*Department of Drug Sciences, Centre of Excellence in Applied Biology, University of Pavia, Pavia,

Italy

†Department of Biomedical Sciences and Biotechnologies, University of Brescia, Brescia, Italy

AbstractZyxin is an adaptor protein recently identified as a novelregulator of the homeodomain-interacting protein kinase 2(HIPK2)-p53 signaling in response to DNA damage. Werecently reported an altered conformational state of p53 intissues from patients with Alzheimer ‘s disease (AD), becauseof a deregulation of HIPK2 activity, leading to an impaired anddysfunctional response to stressors. Here, we examined themolecular mechanisms underlying the deregulation of HIPK2activity in two cellular models, HEK-293 cells and SH-SY5Yneuroblastoma cells differentiated with retinoic acid over-expressing the amyloid precursor protein, focusing on theevidence that zyxin expression is important to maintain HIPK2

protein stability. We demonstrated that both beta-amyloid (Ab)1-40 and 1-42 induce zyxin deregulation, thus affecting thetranscriptional repressor activity of HIPK2 onto its targetpromoter, metallothionein 2A, which is in turn responsible forthe induction of an altered conformational state of p53. Wedemonstrate for the first time that zyxin is a novel target of Abactivities in AD. These results may help the studies on thepathogenesis of AD, through the fine dissection of eventsrelated to beta-amyloid activities.Keywords: Alzheimer’s disease, beta-amyloid peptides, con-formationally altered p53, metallothionein 2A, zyxin.J. Neurochem. (2013) 125, 790–799.

The protein p53 responds to a variety of cellular stresses andis able to sense the intensity of the damage and modulatebiological responses, ranging from transient growth arrest topermanent replicative senescence or apoptosis (Vousden andPrives 2005). One important mechanism that controls p53function is its conformational stability (Joerger and Fersht2007). An altered protein conformational state of p53,independent from point mutations, has been reported intissues from patients with Alzheimer’s disease (AD) (Ubertiet al. 2006; Lanni et al. 2008; Zhou and Jia 2010). Wheninvestigating the mechanism of such alteration, we found thatsoluble nanomolar concentrations of beta-amyloid (Ab) 1-40peptide induced the expression of an unfolded p53 proteinisoform and modulated p53 functions by interfering with thehomeodomain-interacting protein kinase 2 (HIPK2) (Lanniet al. 2007, 2010), a fundamental protein in maintaining wild-type p53 function (Puca et al. 2008). In particular, soluble Ab1-40 inhibited HIPK2 activity, consequently inducting an

altered conformational state of p53, and thus resulting in aninability to properly activate an apoptotic program when cellsare exposed to a noxious stimulus (Lanni et al. 2010).A novel regulator of the HIPK2-p53 signaling in response

to DNA damage, named zyxin, has been recently identified(Crone et al. 2011). Zyxin is primarily localized at the focal

Received January 2, 2013; revised manuscript received January 11,2013; accepted January 11, 2013.Address correspondence and reprint requests to Cristina Lanni,

Department of Drug Sciences, University of Pavia, Viale Taramelli 14,27100 Pavia, Italy. E-mail: cristina.lanni@unipv.itAbbreviations used: bSI, b-secretase inhibitor; cSI, c-secretase

inhibitor; AD, Alzheimer’s disease; APP, amyloid precursor protein;Ab, beta-amyloid; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde3-phosphate dehydrogenase; HEK 293, human embryonic kidney;HIPK2, homeodomain-interacting protein kinase 2; LDH, lactatedehydrogenase; MT2A, metallothionein 2A; PVDF, polyvinylidenedifluoride; TBST, Tris-buffered saline/Tween 20.

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JOURNAL OF NEUROCHEMISTRY | 2013 | 125 | 790–799 doi: 10.1111/jnc.12154

adhesion plaque complex as an adaptor protein (Beckerle1997; Wang and Gilmore 2003) and contains a proline-richdomain at the N-terminus and three LIM domains at the C-terminus that are cysteine-rich motifs involved in protein–protein interactions. Zyxin is involved in regulating celladhesion, spreading, and motility but also in transducingsignals into the nucleus, to regulate gene expression, cellproliferation, differentiation, and apoptosis (Sadot et al.2001; Wang and Gilmore 2001). Consistently, zyxin hasbeen demonstrated to shuttle between the cytosol and thenucleus, where it affects transcriptional activity (Degenhardtand Silverstein 2001). In cancer cells, zyxin has beeninvolved in DNA damage-induced cell fate control throughthe modulation of the HIPK2-p53 signaling. In particular,depletion of endogenous zyxin resulted in proteasome-dependent HIPK2 degradation, thus compromising DNAdamage-induced p53 Ser46 phosphorylation dependent fromHIPK2 (Crone et al. 2011).A deregulation of HIPK2 has also been observed in cells

