Amyloid-β oligomers induce differential gene expression in adult human brain slices

Amyloid-� Oligomers Induce Differential Gene Expression inAdult Human Brain Slices*□S

Received for publication, August 26, 2011, and in revised form, December 27, 2011 Published, JBC Papers in Press, January 10, 2012, DOI 10.1074/jbc.M111.298471

Adriano Sebollela‡1, Leo Freitas-Correa‡, Fabio F. Oliveira‡, Andrea C. Paula-Lima‡2, Leonardo M. Saraiva‡,Samantha M. Martins‡, Louise D. Mota§, Cesar Torres§, Soniza Alves-Leon¶, Jorge M. de Souza�, Dirce M. Carraro§,Helena Brentani§3, Fernanda G. De Felice‡, and Sergio T. Ferreira‡4

From the ‡Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21944-590, Brazil, the§International Center of Education and Research, A. C. Camargo Hospital, São Paulo SP 01509, Brazil, and the ¶Division ofNeurology/Epilepsy Program and the �Division of Neurosurgery, Clementino Fraga Filho University Hospital, Federal University ofRio de Janeiro, Rio de Janeiro RJ 21941-913, Brazil

Background: Soluble A� oligomers (A�Os) have been increasingly proposed as the cause of synapse failure and cognitivedysfunction in Alzheimer disease.Results: Sublethal A�O concentrations induce changes in gene expression in adult human brain slices.Conclusion: A�Os impact transcription in important neuronal pathways preceding neurodegeneration.Significance: Results establish early mechanisms involved in A�O-triggered neuronal dysfunction in a novel human-derivedexperimental model.

Cognitive decline in Alzheimer disease (AD) is increasinglyattributed to the neuronal impact of soluble oligomers of theamyloid-� peptide (A�Os). Current knowledge on the molecu-lar and cellular mechanisms underlying the toxicity of A�Osstems largely from rodent-derived cell/tissue culture experi-ments or from transgenic models of AD, which do not necessar-ily recapitulate the complexity of the human disease. Here, weused DNA microarray and RT-PCR to investigate changes intranscription in adult human cortical slices exposed to sublethaldoses of A�Os. The results revealed a set of 27 genes thatshowed consistent differential expression upon exposure ofslices from three different donors to A�Os. Functional classifi-cation of differentially expressed genes revealed that A�Osimpact pathways important for neuronal physiology and knowntobedysregulated inAD, including vesicle trafficking, cell adhe-sion, actin cytoskeleton dynamics, and insulin signaling. Mostgenes (70%) were down-regulated by A�O treatment, suggest-ing a predominantly inhibitory effect on the correspondingpathways. Significantly, A�Os induced down-regulation of syn-aptophysin, a presynaptic vesiclemembrane protein, suggestinga mechanism by which oligomers cause synapse failure. Theresults provide insight into earlymechanisms of pathogenesis ofAD and suggest that the neuronal pathways affected by A�Os

may be targets for the development of novel diagnostic or ther-apeutic approaches.

Alzheimer disease (AD),5 the most common form of demen-tia in the elderly, currently affects more than 35 million peopleworldwide (1). AD is characterized clinically by early memorydysfunction followed by progressive cognitive impairment (1,2). Soluble oligomers of the amyloid-� peptide (��s) are cur-rently considered central players in the pathogenesis of AD(3–6). A�Os initially were found to act as potent cell surfaceligands, instigating rapid inhibition of long-term potentiationand alterations in neuronal cell signaling (7). Those findingshave been followed by reports on a variety of neurotoxic effectsof A�Os, including Tau hyperphosphorylation (8), calciumdysregulation and oxidative stress (9–13), blockade of fastaxonal transport (14, 15), altered turnover of neuronal recep-tors involved in synaptic plasticity (e.g. 16–20), and synapse loss(17, 19).Although it is now generally accepted that A�Os cause syn-

apse dysfunction, the molecular and cellular mechanismsunderlying their toxicity are still poorly understood. In part,this is because most studies of A�O toxicity have utilizedrodent-derived cell/tissue cultures or transgenic models of AD,which do not necessarily recapitulate the complexity of thehuman disease. Previous studies have indicated changes in neu-ronal gene expression inADbrain (e.g. 21–23) and in transgenicmouse models of AD (e.g. 24, 25). However, a central issue thatremains to be determined is whether the changes in geneexpression detected in those studies represent a late conse-quence of cerebral inflammation and neurodegeneration or adirect and early effect of A�Os on neuronal gene expression.

* This work was supported by grants from the Human Frontiers Science Pro-gram and The John Simon Guggenheim Foundation (to F. G. D.-F.) and theConselho Nacional de Desenvolvimento Cientifico e Tecnológico, Funda-ção de Amparo à Pesquisa do Estado do Rio de Janeiro, and National Insti-tute for Translational Neuroscience (to S. T. F. and F. G. D.-F.).

