blacketer, D., Marshall, V.J.,Irizarry, M., Younkin, L., Good, M.A., Bliss, T.V., Hyman, B.T., Younkin, S.G.,Hsiao, K.K., 1999. Impaired synaptic plasticity and learning in aged amyloidprecursor protein transgenic mice. Nat. Neurosci. 2, 271e276.

Ciccotosto, G.D., Tew, D.J., Drew, S.C., Smith, D.G., Johanssen, T., Lal, V., Lau, T.L.,Perez, K., Curtain, C.C., Wade, J.D., Separovic, F., Masters, C.L., Smith, J.P.,

C. Ripoli et al. / Neurobiology of Aging xxx (2012) 1e13 13

Barnham, K.J., Cappai, R., 2011. Stereospecific interactions are necessary forAlzheimer disease amyloid-b toxicity. Neurobiol. Aging 32, 235e248.

Clementi, M.E., Pezzotti, M., Orsini, F., Sampaolese, B., Mezzogori, D., Grassi, C.,Giardina, B., Misiti, F., 2006. Alzheimer’s amyloid b-peptide (1-42) induces celldeath in human neuroblastoma via bax/bcl-2 ratio increase: an intriguing rolefor methionine 35. Biochem. Biophys. Res. Commun. 342, 206e213.

Cuccurazzu, B., Leone, L., Podda, M.V., Piacentini, R., Riccardi, E., Ripoli, C.,Azzena, G.B., Grassi, C., 2010. Exposure to extremely low-frequency (50 Hz)electromagnetic fields enhances adult hippocampal neurogenesis in C57BL/6mice. Exp. Neurol. 226, 173e182.

D’Amelio, M., Cavallucci, V., Middei, S., Marchetti, C., Pacioni, S., Ferri, A.,Diamantini, A., De Zio, D., Carrara, P., Battistini, L., Moreno, S., Bacci, A., Ammas-sari-Teule, M., Marie, H., Cecconi, F., 2010. Caspase-3 triggers early synapticdysfunction in a mouse model of Alzheimer’s disease. Nat. Neurosci. 14, 69e76.

Di Paolo, G., Kim, T.W., 2011. Linking lipids to Alzheimer’s disease: cholesterol andbeyond. Nat. Rev. Neurosci. 12, 284e296.

Dong, J., Atwood, C.S., Anderson, V.E., Siedlak, S.L., Smith, M.A., Perry, G., Carey, P.R.,2003. Metal binding and oxidation of amyloid-b within isolated senile plaquecores: Raman microscopic evidence. Biochemistry 42, 2768e2773.

Fletcher, T.L., Cameron, P., De Camilli, P., Banker, G., 1991. The distribution of syn-apsin I and synaptophysin in hippocampal neurons developing in culture.J. Neurosci. 11, 1617e1626.

Fusco, S., Ripoli, C., Podda, M.V., Chiatamone Ranieri, S., Leone, L., Toietta, G.,McBurney, M.W., Schütz, G., Riccio, A., Grassi, C., Galeotti, T., Pani, G., 2012.A role for neuronal cAMP responsive element binding (CREB)-1 in brainresponses to calorie restriction. Proc. Natl. Acad. Sci. U. S. A. 109, 621e626.

Galea, E., Feinstein, D.L., Reis, D.J., 1992. Induction of calcium-independent nitricoxide synthase activity in primary rat glial cultures. Proc. Natl. Acad. Sci. U. S. A.89, 10945e10949.

Gaus, K., Zech, T., Harder, T., 2006. Visualizing membrane microdomains by Laurdan2-photon microscopy. Mol. Membr. Biol. 23, 41e48.

Gerber, S.H., Rah, J.C., Min, S.W., Liu, X., de Wit, H., Dulubova, I., Meyer, A.C., Rizo, J.,Arancillo, M., Hammer, R.E., Verhage, M., Rosenmund, C., Südhof, T.C., 2008.Conformational switch of syntaxin-1 controls synaptic vesicle fusion. Science321, 1507e1510.