treated with Ab 1-40 (Lanni et al. 2010); however, themolecular mechanisms underlying HIPK2 proteasomal deg-radation in conditions related to Ab-exposure need to beinvestigated. Considering that an altered proportion betweenAb 1-40/Ab 1-42 as well as an increased production of oneof the two species has been indicated as dangerous for thepathogenesis of AD (Verdile et al. 2004; Pangalos et al.2005; Barten and Albright 2008), our purpose was to firstinvestigate whether a difference in the activation of thispathway exists between the species 1-40 and 1-42, byevaluating the capability of these species to affect thetranscriptional repressor activity of HIPK2 onto its targetpromoter, metallothionein 2A (MT2A). Furthermore, basedon data from literature demonstrating that zyxin expression isimportant in maintaining HIPK2 protein stability (Croneet al. 2011), our second goal was to determine whether amodulation of zyxin is involved in AD pathogenesis, usingtwo cellular models of altered Ab production. In particular,since in AD an altered metabolism of the amyloid precursorprotein (APP) occurred, in turn leading to an aberrantproduction of Ab peptides (Verdile et al. 2004), our purposewas also to investigate the effect of Ab peptides on zyxinmRNA and protein levels. The results showed here may helpto better understand the pathogenesis of AD, through the finedissection of events related to Ab activities. The character-ization of Ab activity on zyxin-HIPK2 signaling pathwayrepresents a relevant characteristic, since it concerns aresearch field yet unexplored in a neurodegenerative context.

Materials and methods

Reagents

All culture media, Fetal Bovine Serum (FBS), and supplements werepurchased from Euroclone (Life Science Division, Milan, Italy).Electrophoresis reagents were acquired from Bio-Rad (Hercules,

CA, USA). All other reagents were of the highest grade availableand were obtained from Sigma Chemical Co. (St. Louis, MO, USA)unless otherwise indicated. Ab 1-40, Ab 1-42, and Ab 40-1 reversepeptide were solubilized in dimethyl sulfoxide (DMSO) at theconcentration of 100 lM and frozen in stock aliquots. For eachexperimental setting, one stock aliquot was diluted at the finalconcentration of 10 nM. The Ab concentration was chosen follow-ing dose–response experiments (data not shown), where maximalmodulation of p53 structure and its transcriptional activity (Ubertiet al. 2007) was obtained at 10 nM. All the experiments performedwith Ab were made in 1% of serum. H2O2 was diluted to workingconcentration (1 mM) in phosphate buffer saline (PBS) at themoment of use. Mouse monoclonal anti-a-tubulin was purchasedfrom Sigma-Aldrich (St. Louis, MO, USA). Host specific peroxidaseconjugated IgG secondary antibodies were obtained from Pierce(Rockford, IL, USA).

Cell cultures

Human embryonic kidney (HEK) 293 cells from European Collec-tion of Cell Cultures (ECACC No. 85120602) were cultured inEagle’s minimum essential medium containing 10% FBS, glutamine(2 mM), penicillin/streptomycin (2 mM), at 37°C in 5%CO2/95%air (Uberti et al. 2007). The HEK-293 cells stably transfected withAPP751 were obtained as previously described (Uberti et al. 2007)and maintained in G418 at a final concentration of 400 lg/mL.

Human neuroblastoma SH-SY5Y cell line from EuropeanCollection of Cell Cultures (ECACC No. 94030304) were culturedin medium with equal amount of Eagle’s minimum essentialmedium and Nutrient Mixture Ham’s F-12, supplemented with 10%fetal bovine serum, glutamine (2 mM), penicillin/streptomycin, non-essential amino acids at 37°C in 5% CO2/95% air. The SH-SY5Ycells were also stably transfected with APP751 and maintained inG418 at a final concentration of 400 lg/mL. For the differentiation,cells were plated at a density of 5 9 103 cells/cm2 in 60-mmdiameter culture dishes using cell medium with 10% FBS. Fromday 1 after plating, cells were differentiated in the presence of10 lM all trans-retinoic acid in the cell medium containing 1%FBS. The medium was replaced every 2 days for 6 days to achievecell differentiation. Differentiation was considered completed con-sidering the length of growth cone-terminated neurites versus cellbody diameter: the growth cone-terminated neurites of differentiatedcells appeared to be up to three times longer than the diameter of thecorresponding cell body as compared with non-differentiated cells,where the neurites were shorter than the diameter of the corre-sponding cell body.