□S This article contains supplemental Figs. 1– 4 and Tables 1– 4.1 Recipient of a postdoctoral fellowship from the Conselho Nacional de

Desenvolvimento Cientifico e Tecnologico. To whom correspondencemay be addressed. E-mail: [email protected].

2 Present address: Center for Molecular Studies of the Cell, Faculty of Den-tistry, University of Chile.

3 Present address: Inst. of Psychiatry-LIM 23, University of Sao Paulo School ofMedicine, Sao Paulo, Brazil.

4 To whom correspondence may be addressed. E-mail: [email protected].

5 The abbreviations used are: AD, Alzheimer disease; A�, amyloid-�; A�O,amyloid-� oligomer; qRT-PCR, quantitative reverse transcriptase PCR; MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GFAP, glialfibrillary acidic protein; LTD, long-term depression.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 10, pp. 7436 –7445, March 2, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

7436 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 10 • MARCH 2, 2012

http://www.jbc.org/cgi/content/full/M111.298471/DC1

Here, we report an investigation of changes in gene expres-sion induced by A�Os using human adult cortical slices as anovel experimental model to study the neuronal impact ofA�Os. Using a combination of DNA microarray analysis andquantitative reverse transcriptase PCR (qRT-PCR), we foundthat sublethal concentrations of A�Os induce changes inexpression of genes classified into several pathways importantfor neuronal physiology, including synaptophysin, a presynap-tic vesicle membrane protein that has been implicated in syn-aptic failure in AD (26–28). A detailed understanding of themechanisms by which A�Os affect neuronal gene expressionand function may uncover early biomarkers of AD pathologyand lead to the development of novel and effective therapeuticsto prevent or halt the progression of AD.

EXPERIMENTAL PROCEDURES

Adult Human Cortical Slices—Adult human brain corticalfragments were obtained frompatients with pharmacoresistanttemporal lobe epilepsy submitted to hippocampectomy andanterior temporal lobectomy for removal of epileptic focidetected by magnetic resonance imaging (MRI). Only healthycortical tissue removed from the area approached as surgicalaccess to the target hippocampus was used for culture pur-poses. The health of cortical tissues was determined based onthe structural/anatomical integrity revealed by MRI (supple-mental Fig. 1), macroscopic appearance, and lack of correlationwith epileptic activity in scalp video-EEG monitoring. Donors(six men and four women) were 34 � 9 years old and gavewritten informed consent for use of brain tissue that wouldotherwise have been discarded. All procedures were approvedand regulated by the National Committee for Research Ethics(CONEP) of the Brazilian Ministry of Health (Protocol0069.0.197.000-05).Tissue was placed immediately in transport medium (50%

(v/v) Hanks’ balanced salt solution containing 10 mMHEPES, 3mg/ml glucose, and 50�g/ml gentamicin diluted inNeurobasalA medium supplemented previously with 2% B27 and 0.5 mM

glutamine) and transported to the laboratory within 30 min ofremoval. Under sterile conditions, meninges were carefullyremoved, and 0.3-mm slices were prepared using a McIlwaintissue chopper. Slices were plated on 24-well plates (2–3 slices/well) in Neurobasal A/B27 (Invitrogen) medium with antibiot-ics. One-third of the medium was replaced after 3 days in cul-ture. After 5 days at 37 °C in a 5% CO2 atmosphere, cultureswere treated with vehicle or A�Os (500 nM) and further incu-bated at 37 °C for 12 h. Cell viability in cultured slices was eval-uated by Live/Dead and MTT reduction assays as describedbelow.DissociatedHippocampalNeuronalCultures—Hippocampal

neuronal cultures were prepared from 18-day-old rat embryosas described previously (29, 30). Briefly, hippocampi were dis-sected in PBS-glucose and mechanically dissociated, and cellswere plated onto poly-L-lysine-coatedwells at a density of 1.5�106 cells/well (for 35-mm wells) in Neurobasal/B27 mediumwith antibiotics. All procedures were approved by and followedthe guidelines of the Institutional Animal Care and UtilizationCommittee of the Federal University of Rio de Janeiro (ProtocolIBQM 022). After 21 days at 37 °C in a 5% CO2 atmosphere,

cultures were treated with vehicle or A�Os and further incu-bated at 37 °C for 12 or 24 h.A�O Preparation—A�Os were prepared in PBS (31) with