Halpain, S., Hipolito, A., Saffer, L., 1998. Regulation of F-actin stability in dendriticspines by glutamate receptors and calcineurin. J. Neurosci. 18, 9835e9844.

Hsia, A.Y., Masliah, E., McConlogue, L., Yu, G.Q., Tatsuno, G., Hu, K., Kholodenko, D.,Malenka, R.C., Nicoll, R.A., Mucke, L., 1999. Plaque-independent disruption ofneural circuits in Alzheimer’s disease mouse models. Proc. Natl. Acad. Sci.U. S. A. 96, 3228e3333.

Jiao, S., Li, Z., 2011. Nonapoptotic function of BAD and BAX in long-term depressionof synaptic transmission. Neuron 70, 758e772.

Jo, J., Whitcomb, D.J., Olsen, K.M., Kerrigan, T.L., Lo, S.C., Bru-Mercier, G.,Dickinson, B., Scullion, S., Sheng, M., Collingridge, G., Cho, K., 2011. Ab(1-42)inhibition of LTP is mediated by a signaling pathway involving caspase-3, Akt1and GSK-3b. Nat. Neurosci. 14, 545e547.

Kelly, B.L., Vassar, R., Ferreira, A., 2005. b-amyloid-induced dynamin 1 depletion inhippocampal neurons. A potential mechanism for early cognitive decline inAlzheimer disease. J. Biol. Chem. 280, 31746e31753.

Kessels, H.W., Nguyen, L.N., Nabavi, S., Malinow, R., 2010. The prion protein asa receptor for amyloid-b. Nature 466, E3eE4.

Knobloch, M., Farinelli, M., Konietzko, U., Nitsch, R.M., Mansuy, I.M., 2007. Abetaoligomer-mediated long-term potentiation impairment involves protein phos-phatase 1-dependent mechanisms. J. Neurosci. 27, 7648e7653.

Kuo, Y.M., Kokjohn, T.A., Beach, T.G., Sue, L.I., Brune, D., Lopez, J.C., Kalback, W.M.,Abramowski, D., Sturchler-Pierrat, C., Staufenbiel, M., Roher, A.E., 2001.Comparative analysis of amyloid-b chemical structure and amyloid plaquemorphology of transgenic mouse and Alzheimer’s disease brains. J. Biol. Chem.276, 12991e12998.

Lacor, P.N., Buniel, M.C., Chang, L., Fernandez, S.J., Gong, Y., Viola, K.L., Lambert, M.P.,Velasco, P.T., Bigio, E.H., Finch, C.E., Krafft, G.A., Klein, W.L., 2004. Synaptic tar-geting by Alzheimer’s-related amyloid b oligomers. J. Neurosci. 24,10191e10200.

LaFerla, F.M., Oddo, S., 2005. Alzheimer’s disease: Ab, tau and synaptic dysfunction.Trends Mol. Med. 11, 170e176.

Li, S., Jin, M., Koeglsperger, T., Shepardson, N.E., Shankar, G.M., Selkoe, D.J., 2011.Soluble Ab oligomers inhibit long-term potentiation through a mechanisminvolving excessive activation of extrasynaptic NR2B-containing NMDA recep-tors. J. Neurosci. 31, 6627e6638.

Li, Z., Jo, J., Jia, J.M., Lo, S.C., Whitcomb, D.J., Jiao, S., Cho, K., Sheng, M., 2010. Caspase-3 activation via mitochondria is required for long-term depression and AMPAreceptor internalization. Cell 141, 859e871.

Maiti, P., Piacentini, R., Ripoli, C., Grassi, C., Bitan, G., 2011. Surprising toxicity andassembly behaviour of amyloid b-protein oxidized to sulfone. Biochem. J. 433,323e332.

Malinow, R., Hsieh, H., Wei, W., 2008. Impact of beta amyloid on excitatory synaptictransmission and plasticity. In: Selkoe, D.J., Triller, A., Christen, Y. (Eds.), SynapticPlasticity and the Mechanism of Alzheimer’s Disease. Springer-Verlag, BerlinHeidelberg, pp. 63e68.