Cell treatment

Cells were used at 80% confluent monolayers. Doxorubicin wasadded to the medium at the concentration of 50 lmol/L and thencells were cultured for additional 24 h. For H2O2 exposure, culturecells were washed with PBS and treated with 1 mM H2O2 for10 min. After washing, cells returned to fresh medium for additionaltime according to the experiments. For each cell line, theexperiments were repeated at least three times.

Cell viability

Cell viability was evaluated 24 h after the addition of the cytotoxicagent to the media by measuring lactate dehydrogenase (LDH)

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activity using Cytoxicity Detection Kit (Boehringer, Mannheim,Germany) and an ELISA reader (340 ATC; SLT LabInstruments,Salzburg, Austria). Cytotoxicity was evaluated as percentage ofmaximum amount of releasable LDH enzyme activity, which isdetermined by lysing the cells with 1% of TritonX-100.

RNA extraction and reverse transcription-PCR (RT-PCR)

Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA,USA), following the manufacturer’s instructions. The first strandcDNA was synthesized by reverse-transcribing mRNA, as previ-ously described (Lanni et al. 2010). Semiquantitative RT-PCR wasperformed by using Hot-Master Taq (Eppendorf, Milan, Italy) with2 lL cDNA reaction and genes specific oligonucleotides underconditions of linear amplification.

Real-Time PCR

For mRNA extraction 2 9 106 cells were used in a 60 mm2 Petriplate. Total RNA was extracted using RNeasy Plus Mini Kit(Qiagen, Valencia, CA, USA) following manufacturer’s instruc-tions. QuantiTect reversion transcription kit and QuantiTect SyberGreen PCR kit (Qiagen) were used for cDNA synthesis and geneexpression analysis following manufacturer’s specifications. Quan-tiTect primer assay for MT2A and glyceraldehyde 3-phosphatedehydrogenase (GAPDH) were provided by Qiagen. GAPDH RNAtranscription was used as endogenous reference and the quantifica-tion of the transcripts was performed by the DDCT method.

Immunodetection of zyxin

Cell monolayers were washed twice with ice-cold PBS, lysed on thetissue culture dish by addition of ice-cold lysis buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 50 mM EDTA, 0.2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF),20 lg/mL leupeptin, 25 lg/mL aprotinin, 0,5 lg/mL pepstatin Aand 1% Triton X-100), and an aliquot was used for protein analysiswith the Pierce Bicinchoninic Acid kit, for protein quantification.Cell lysates were diluted in sample buffer (62.5 mM Tris/HCl pH6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% Brom-ophenol blue) and subjected to western blot analysis. Proteins weresubjected to SDS-PAGE (8%) and then transferred onto polyviny-lidene difluoride (PVDF) membrane 0.45 lm (Immobilion; Milli-pore Corp, Bedford, MA, USA). The membrane was blocked for 1 hwith 5% non-fat dry milk in Tris-buffered saline containing 0.1%Tween 20 (TBST). Membranes were immunoblotted with the rabbitanti-human zyxin polyclonal antibody (at 1 : 1000 dilutions in 5%non-fat dry milk, from Cell Signaling Technology, EuroClone,Milan, Italy). The detection was carried out by incubation withhorseradish peroxidase conjugated goat anti-rabbit IgG (1 : 5000dilutions in 5% non-fat dry milk, from Pierce, Rockford, IL, USA)for 1 h. The blots were then washed extensively and the proteins ofinterest were visualized using an enhanced chemiluminescentmethod (Pierce). Tubulin was also performed as a normal controlof proteins.

p53 conformational immunoprecipitation

p53 conformational state was analyzed by immunoprecipitation. Inparticular, cells were lysed in immunoprecipitation buffer (10 mMTris, pH 7.6; 140 mM NaCl; and 0.5% NP40 including proteaseinhibitors) for 20 min on ice, and cell debris was cleared by

centrifugation. Hundred microgram of total cell extracts were usedfor immunoprecipitation experiments performed in a volume of500 lL. To prevent non-specific binding, the supernatant ofimmunoprecipitated samples was pre-cleared with 10% (w/v)protein A/G (50 lL) (SantaCruz Biotechnology Inc., Heidelberg,Germany) for 20 min on ice, followed by centrifugation. Forimmunoprecipitation of p53, 1 lg of the conformation-specificantibodies PAb1620 (wild-type specific) (Calbiochem, EMB Bio-science, La Jolla, CA, USA), or PAb240 (mutant specific)(Neomarkers-Lab Vision, Fremont, CA, USA) was added to thesamples and incubated overnight at 4°C. Immunocomplexes wereseparated by 10% SDS-PAGE and immunoblotting was performedwith polyclonal anti-p53 antibody CM1 (Novocastra, Newcastle,UK). Immunoreactivity was detected with an enhanced chemilumi-nescent method (Pierce).