minor modifications as described previously (32) using A�42peptide from Bachem (Torrance, CA). The preparation wascentrifuged at 14,000 � g for 10 min at 4 °C to remove anyinsoluble aggregates, and the supernatant containing solubleA�Oswas transferred to clean tubes and stored at 4 °C. Proteinconcentration was determined using the BCA assay (Pierce).Oligomer solutions were used within 24 h of preparation. Rou-tine characterization of oligomer preparations was performedby size-exclusion chromatography andWestern blot and, occa-sionally, by transmission electron microscopy (supplementalFig. 2). Collectively, the results indicate that our preparationcomprises soluble oligomeric species including dimers, trimers,tetramers, and higher molecular mass oligomers of �50–180kDa, ranging in diameter from �1.5 to 3.5 nm.Live/Dead Viability Assay—Cell viability in cultured slices

from three different donors was determined using the Live/Dead assay (Invitrogen). At different days in vitro, slices wereexposed to dyes (4 �M calcein and 2 �M ethidium homodimer)for 40min, washed twice with ice-cold PBS, and cut into 12-�msections for imaging on a Nikon Eclipse TE300 microscope.Live or dead neurons were identified by green calcein fluores-cence or red ethidium fluorescence, respectively. Percentagesof live neurons are expressed relative to the total number ofcells (determined by DAPI staining) in each slice.Immunohistochemistry—After 4 days in culture, brain slices

were fixed in 4% paraformaldehyde and double stained formature neurons (mouse anti-NeuN, Chemicon; 1:100) andastrocytes (rabbit anti-GFAP, Dako; 1:200). Alexa 488 andAlexa 594 conjugates were used as secondary antibodies(Molecular Probes; 1:200). DAPI (Sigma) was used to visualizenuclei. Antibody incubations were performed using free float-ing sections (treated previously with 0.1 M citrate buffer, pH 6,at 60 °C for 5 min and blocked with 5% BSA, 5% normal goatserum, and 1% Triton X-100 for 6 h at room temperature) inPBS containing 1% Triton X-100. Primary antibodies werediluted in blocking solution and incubated for 48 h at 4 °C,followed by incubation with Alexa-conjugated secondary anti-bodies for 24 h at 4 °C. Tissue autofluorescence was quenchedby incubation with 0.06% potassium permanganate for 10 minat room temperature.MTT Assay—Hippocampal slices were incubated for 24 h in

the presence of A�Os at different concentrations, and cell via-bility was analyzed by the MTT reduction assay carried out asdescribed previously (13). Briefly, slices were incubated with a0.5mg/ml solution ofMTTat 37 °C for 4 h to allow reduction toformazan blue by metabolically active cells. Cells were thenlysed, and formazan crystals were solubilized by incubation in0.01 NHCl containing 10%SDS for 24 h under agitation at roomtemperature. Optical density at 570 nm was measured in aThermoMax Microplate reader.RNA Extraction and Amplification—Total RNA from hip-

pocampal cultures was extracted with TRIzol (Invitrogen) fol-lowing themanufacturer’s instructions.Onemilliliter of TRIzolwas used to extract RNA from 1.5 � 106 cells. The purity andintegrity of RNApreparations were checked by the 260/280 nm

A�O-induced Changes in Gene Expression in Human Brain Slices

MARCH 2, 2012 • VOLUME 287 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7437





absorbance ratio and by agarose gel electrophoresis. Only prep-arations with 260/280 nm ratios � 1.8 and no signs of RNAdegradation were used. mRNA from human cortical slices wasobtained using an RNA amplification protocol. After treatmentwith A�Os, slices were washed twice with PBS and kept inRNAlater (Ambion) at �20 °C until use. For total RNA extrac-tion, tissue was ground using an electric homogenizer, andRNA was purified using an RNeasy mini kit (Qiagen). TotalRNA was then used as a template in a two-round linear ampli-fication procedure based on T7-driven amplification asdescribed previously (33). The quality of amplified RNAs waschecked by agarose gel electrophoresis upon visualization withethidium bromide (supplemental Fig. 3). All samples presenteda smear between 300 and 700 bp and no signs of RNAdegradation.Microarray Analysis—Ten �g of amplified RNA from each

sample (three different donors, each used for both control andA�O-treated conditions, corresponding to six paired samplesin total) were used as template for cDNA synthesis. cDNA sam-ples were labeled indirectly with Alexa Fluor� 555 or AlexaFluor� 647 reactive dye (Invitrogen) and IMPROM II reversetranscriptase (Promega) in a reverse transcriptase reaction andpurified as described (34). Hybridization reactions were per-formed in duplicatewith dye swapping using a humanuniversal4,800 chip (35), with excellent agreement between duplicates.After washing, slides were scanned on a confocal laser scanner(ScanArray Express, PerkinElmer Life Sciences), and data wereextracted with ScanArray Express software. Self-self hybridiza-tion was utilized to define cutoff limits for differential geneexpression (36). The percentile used was 0.95.A list of differentially expressed genes induced byA�O treat-