Naslund, J., Schierhorn, A., Hellman, U., Lannfelt, L., Roses, A.D., Tjernberg, L.O.,Silberring, J., Gandy, S.E., Winblad, B., Green Gard, P., Nordstedt, C., Terenius, L.,1994. Relative abundance of Alzheimer Ab amyloid peptide variants in Alz-heimer disease and normal aging. Proc. Natl. Acad. Sci. U. S. A. 91, 8378e8382.

Oliveira, T.G., Chan, R.B., Tian, H., Laredo, M., Shui, G., Staniszewski, A., Zhang, H.,Wang, L., Kim, T.W., Duff, K.E., Wenk, M.R., Arancio, O., Di Paolo, G., 2010.Phospholipase d2 ablation ameliorates Alzheimer’s disease-linked synapticdysfunction and cognitive deficits. J. Neurosci. 30, 16419e16428.

Olsen, K.M., Sheng, M., 2012. NMDA receptors and BAX are essential for Abimpairment of LTP. Sci. Rep. 2, 225.

Parodi, J., Sepúlveda, F.J., Roa, J., Opazo, C., Inestrosa, N.C., Aguayo, L.G., 2010. b-amyloid causes depletion of synaptic vesicles leading to neurotransmissionfailure. J. Biol. Chem. 285, 2506e2514.

Penzes, P., Cahill, M.E., Jones, K.A., VanLeeuwen, J.E., Woolfrey, K.M., 2011. Dendriticspine pathology in neuropsychiatric disorders. Nat. Neurosci. 14, 285e293.

Piacentini, R., Civitelli, L., Ripoli, C., Marcocci, M.E., De Chiara, G., Garaci, E.,Azzena, G.B., Palamara, A.T., Grassi, C., 2011. HSV-1 promotes Ca2þ-mediatedAPP phosphorylation and Ab accumulation in rat cortical neurons. Neurobiol.Aging 32, 2323.e13e2323.e26.

Piacentini, R., Gangitano, C., Ceccariglia, S., Del Fà, A., Azzena, G.B., Michetti, F.,Grassi, C., 2008a. Dysregulation of intracellular calcium homeostasis isresponsible for neuronal death in an experimental model of selective hippo-campal degeneration induced by trimethyltin. J. Neurochem. 105, 2109e2121.

Piacentini, R., Ripoli, C., Leone, L., Misiti, F., Clementi, M.E., D’Ascenzo, M.,Giardina, B., Azzena, G.B., Grassi, C., 2008b. Role of methionine 35 in theintracellular Ca2þ homeostasis dysregulation and Ca2þ-dependent apoptosisinduced by amyloid b-peptide in human neuroblastoma IMR32 cells.J. Neurochem. 107, 1070e1082.

Podda, M.V., D’Ascenzo, M., Leone, L., Piacentini, R., Azzena, G.B., Grassi, C., 2008.Functional role of cyclic nucleotide-gated channels in rat medial vestibularnucleus neurons. J. Physiol. 586, 803e815.

Priller, C., Dewachter, I., Vassallo, N., Paluch, S., Pace, C., Kretzschmar, H.A., VanLeuven, F., Herms, J., 2007. Mutant presenilin 1 alters synaptic transmission incultured hippocampal neurons. J. Biol. Chem. 282, 1119e1127.

Priller, C., Mitteregger, G., Paluch, S., Vassallo, N., Staufenbiel, M., Kretzschmar, H.A.,Jucker, M., Herms, J., 2009. Excitatory synaptic transmission is depressed incultured hippocampal neurons of APP/PS1 mice. Neurobiol. Aging 30, 1227e1237.

Puzzo, D., Privitera, L., Fa’, M., Staniszewski, A., Hashimoto, G., Aziz, F., Sakurai, M.,Ribe, E.M., Troy, C.M., Mercken, M., Jung, S.S., Palmeri, A., Arancio, O., 2011.Endogenous amyloid-b is necessary for hippocampal synaptic plasticity andmemory. Ann. Neurol. 69, 819e830.