Densitometry and statistics

Following acquisition of the western blot image through an HPScanjet G4010 scanner and analysis by means of the Image 1.47program (Wayne Rasband, NIH, Research Services Branch, NIMH,Bethesda, MD, USA), the relative densities of the bands wereexpressed as arbitrary units and analyzed as described previously(Lanni et al. 2004). Data were analyzed using the analysis ofvariance test followed, when significant, by an appropriate post hoccomparison test as indicated in figure legend. A p-value < 0.05 wasconsidered statistically significant. Data reported are expressed asmeans � SD of at least three independent experiments.

Results

Ab 1-40 and Ab 1-42 peptides impair p53

transcriptional activity

HEK-293 cells were evaluated for their vulnerability to agenotoxic insult. To this purpose, HEK-293 cells weretreated with soluble Ab 1-40 and 1-42 at the concentration of10 nM for 48 h and then exposed for 24 h to 50 lmol/Ldoxorubicin, a cytotoxic agent able to induce DNA damageand apoptosis in a p53-dependent manner (Wang et al.2004). Cell viability was evaluated measuring LDH releasein the medium. Doxorubicin reduced cell viability of about50% in HEK cells (Fig. 1a). On the other hand, 10 nM Ab1-40 and 1-42 pre-treated cells challenged with the sameconcentration of the genotoxic agent were found to be lessvulnerable to doxorubicin-induced cytotoxicity, as indicatedby a smaller reduction of cell viability (about 15%, Fig. 1a).We then analyzed the conformational state of p53. HEK cellswere treated with 10 nM Ab 1-40 or Ab 1-42 for 48 h andsubsequently processed for immunoprecipitation with twoconformational-specific antibodies, PAb1620 and PAb240,which discriminate folded versus unfolded p53 tertiarystructure (M�eplan et al. 2000). Ab 1-40 and Ab 1-42treatment induced the expression of a conformationallyaltered p53 phenotype that reacted with PAb 240 antibody(Fig. 1b).As previously reported, a mechanism, through which

p53 conformation is affected, involves HIPK2 activity on

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metallothionein 2A (MT2A), a potent chelator in removingzinc from p53 in vitro (Puca et al. 2009). In particular,HIPK2 deregulation has been recently involved in p53misfolding through MT2A up-regulation in AD cellularmodels and fibroblasts from AD patients (Lanni et al. 2010).Consistent with these data, analysis of mRNA showed thatMT2A expression was up-regulated in both Ab 1-40 and Ab1-42 treated cells, which is an index for HIPK2 deregulationin p53 misfolding, whereas treatment with the reversepeptide Ab 40-1 failed to do so (Fig. 1c).

Ab 1-40 and 1-42 peptides are responsible for zyxin

deregulation

Following data indicating that both Ab 1-40 and 1-42peptides were able to affect the transcriptional repressoractivity of HIPK2 onto its target promoter MT2A and thatzyxin expression is important to maintain HIPK2 proteinstability (Crone et al. 2011), we investigated whether zyxinexpression was somehow compromised by nanomolar con-centrations of soluble Ab peptides. We evaluated zyxinmRNA expression and protein level in HEK-293 cells after

treatment with Ab 1-40 and 1-42. As shown in Fig. 2a, theanalysis of RT-PCR following normalization to GAPDHexpression revealed that no differences in zyxin mRNAexpression were observed when comparing treated tountreated HEK-293 cells. Next, zyxin protein levels wereevaluated by western immunoblotting. As shown in Fig. 2b,Ab 1-40 and 1-42 treatment significantly reduced zyxinprotein levels in HEK-293 cells, compared to vehicle or Ab40-1 treatment. Treatment with nanomolar concentrations ofAb 1-40 and 1-42 in low serum condition provided evidencethat both peptides entered into the cells (data not shown),consistently with previous data from literature (Uberti et al.2007).Altogether, these results show that nanomolar concentra-

tions of soluble Ab 1-40 and 1-42 peptides can modulatezyxin protein levels.