mentwas used to identify the affected biological pathways usingthe Gene Set Analysis Toolkit V2 (37). The gene list used asinput for the Kegg pathway analysis comprised both up- anddown-regulated genes present at the intersections between atleast two samples. The parameters used were as follows: orga-nism, Homo sapiens; ID type, gene_symbol; reference set,Entrez Gene; significance level, 0.05; statistics test, hypergeo-metric;multiple test correction, Benjamini andHochberg;min-imum genes per category, 2.Quantitative RT-PCRAssays—Quantitative expression anal-

ysis of genes of interestwas performed by quantitative real-timePCRon anApplied Biosystems 7500 real-time PCR systemwiththe Power SYBR kit (Applied Biosystems). For human tissue,cDNAconverted fromamplifiedRNAwas used as the template.This procedure has been found not to introduce any bias onrelative gene expression measurement (38). Three referencehuman genes, glyceraldehyde-3-phosphate dehydrogenase(GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT),and �-actin (ACTB), were used to generate a single normaliza-tion factor as described (39).For rat neuronal cultures, template cDNAwas prepared from

total RNA (1 �g) using 50 pmol of oligo dT20 and the Super-script III First Strand cDNA kit (Invitrogen), and ACTB orGAPDH was used as the endogenous control. Cycle threshold(Ct) values were used to calculate -fold changes in gene expres-sion using the 2��Ct method (40). In all cases, reactions wereperformed in 20-�l reaction volumes.

Western Blotting—Mature rat embryonic hippocampal cul-tures were treated with A�Os (500 nM) or vehicle for 12 or 24 h,rinsed with PBS, and lysed in buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, 1%Triton X-100, 10% glycerol, and HaltTM protease inhibitorsmixture (Thermo Fisher Scientific, Rockford, IL). Human brainslice extracts were prepared using the same buffer in the ratio of50 �l/slice. The protein content in the extracts was determinedusing the BCATM protein assay kit (Thermo Fisher Scientific).Extracts (100 �g of protein/lane) were resolved on 12% SDS-PAGE. Proteins were transferred to nitrocellulose membranes(HybondTM-C Extra, AmershamBiosciences) for 90min at 100V. Membranes were blocked with 3% BSA in Tris-bufferedsaline/Tween 20 (TBS-T: 10 mM Tris, pH 7.2, 150 mM NaCl,and 0.1% Tween 20), followed by overnight incubation withanti-synaptophysin monoclonal antibody (5 �g/ml; Sigma-Al-drich) and polyclonal anti-cyclophilin B (0.01�g/ml; Abcam) at4 °C. After washing with TBS-T, immunoreactivity was visual-ized using peroxidase-conjugated anti-rabbit IgG secondaryantibody (1:10,000 dilution; Zymed Laboratories Inc., Carlsbad,CA) for cyclophilin B detection (used as a loading control) andanti-mouse IgG secondary antibody (1:50,000 dilution; Amer-sham Biosciences) for synaptophysin detection. SuperSignal�West Pico chemiluminescent detection (Thermo Fisher Scien-tific)wasused forvisualization.Densitometric scanningandquan-tification were carried out using NIH ImageJ (Windows version).

RESULTS

Exposure to a Sublethal A�O Concentration Alters GeneExpression in Human Cortical Slices—Organotypic cultures ofpost-mortem human cortex have been previously prepared(41). Here, starting from ex vivo human cortical tissue, wedeveloped a novel experimental model to investigate the neu-ronal impact of A� oligomers. Slices from healthy cortical tis-sue obtained from adults submitted to surgical removal of hip-pocampal epileptic foci weremaintained successfully in culturefor up to 25 days with cell viability greater than 50% (Fig. 1).Semiquantitative analysis of cell types in slices from three dif-ferent donors revealed an average of 60% neurons and 21%GFAP-positive cells in cortical human slices cultured for 4 days.Previous studies have shown that exposure of dissociated neu-ronal cultures (for up to 24 h) to A�Os at submicromolar con-centrations causes neuronal dysfunction in the absence of celldeath (8, 17) (reviewed in Ref. 5). Consistent with those find-ings, no change in cell viability was detected in human corticalslices exposed to A�Os (500 nM) for 24 h compared with con-trol, vehicle-treated slices (supplemental Fig. 4).