Puzzo, D., Privitera, L., Leznik, E., Fà, M., Staniszewski, A., Palmeri, A., Arancio, O.,2008. Picomolar amyloid-b positively modulates synaptic plasticity andmemory in hippocampus. J. Neurosci. 28, 14537e14545.

Pyott, S.J., Rosenmund, C., 2002. The effects of temperature on vesicular supply andrelease in autaptic cultures of rat and mouse hippocampal neurons. J. Physiol.539, 523e535.

Querfurth, H.W., LaFerla, F.M., 2010. Alzheimer’s disease. N. Engl. J.Med. 362, 329e344.Rahimi, F., Maiti, P., Bitan, G., 2009. Photo-induced cross-linking of unmodified

proteins (PICUP) applied to amyloidogenic peptides. J. Vis. Exp. pii:1071.Resende, R., Ferreiro, E., Pereira, C., Resende de Oliveira, C., 2008. Neurotoxic effect

of oligomeric and fibrillar species of amyloid-beta peptide 1-42: involvement ofendoplasmic reticulum calcium release in oligomer-induced cell death.Neuroscience 155, 725e737.

Rönicke, R., Mikhaylova, M., Rönicke, S., Meinhardt, J., Schröder, U.H., Fändrich, M.,Reiser, G., Kreutz, M.R., Reymann, K.G., 2011. Early neuronal dysfunction byamyloid b oligomers depends on activation of NR2B-containing NMDA recep-tors. Neurobiol. Aging 32, 2219e2228.

Schell, M.J., Irvine, R.F., 2006. Calcium-triggered exit of F-actin and IP3 3-kinase Afrom dendritic spines is rapid and reversible. Eur. J. Neurosci. 24, 2491e2503.

Selkoe, D.J., 2002. Alzheimer’s disease is a synaptic failure. Science 298, 789e791.Selkoe, D.J., 2008. Soluble oligomers of the amyloid b-protein impair synaptic

plasticity and behavior. Behav. Brain Res. 192, 106e113.Sondag, C.M., Dhawan, G., Combs, C.K., 2009. Beta amyloid oligomers and fibrils

stimulate differential activation of primary microglia. J. Neuroinflammation 6, 1.Stern, E.A., Bacskai, B.J., Hickey, G.A., Attenello, F.J., Lombardo, J.A., Hyman, B.T.,

2004. Cortical synaptic integration in vivo is disrupted by amyloid-b plaques.J. Neurosci. 24, 4535e4540.

Stine Jr., W.B., Dahlgren, K.N., Krafft, G.A., LaDu, M.J., 2003. In vitro characterizationof conditions for amyloid- b peptide oligomerization and fibrillogenesis. J. Biol.Chem. 278, 11612e11622.

Ting, J.T., Kelley, B.G., Lambert, T.J., Cook, D.G., Sullivan, J.M., 2006. Amyloidprecursor protein overexpression depresses excitatory transmission throughboth presynaptic and postsynaptic mechanisms. Proc. Natl. Acad. Sci. U. S. A.104, 353e358.

Wang, Q., Walsh, D.M., Rowan, M.J., Selkoe, D.J., Anwyl, R., 2004. Block of long-termpotentiation by naturally secreted and synthetic amyloid b-peptide in hippo-campal slices is mediated via activation of the kinases c-Jun N-terminal kinase,cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well asmetabotropic glutamate receptor type 5. J. Neurosci. 24, 3370e3378.

Wei, W., Nguyen, L.N., Kessels, H.W., Hagiwara, H., Sisodia, S., Malinow, R., 2009.Amyloid b from axons and dendrites reduces local spine number and plasticity.Nat. Neurosci. 13, 190e196.

Yan, Y., Wang, C., 2006. Ab42 is more rigid than Ab40 at the C terminus: implicationsfor Ab aggregation and toxicity. J. Mol. Biol. 364, 853e862.

Zhang, C., Wu, B., Beglopoulos, V., Wines-Samuelson, M., Zhang, D., Dragatsis, I.,Südhof, T.C., Shen, J., 2009. Presenilins are essential for regulating neurotrans-mitter release. Nature 460, 632e636.