Endogenous Ab 1-40 and 1-42 peptides negativelyaffect zyxin protein levels

To better evaluate the role of intracellular beta-amyloidpeptides in zyxin deregulation, we also used HEK-293 cells

Fig. 1 Beta-amyloid (Ab) peptides impair p53 transcriptional activity.(a) Human embryonic kidney (HEK)-293 cells were treated with 10 nM

Ab 1-40 or 1-42 for 48 h and then exposed to 50 lM doxorubicin for24 h. Cell death was detected measuring lactate dehydrogenaserelease in the media. Data were expressed as % of cell viability versus

control. ***p < 0.001 versus control, ###p < 0.001 versus doxorubicintreatment; Bonferroni Multiple Comparison test. (b) HEK cells wereexposed to 10 nM Ab 1–40 or Ab 1–42 for 48 h and then cell lysateswere immunoprecipitated with PAb240 or PAb1620 antibody. Immu-

noprecipitates were analyzed by western blot with the CM1 polyclonal

anti-p53 antibody. Antibodies for immunoprecipitation were omitted incontrol (blk) samples. After densitometric analysis data were

expressed as integrated density of ratio PAb240/PAb1620 antibodiessignal over blank and represent means � SD of at least three differentexperiments. **p < 0.01 and ***p < 0.001 versus untreated cells;

Bonferroni Multiple Comparison test. (c) MT2A mRNA expression wasdetermined by real-time PCR in HEK-293 cells treated with 10 nM Ab1-40 or 1-42 or 40-1 for 48 h. **p < 0.01 and ***p < 0.001 versusCTR; Dunnett’s Multiple Comparison Test.

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stably transfected with wild-type APP751 (HEK-APP) thatexpress high levels of full length APP (Uberti et al. 2007).HEK-APP cells have been previously demonstrated toproduce and release elevated amounts of Ab peptides, whereAb 1-40 was the most abundant isoform (Lanni et al. 2010).In HEK-APP cells, MT2A mRNA was up-regulated com-pared to HEK-293, in a similar way to the treatment with Abpeptides (Fig. 3a), thus subtending an impairment of HIPK2activity. To acquire more insight into the contribution of thedifferent APP processing products, HEK-APP cells were alsotreated with b- (bSI) and c- (cSI) secretase inhibitors toprevent amyloidogenic APP metabolism. Zyxin proteinlevels were then evaluated in HEK-293 cells and HEK-APP cells that were also treated with the bSI and cSI. Asshown in Fig. 3b (upper panel), zyxin protein levels werestrongly decreased in HEK-APP cells, compared to untrans-fected counterparts, as is also shown by quantitative analysisof zyxin/tubulin ratio (Fig. 3b, lower panel). Beta- andgamma-secretase inhibitors treatment restored zyxin proteinlevels to those of control cells (Fig. 3b), strongly suggestingthat APP amyloidogenic metabolites may indeed affect zyxinprotein levels.We then tested the ability of the conditioned medium of

HEK-APP cells to deregulate zyxin. To this aim, HEK-293cells were cultured with conditioned medium of HEK-APPcells that, as shown in Fig. 4, decreased zyxin protein levels.To further discriminate the effect of intracellular fromsecreted Ab peptides, HEK cells were treated with HEK-APP conditioned medium for 48 h in the presence or absenceof the specific neutralizing antibody 6E10, that recognizesthe first 17 aa of the Ab sequence. Cells were then processed

for western blot experiment carried out with zyxin antibody.Protein extracts from HEK cells exposed to HEK-APPconditioned medium showed reduced zyxin protein levels,compared to vehicle (Fig. 4). The reduction in zyxin proteinlevels found after treatment of HEK cells with HEK-APPconditioned medium was reverted by 6E10 antibody (Fig. 4).On the contrary, treatment with 6E10 antibody failed torestore zyxin protein levels in HEP-APP cells (Fig. 4).

Neuronal cells show impairment in zyxin-HIPK2 signaling

Parallely to HEK-APP, we also used SH-SY5Y cells stablytransfected with wild-type APP751 (SY-APP) that expressincreased APP mRNA and protein levels and showed anincreased APP metabolism toward the amyloidogenic path-way (data not shown) when compared to untransfected cells.SH-SY5Y and SY-APP cells were differentiated with 10 lMretinoic acid, to have a closer neuronal model. First, SH-SY5Y and SY-APP cells were evaluated for their vulnera-bility to an oxidative insult after differentiation with retinoicacid. To this purpose, SH-SY5Y and SY-APP cells wereexposed to 1 mM H2O2 for 10 min and cell viability wasevaluated measuring LDH release in the medium. H2O2

reduced cell viability of about 50% in SH-SY5Y cells(Fig. 5a). On the other hand, SY-APP cells, after exposure tothe same pulse of H2O2, were found to be less vulnerable toH2O2-induced cytotoxicity, as indicated by a smaller reduc-tion of cell viability (about 20%, Fig. 5a). These data areconsistent with those obtained in HEK cells pre-treated withsublethal concentrations of soluble Ab 1-40 and 1-42(Fig. 1a). We then characterized this cellular model in termof HIPK2 and zyxin expression. Moreover, to better