Following treatment with A��, mRNA was extracted fromslices and hybridization was performed on a customized cDNAplatform containing 4608 human genes (35). Microarray anal-ysis revealed significant alterations in gene expression inducedby A�Os compared with paired vehicle-treated slices from thesame donors (Fig. 2). Considering all of the genes identified asup- or down-regulated that were present in at least two inde-pendent donors, a total of 345 genes were found to be differen-tially expressed upon A�O treatment (72% of which weredown-regulated (Fig. 2A)), corresponding to �7% of the totalnumber of genes represented on the chip. When the intersec-





tion between the three independent donors was taken intoaccount, 27 genes showed consistent alteration in expression(Fig. 2B). Again, most of those genes (70%) were down-regu-lated (Fig. 2C). Complete lists of up- and down-regulated genes,with -fold change values for each independent donor, areshown in supplemental Tables 1 and 2, respectively.Functional Classification of Differentially Expressed Genes in

A�O-treated Slices—To assign biological functions to the list ofA�O-induced differentially expressed genes, we mapped bothup- and down-regulated genes present in hybridizations fromat least two donors to specific biological Kegg pathways (42), anapproach known to add confidence to the results found forexpression of individual genes (43). This analysis revealed 18pathways that were significantly over-represented (p � 0.05) inA�O-treated tissue (Fig. 3 and supplemental Table 3). Signifi-cantly, six of those pathways (“SNARE interaction in vesiculartransport,” “Alzheimer’s disease,” “axon guidance,” “long-termdepression,” “regulation of actin cytoskeleton,” and “focal adhe-sion”) have been implicated in themechanisms of pathogenesisin the central nervous system. Of considerable interest wasfinding the “insulin-signaling pathway” among the over-repre-sented pathways, as impaired neuronal insulin signaling hasbeen linked recently toADpathogenesis and, specifically, to theimpact of A�Os (18, 19). Another over-represented pathwaypreviously associated with energy metabolism and AD patho-genesis is the “adipocytokine-signaling pathway”, as leptin, anadipocytokine involved in fatty acid metabolism, has recentlybeen reported to control A� levels (44). It is noteworthy that,among the 41 distinct genesmapped to the 18 over-representedpathways (representing 12% of the total number of differen-

tially expressed genes), 63% were down-regulated by A�Os.Functional classification analysis thus suggests that A�Osdown-regulate expression of genes crucial for proper neuronalfunction and plasticity, establishing organotypic cultures ofadult human brain cortex as a novel experimental model thateffectively recapitulates the association between molecularabnormalities and clinical manifestations of AD.Validation of A�O-induced Changes in Gene Expression—

Selected differentially expressed genes identified bymicroarrayanalysis were validated by qRT-PCR using the same set of cor-tical slice samples plus four extra pairs of A�O- and vehicle-treated samples obtained from independent donors notincluded in the microarray analysis. The basic criterion used toselect genes for qRT-PCR analysis was that they should be pres-ent at the intersection between the three donors used in themicroarray analysis (Fig. 2C and supplemental Tables 1 and 2).mRNA levels of 4 of 6 up-regulated annotated genes and 11 of17 down-regulated annotated genes were determined directlyby qRT-PCR (Fig. 4), corresponding to 65% of the differentiallyexpressed genes identified at the intersection among threedonors in the microarray data (Fig. 2C). Two additional genestested (rbed and prlr) rendered inconclusive results because ofvery low basal expression levels. Primer sequences for all of thegenes can be found in supplemental Table 4. Differentialexpressionwas confirmed by qRT-PCR for five down-regulatedgenes and one up-regulated gene (Fig. 4).Synaptophysin Down-regulation atmRNAand Protein Levels

in A�O-treated Hippocampal Neuronal Cultures and HumanBrain Slices—Because synaptophysin has been implicated insynapse loss in AD (26, 45, 46) and its expression was markedly

FIGURE 1. Cell viability in cultured slices from ex vivo adult human cortex. After 25 days in vitro, slices were incubated with calcein and ethidium homodimerand imaged. A–C, representative images of a slice. A, bright field. Scale bar: 400 nm. B, ethidium homodimer � DAPI merged image. C, calcein � ethidiumhomodimer merged image. Dead cells can be readily identified among total (DAPI-stained) cells by red ethidium homodimer staining (B), in contrast with greencalcein fluorescence emitted by live cells (C). D, cell viability in slices obtained from a single donor. Viability averaged 62 � 1.4% for this particular experiment.E, no significant reduction in viability was noted after several days in culture using slices prepared from three different donors. F, immunohistochemistry ofslices after 4 days in culture showing neurons (green, labeled using neuronal marker NeuN) and astroglial cells (red, labeled using anti-GFAP antibody). Scale bar:40 nm. Semiquantitative analysis of cell types in slices from three different donors revealed an average of 60% neurons and 21% GFAP-positive cells in corticalhuman slices cultured for 4 days.







down-regulated byA�Os (�50%decrease comparedwith vehi-cle-treated slices (Fig. 4)), we further investigated the effect ofshort-term exposure to A�Os (500 nM) on synaptophysin levelsusing mature rat hippocampal neuronal cultures. Consistentwith the finding in human brain slices, exposure of neuronalcultures to A�Os for 12 h caused a significant reduction insynaptophysin mRNA level (Fig. 5A). Although synaptophysinprotein levels were not affected by A�Os at 12 h of exposure, alonger (24 h) incubation caused a significant reduction in syn-aptophysin (Fig. 5B), indicating that A�O-induced down-regu-lation of synaptophysin mRNA is followed by a comparablereduction in the protein level. Importantly, corroborating ourmicroarray/qPCR findings, we also detected down-regulationof synaptophysin protein levels in A�O-treated human brainslices obtained from three new independent donors (Fig. 5C).

DISCUSSION

Changes in gene expression have been reported as part of ADpathology, but a general consensus is still lacking as to whichgenes or cellular pathways are primarily affected, especially at

early stages of the disease. Previous studies have relied largelyon the analysis of post-mortem material, making it difficult todistinguish whether changes in transcription were eliciteddirectly byA�� toxic signaling orwere a consequence of eventstaking place at later stages of the disease, including brain

FIGURE 2. Differentially expressed genes in A�O-treated human corticalslices. A, a total of 345 genes (72% down-regulated) were found to be differ-entially expressed in A�O-treated slices. B, Venn diagrams showing the totalnumber of up- and down-regulated genes in samples from three differentdonors (labeled S1, S2, and S3). C, heat map for genes found to be up- (red) ordown-regulated (green) by A�Os in samples from the three independentdonors (S1–S3). The color code (see scale bar) reflects -fold change inexpression.

FIGURE 3. Functional classification of up- and down-regulated genes inA�O-treated human cortical slices. Up- and down-regulated genes at theintersection of at least two donors were used to identify over-representedKegg pathways (p � 0.05). Genes classified into each over-represented path-way (listed on the right) are marked with the same symbol as the correspond-ing pathway.

FIGURE 4. qRT-PCR analysis of A�O-induced differential gene expression.cDNA from the three donors used in the microarray analysis plus four samplesfrom additional independent donors were used. Expression levels weredetermined by relative expression, calculated using the gnorm method (see“Experimental Procedures”). Three different normalizer genes were used(ACTB, HPRT, and GAPDH). Each data point (gene or donor) reflects the meanvalue from an independent determination (for an individual donor) per-formed at least in triplicate. Down-regulated genes (indicated by microarrayanalysis) are represented by green symbols: synaptophysin (SYP), cullin 3(CUL3), vaccinia-related kinase 3 (VRK3), heterogeneous nuclear ribonucleo-protein A1 (HNRPA1), C2 and WW domain-containing E3 ubiquitin proteinligase 1 (HECW1), casein kinase 2 (CSNK2A2), asparaginyl tRNA synthetase 2(NARS2), Bcl-2-modifying factor (BMF), HEAT repeat containing 5A (HEATR5A),neurochondrin (NCDN), and adaptor-related protein complex 3, beta 2 sub-unit (AP3B2). Up-regulated genes (indicated by microarray analysis) are rep-resented by red symbols: cytoplasmic FMR1-interacting protein 1 (CYF1P1),cell division cycle 14 (CDC14A), taste receptor 2 (TAS2R49), and vesicle-asso-ciated membrane protein 3 (VAMP3). Medians are denoted by horizontal solidlines.



inflammation and neurodegeneration. In the present study, wepresent the first demonstration that A��s (used at a sublethalconcentration) markedly affect gene expression in the absenceof overt neurodegeneration in adult human brain slices inculture.Analyses of changes in gene expression in post-mortem AD

brain have yielded controversial results. For example, up-regu-lation of tumor suppressor genes (21) and down-regulation ofretromer trafficking complex genes (22) have each beenreported independently as the most significant transcriptionalalteration inADhippocampus. Similarly, in cortical tissue fromAD patients, expression of both immunity-relatedMHC II (47)and calcium-signaling genes (48) were found to be dysregu-lated. More recently, an elegant study employing cultivatedslices from post-mortem AD brain (23) described significantalterations in the expression of genes related to synaptic activityand �-amyloid processing in prefrontal cortex in the presymp-tomatic stage, when plaque pathology was not detected. Inaddition to post-mortem AD brain, transgenic mouse modelsof AD have been used to detect gene expression alterationsrelevant to AD pathogenesis. Again, there is no consensusamong transcriptional alterations reported in those studies. Forexample, Reddy et al. (25) found up-regulation of genesinvolved in mitochondrial energy metabolism in animalsexpressing human APP harboring an AD-associated mutation(Tg2576 mice), whereas the down-regulation of genes impor-tant for memory consolidation was reported in a study using adouble transgenic mouse model (APP � PS1 human trans-genes) (24). In addition to technical considerations (e.g. differ-ent mouse models or DNAmicroarray platforms employed), itis likely that differences between specificmolecular and cellularevents underlying AD pathogenesis and findings in animalmodels or in post-mortem tissue contributed to the differencesin outcome in the studies mentioned above.Animal models do notmirror the full range of AD symptoms