Fig. 2 Beta-amyloid (Ab) peptides are responsible for zyxin deregu-lation. (a) Zyxin mRNA expression was determined by real-time PCR in

human embryonic kidney (HEK)-293 cells treated with 10 nM Ab 1-40or Ab 1-42 or Ab 40-1 for 48 h. (b) Total cell extracts of HEK-293 cells,treated with 10 nM Ab 1-40 or Ab 1-42 or Ab 40-1 for 48 h, were

analyzed for zyxin expression. Anti-tubulin was used as protein loadingcontrol. A representative experiment of at least three independent

experiments was shown (upper panel). Results are shown as ratiozyxin/tubulin � SD (lower panel). *p < 0.05 versus CTR (BonferroniMultiple Comparison test).

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understand the role of beta-amyloid peptides in the dereg-ulation of this pathway in neuronal cells, we also exposeddifferentiated SH-SY5Y to 10 nM Ab 1-40 and Ab 1-42 for

48 h. Analysis of mRNA showed that MT2A expression wasup-regulated in both Ab 1-40 and Ab 1-42 treated cells, aswell as in differentiated SY-APP cells, suggesting thatHIPK2 deregulation is involved in p53 misfolding, throughMT2A up-regulation, as previously shown (Lanni et al.2010) (Fig. 5b). When evaluating zyxin protein level indifferentiated SH-SY5Y cells after treatment with Ab 1-40and 1-42, both Ab 1-40 and Ab 1-42 treatment significantlyreduced zyxin protein levels, compared to vehicle or Ab 40-1treatment (Fig. 5c). Furthermore, as shown in Fig. 5c, zyxinprotein levels were also strongly decreased in differentiatedSY-APP cells, compared to untransfected counterparts.Parallel to data on HEK-APP cells, these data demonstratedthat APP amyloidogenic metabolites may affect zyxin proteinlevels and suggest that p53-zyxin-HIPK2 signaling pathwayis affected also in neuronal-like cells.

Discussion

We describe here a link between zyxin-HIPK2-p53 signalingpathway and Alzheimer’s disease. We previously demon-strated the existence of Ab-dependent HIPK2 deregulationresponsible for the induction of an unfolded state of p53protein in fibroblasts from AD patients leading to animpaired and dysfunctional response to stressor (Ubertiet al. 2002; Lanni et al. 2008). On the contrary, the non-amyloidogenic product of APP metabolism, sAPPalpha, wasdemonstrated to be not involved in the regulation of thepathway resulting in conformationally altered p53 (Ubertiet al. 2007). Here, we examined the molecular mechanisms

Fig. 3 Endogenous beta-amyloid (Ab) 1-40 and 1-42 peptides nega-tively affect zyxin expression. (a) MT2A mRNA expression was

determined in 10 nM Ab 1-40 or Ab 1-42 pre-treated human embryonickidney (HEK)-293 and in HEK-APP compared to mock cells byreverse-transcriptase (RT)-PCR. GAPDH was used as loading control.

(b) Western blot experiments were performed with anti-zyxin antibody

on HEK-293 and HEK-APP cells that were also treated with b- and c-secretase inhibitors at 10 lmol/L for 48 h. Anti-tubulin was used as

protein loading control. A representative experiment of at least threeindependent experiments was shown (upper panel). Results areshown as ratio zyxin/tubulin � SD (lower panel). *p < 0.05 versus

HEK-293 (Bonferroni Multiple Comparison test).

Fig. 4 Intracellular beta-amyloid (Ab) is responsible for zyxin dereg-

ulation. Human embryonic kidney (HEK) cells were treated withconditioned medium of HEK-APP cells in the presence or absence ofthe monoclonal antibody 6E10. HEK-APP cells were also treated with6E10 antibody. Western blot experiments were performed with

anti-zyxin antibody. A representative experiment of at least threeindependent experiments was shown (upper panel). Results areshown as ratio zyxin/tubulin � SD (lower panel). *p < 0.05 versus

HEK-293 (Dunnett’s Multiple Comparison test).