(49, 50), and studies with post-mortem human brain samplesare often hampered by difficulties in determining the effect offactors such as the post-mortem interval, brain pH, age, diseasestatus, and other neurological conditions affecting tissue qual-ity (51, 52). As a novel approach to gain insight into clinically

relevant diseasemechanisms that cannot be properly addressedusing post-mortemor animalmodel brain tissue, herewe intro-duced the use of cultured adult human brain slices as amodel toinvestigate A��-induced AD pathology. To date, a singlestudy has investigated the effects of A�� treatment on tran-scription (53) and revealed down-regulation of genesinvolved in protein modification and degradation. However,the possible physiopathological significance of those find-ings appears questionable, as the concentration of A�Osused (50 �M) was orders of magnitude higher than the con-centrations found in the diseased human brain. Moreover,that study made use of an immortalized cell line (rather thanneurons), further complicating correlation of the findingswith human brain neuropathology.The presentwork constitutes the first description of the early

effects of A�Os at low concentrations on gene expression inadult human neurons under conditions in which cell viability isnot impaired. The investigation of the impact of A�Os onhuman brain tissue may provide insight into mechanisms thatare directly relevant to AD pathogenesis but might not beapparent in studies based on transgenic rodent models or dis-sociated cell cultures.Quantitative RT-PCR was used to validate microarray

results. The percentage of validation of differential expressionwas higher in the set of down-regulated genes, consistent withthe finding that A�Os predominantly induced down-regula-tion rather than up-regulation of gene expression (Fig. 2A).Moreover, down-regulation of synaptophysin (SYP) and vac-cinia-related kinase 3 (VRK3), two of the three genes with thehighest level of down-regulation in themicroarray analysis (Fig.2C), was validated by qPCR (Fig. 4), confirming that the mostsignificant alterations detected in the microarray analysisindeed reflect gene expression changes triggered by A�O treat-ment. qPCR data also validated the up-regulation of cytoplas-mic FMR1-interacting protein 1 (CYFIP1), initially detected inthe microarray analysis (Fig. 2C). CYFIP1, along with EIF4E,forms a complex with Fragile X mental retardation protein(FMR1), a key regulator of translation in dendritic spinesknown to be down-regulated in the Fragile X syndrome (54). Ithas been proposed recently that dementia in AD and mental

FIGURE 5. A�Os induce down-regulation of synaptophysin protein levels in rat hippocampal cultures and human brain slices. A, down-regulation ofsynaptophysin mRNA was observed in hippocampal cultures exposed to A�Os (500 nM). qPCR analysis was performed using two different gene normalizers(ACTB and GAPDH). *, p 0.09; **, p 0.05, Student’s t test. B, synaptophysin (Syp) down-regulation was also detected at the protein level (determined byWestern blot analysis) after a 24-h treatment with A�Os (*, p 0.05 compared with control, indicated by the dashed line). qPCR and Western blot results arepresented as means � S.E. from at least three independent cultures. C, human brain slices were exposed to 500 nM A�Os for 12 h and protein levels(synaptophysin and cyclophilin (Cyc)) were determined by Western blots. Significant down-regulation (*, p 0.02, Student’s t test) of synaptophysin wasverified compared with vehicle. Results are means � S.E. from experiments using material from three independent donors. A representative blot is shown atthe top.



retardation in Fragile X syndrome share common pathogenicfeatures andmay both be consequences of synaptic dysfunctioncaused by loss of or decreased FMR1 activity (6, 55). Therefore,it is possible that A�O-induced up-regulation of CYFIP1 isrelated to disruption of FMR1-mediated control of translationin AD.Synaptophysin loss has been extensively associated with AD

pathology. Synaptophysin mRNA and protein levels arereduced in the post-mortem AD brain (45, 46), and proteinlevels are also found to be down-regulated in animal modelbrains (56, 57). Synaptophysinwas themost significantly down-regulated gene in our analysis, establishing a clear correlationbetween our findings and a typical AD-type pathology. Inter-estingly, whereas previous studies have investigated synapto-physin as a marker of AD pathology rather than a direct targetof A�Os, we now show that significant reductions in synapto-physin mRNA and protein levels are induced by short-termA�O treatment (Fig. 5). These results implicate synaptophysindown-regulation as an early abnormality triggered by A�Otoxic signaling and synapse loss.Synaptophysin plays an important role in intraneuronal ves-

icle transport, namely in the processes of presynaptic vesiclemembrane fusion and neurotransmitter release in the synapticcleft (58). Interestingly, current results indicated that “SNAREinteractions in vesicle transport” was the most significantlyoverrepresented pathway in A�O-treated slices (supplementalTable 3). Two additional important SNARE proteins in whichgene expression was affected by A�Os are VAMP and syntaxin(Fig. 3), both involved in synaptic vesicle fusion to the plasmamembrane and neurotransmitter release (59). Along with pre-vious reports of AD-associated alterations in neuronal vesicletransport machinery at the mRNA level (22), current data indi-cate that A�Os promote a rapid and specific attack on the neu-ronal vesicle transport system, which likely culminates in syn-aptic dysfunction.Increasing evidence supports a recently proposed link

between human brain insulin resistance and AD pathology (18,60–65). At the cellular level, A�Os have been shown to induceinsulin receptor internalization and the impairment of neuro-nal insulin signaling in hippocampal cultures (18, 19). In linewith those studies, we found here that the insulin-signalingpathway is one of the pathways targeted byA�Os (Fig. 3). Inter-estingly, among the genes identified in the present study aretwo key components of insulin signaling, AMP-activated kinase(AMPK) and mTOR (Fig. 3, gene symbols prkag1 and frap1,respectively). The possible involvement of mTOR in the patho-physiology of AD is still a controversial matter. For example,Spilman et al. (66) show that pharmacological inhibition ofmTOR by rapamycin decreases A�42 levels and rescues thecognitive function in a transgenicmousemodel of AD, suggest-ing that mTOR activity may be increased in A�-affected tissue.Conversely,Ma et al. (67), also usingAD transgenicmice (albeita different strain from that used by Spilman et al. (66)), foundthat mTOR signaling was inhibited both in cultured neuronsand hippocampal slices from AD-transgenic mice and in wild-type hippocampal slices exposed to exogenous A�1–42 andthat this mTOR dysregulation correlated with impairment insynaptic plasticity. It is thus possible that different genetic

backgrounds may lead to distinct responses in terms of mTORactivity in transgenic mice. The present observation of altera-tions in the expression ofmTOR and other proteins involved ininsulin signaling in A�O-treated human tissue may reflect anearly pathological response not apparent in transgenic mice(which exhibit exacerbated amyloid pathology) or AD post-mortem samples. Taken together, the current results, alongwith recent and discrepant literature reports, warrant furtherstudies to elucidate the role of mTOR and functionally associ-ated proteins inAD-linked insulin resistance, particularly in theearly stages of the disease. In addition, the results suggest thattranscriptional dysregulation of genes responsive to insulin sig-nalingmay be part of themechanism bywhich neuronal insulinresistance is induced by A�Os.Ubiquitin-mediated proteolysis, a pathway found to be

altered by A�O treatment (Fig. 3), has also been described aspart of AD pathogenesis (22, 68). One of the genes grouped inthis pathway is cullin 3, which encodes an essential componentof the ubiquitin E3 ligase complex. We found a significantdown-regulation of cullin 3 in A�O-treated slices, in accordwith results reported using post-mortem AD hippocampal tis-sue (21).Down-regulation of the ubiquitin systemat the proteinlevel in AD has also been reported previously (69), strengthen-ing the possibility that ubiquitin-mediated systems are specifictargets of A�O toxicity.

Cell adhesion molecules are well known players in the regu-lation of synaptic plasticity, learning, and memory (70) andhave been suggested to be involved in A�-mediated neurotox-icity (71, 72).We found that the cell adhesion-related pathways“cell adhesion molecules” and “focal adhesion”, as well as “reg-ulation of actin cytoskeleton” (a crucial intracellular compo-nent of the cell adhesion machinery), were among the A��-targeted pathways in human slices (Fig. 3). Furthermore,expression of the acting-binding protein CYFIP1, a componentof the complex that regulates cytoskeleton remodeling in neu-rons (73), was up-regulated in A�O-treated slices from five ofthe seven donors tested by qPCR (Fig. 4). These results areconsistent with the notion that alterations in cell adhesion areinvolved in synaptic failure in AD.A� has been shown to impede the reversal of long-term

depression (LTD) in rat hippocampal slices (74, 75). Here wefound that A�Os disrupt the expression of genes associatedwith LTD (Fig. 3). Facilitation of LTD has been proposed as amechanism by which A�Os cause synapse failure in AD (20,76), and it is possible that altered expression of genes involvedin LTDmay underlie the dysfunctional synaptic plasticity insti-gated by A�Os.In conclusion, we found that short-term exposure to a sub-

lethal concentration of pathologically relevant A�Os disruptsgene expression in adult human brain slices, causing, in mostcases, the down-regulation of genes important for neuronalphysiology. Changes in gene expression found in the presentstudy shed light onmechanisms bywhichA�Os affect neuronalgene expression and function and may be considered earlyevents in AD pathology and, therefore, potential targets for thedevelopment of novel diagnostic or therapeutic approaches inAD.





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Amyloid-β oligomers induce differential gene expression in adult human brain slices

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