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underlying the deregulation of HIPK2 activity in two cellularmodels, HEK-293 and SH-SY5H neuroblastoma cells dif-ferentiated with retinoic acid over-expressing the amyloidprecursor protein. Recent findings show that zyxin expres-sion is important to maintain HIPK2 protein stability (Croneet al. 2011). Our data suggest that intracellular Ab peptidesmay be responsible for zyxin deregulation. This is supportedby the observation that Ab peptides down-regulated zyxinprotein levels, compromising HIPK2 stability and thusleading to HIPK2 disappearance from target promoters suchas MT2A. In agreement, MT2A mRNA up-regulation wasfound in HEK-APP cells that over-express APP751. Theinduction of MT2A, depending on HIPK2 knockdown hasbeen reported to be responsible for p53 misfolding andinhibition of p53 transcriptional activity (Puca et al. 2009);therefore, the present data suggest that zyxin deregulationinduced by Ab peptides might be involved in HIPK2degradation and in p53 misfolding via MT2A up-regulationin HEK-APP cells. This signaling pathway is further affectedin a similar way by both soluble Ab 1-40 and 1-42 atsublethal concentrations.To investigate the contribution of APP metabolic products

in the modulation of zyxin expression, we used HEK cells

over-expressing wild-type APP able to generate high levelsof Ab 1-40 and Ab 1-42 both intracellularly and secreted inthe medium (Uberti et al. 2007). We found that reducingAPP amyloidogenic metabolism by treating HEK-APP cellswith b- and c-secretase inhibitors (Lanni et al. 2010)prevented the deregulation of zyxin. It is worth to note thatthe conditioned medium of HEK-APP cells was able to affectuntransfected HEK-293 cells recapitulating the HEK-APPphenotype, in terms of zyxin deregulation.The data presented in this study suggest that the modulator

effects of Ab peptides on zyxin deregulation are, at least inpart, due to the intracellular peptides. Different observationssupport this conclusion. First, considering that internalizationof Ab can be prevented under experimental conditions that donot allow endocytosis (Knauer et al. 1992), the conditionedmedia of HEK-APP cells treated with the specific neutraliz-ing antibody 6E10, that recognizes the first 17 aa of the Absequence, were unable to influence zyxin protein levels inHEK-293 cells. Second, we found that the synthetic Abpeptides added to the cells crossed the plasma membrane, aspreviously demonstrated (Uberti et al. 2007).The deregulation of zyxin was further analyzed in

SH-SY5Y neuroblastoma cells and their counterpart

Fig. 5 Neuronal cells show impairment in homeodomain-interactingprotein kinase 2 (HIPK2) signaling and down-regulation of zyxin. (a)

SH-SY5Y and SY-APP cells, treated or not with 10 lM retinoic acid,were exposed to 1 mM H2O2 for 10 min. Cell death was detectedmeasuring lactate dehydrogenase release in the media. Data were

expressed as % of cell viability versus control. ***p < 0.001 versusSHSY and **p < 0.01 versus SY-APP; ###p < 0.001 versus differen-tiated SHSY and ##p < 0.01 versus differentiated SY-APP; BonferroniMultiple Comparison test. (b) MT2A mRNA expression was determined

in 10 nM beta-amyloid (Ab) 1-40 or Ab 1-42 pre-treated SHSY and inSY-APP, after differentiation with retinoic acid, compared to mock cells

by reverse-transcriptase (RT)-PCR. GAPDH was used as loadingcontrol. Results are shown as ratio mRNA MT2A/GAPDH � SD.

***p < 0.001 versus differentiated SHSY cells (Dunnett MultipleComparison test). (c) Total cell extracts of differentiated SY-APP andSH-SY5Y cells, treated with 10 nM Ab 1-40 or Ab 1-42 or Ab 40-1 for

48 h, were analyzed for zyxin expression. Anti-tubulin was used asprotein loading control. A representative experiment of at least threeindependent experiments was shown (upper panel). Results areshown as ratio zyxin/tubulin � SD (lower panel). *p < 0.05 and

**p < 0.01 versus differentiated SHSY cells (Dunnett Multiple Com-parison test).

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over-expressing wild-type APP differentiated with retinoicacid. When treating differentiated SH-SY5Y cells withsoluble Ab peptides, both Ab 1-40 and 1-42 were found toderegulate HIPK2 and decrease zyxin protein levels. Fur-thermore, also differentiated SY-APP cells are characterizedby a decrease in zyxin protein levels, besides showing anHIPK2 down-regulation, thus recapitulating the HEK-APPphenotype in terms of zyxin and HIPK2 deregulation.Zyxin is preferentially expressed in developing brain and

various adult tissues, including lungs, spleen, and testis(Fujita et al. 2009). Depending on its subcellular location,zyxin can have anti-apoptotic or pro-apoptotic function, sincezyxin has been shown to promote cell death downstream ofapoptotic stimuli such as UV-C irradiation (Hervy et al.2010), but it has also been implicated in Akt-dependentcardiomyocyte survival pathways (Kato et al. 2005). Zyxinhas been extensively studied within a tumoral context,whereas very limited information is present at this time in theliterature concerning its putative role in neurodegeneration.In this context, zyxin has been recently identified as aninteracting partner for a protein, SIRT1, involved in protec-tion from neurotoxicity in cell-based models for AD/tauopathies, amyotrophic lateral sclerosis and Walleriandegeneration, showing that the interaction of these proteinscould be implicated in cellular survival, especially in thebrain and heart, during physiological senescence (Fujitaet al. 2009). Here, we established for the first time a linkbetween zyxin and Alzheimer’s disease. This observation isintriguing, since recent data from the literature support theconcept that one or more common molecular mechanismsmay be involved in the development of both neurodegener-ative diseases and many cancers (Jope et al. 2007; Li et al.2007; Lu and Zhou 2007; Alves da Costa and Checler2011; Lanni et al. 2012). Taking into account that cancer andAD share common signaling pathways directing cell fatetoward either death or survival, the identification of theputative common mechanisms may be useful to directneurodegeneration studies toward the same intracellularpathways that have been successfully studied and targetedin cancer.In summary, we hypothesize that soluble Ab 1-40 and 1-42

may be responsible for important modulatory effects atcellular level before triggering the amyloidogenic cascade.For the first time, we demonstrated that soluble Ab 1-40 and 1-42 modulate zyxin protein levels, fundamental in maintainingHIPK2 stability and in turn p53 activity. When zyxin is down-regulated by Ab peptides, HIPK2 activity is inhibited, withMT2A up-regulation, in turn responsible for the induction ofan altered conformational state of p53. As a result of thisconformational change, p53 lost its transcriptional activity andwas unable to properly activate an apoptotic program whencells were exposed to a noxious stimulus (Fig. 6). The reasonwhy both Ab 1-40 and Ab 1-42 have the same effects on thispathway is at the moment under investigation in our labora-

tory. It is well known that Ab 1-42 has been reported toaggregate faster than Ab 1-40 and thus it is considered themost neurotoxic species (Verdile et al. 2004). Moreover,physiologically the 40-amino acid long peptide is the mostabundant form (Terai et al. 2001; Kamenetz et al. 2003;Walsh and Selkoe 2007), since the concentration of secretedAb 1-42 is about 10% that of Ab 1-40 (Bitan et al. 2003).However, in pathological conditions, the ratio of theirproduction may be altered, as observed in familial AD cases(Mayeux et al. 1999). On the basis of this observation, one ofthe issues we should investigate is the differential modulationof this pathway by different concentration ratios of these twospecies. The sequence of events driven by beta-amyloid heredescribed might contribute to AD pathogenesis since it mayresult in the presence of dysfunctional cells. Consistently,Yang and co-workers demonstrated the existence of aberrantneurons in AD brain by showing that neurodegeneration iscorrelated with neurons reentering a lethal cell cycle (Yanget al. 2001; Copani et al. 2007, 2008), which suggests thatdysfunctional p53 in non-dividing cells may play a role inaberrant cell cycle progression. Furthermore, the observationthat in AD are involved proteins controlling the duplicationand cell cycle control leads to the speculation that, in senescentneurons, derangements in proteins commonly dealing withcell cycle control and apoptosis could affect neuronalplasticity and functioning rather than cell duplication.

Fig. 6 Working hypothesis for a putative role of zyxin in Alzheimer’sdisease pathogenesis. The Figure indicates zyxin deregulation med-iated by soluble beta-amyloid (Ab) peptides. When zyxin is down-

regulated by sublethal concentrations of Ab 1-40 or Ab 1-42,homeodomain-interacting protein kinase 2 (HIPK2) expression andactivity are inhibited through degradation via the proteasome system.HIPK2 deregulation results in the induction of metallothionein 2A

(MT2A), that exerts its Zn2+ chelator function. As a consequence, p53changes the wild-type conformation to a conformationally alteredstatus, with subsequent abolishment of wild-type p53 transcriptional

activity.

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 125, 790--799

Zyxin deregulation in Alzheimer pathogenesis 797

Acknowledgements

This study was supported by the contribution from the UNIPV-Regione Lombardia grant to C.L., and from the Ministry ofUniversity and Research (MIUR, Grant #2009B7ASKP to S.G.).None of the authors has any conflicting financial interest.

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