Journal of Alzheimer’s Disease 30 (2012) 393–405DOI 10.3233/JAD-2012-111961IOS Press

393

A�PP Intracellular C-Terminal DomainFunction is Related to its DegradationProcesses

Erica Buosoa, Fabrizio Biundoa, Cristina Lannia, Gennaro Schettinib, Stefano Govonia

and Marco Racchia,∗aDepartment of Drug Sciences, Pharmacology Unit, Center of Excellence in Applied Biology,University of Pavia, Pavia, ItalybDepartment of Oncology, Biology and Genetics, University of Genova, Genova, Italy

Accepted 10 February 2012

Abstract. The amyloid-� protein precursor (A�PP) can be processed by either the amyloidogenic or the non-amyloidogenicpathway; both pathways lead to release of the A�PP intracellular C-terminal domain (AICD). AICD involvement in signaltransduction within Fe65/Tip60 complex is one of the most discussed mechanisms, and different models have been hypothesizedto explain the role of AICD within this complex. The analysis of these models in relation to the degradation processes highlightsthe discrepancy among AICD localization, function, and degradation, leading to the hypothesis that a signaling mechanismmay exist which allows A�PP proteolysis to generate either a transcriptionally active fragment or an inactive one with differentinvolvement of proteasome and IDE (insulin-degrading enzyme). Our work aimed to analyze the functional role of AICD withinthe Fe65/Tip60 complex considering the AICD degradation processes. Our data suggest a correlation between the role of AICDin gene regulation and its removal operated by proteasome activity. Moreover, treatments with IDE inhibitor underlined thepresence of an alternative mechanism involved in AICD removal when the latter is not exerting nuclear activity, thus providingclearer support for the existence of at least two mechanisms as previously suggested.

Keywords: A�PP, AICD, Fe65, IDE, proteasome, Tip60

INTRODUCTION

Amyloid-� protein precursor (A�PP) is a typeI transmembrane protein with a large extracellu-lar portion, a membrane anchoring domain, and ashort intracellular C-terminal tail. A�PP can be pro-cessed by two distinct proteolytic pathways. Thenon-amylodogenic pathway involves cleavage bythe enzyme �-secretase, which cuts A�PP withinthe amyloid-� (A�) sequence thereby preventing the

∗Correspondence to: Marco Racchi, PhD, Department of DrugSciences, Pharmacology Unit, University of Pavia, Viale Taramelli14, 27100 Pavia, Italy. Tel.: +39 0382 987738; Fax: +39 0382987405; E-mail: racchi@unipv.it.

formation of A� [1]. The amyloidogenic pathwayinvolves cleavage of A�PP by an enzyme referred toas �-secretase which cleaves A�PP at the N-terminalside of the A� sequence. Following cleavage by �-or �-secretase, the �-secretase complex cleaves insidethe membrane the remaining C-terminal fragments ofA�PP, respectively C83 and C99, via a mechanismreferred to as regulated intramembrane proteolysis.C83 and C99 cleavage generates p3 and A� respec-tively together with A�PP intracellular C-terminaldomain (AICD) [2].

AICD levels are detectable in membrane fractionsof murine total brain homogenates and increase signifi-cantly in mice overexpressing the Swedish mutation ofhuman A�PP [3]. The potential importance of AICD

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394 E. Buoso et al. / AICD Function and its Degradation Processes

has been emphasized by the recognition of similaritiesbetween A�PP and another type I transmembrane pro-tein called Notch [4]. This analogy of A�PP processingto Notch receptor signaling suggested a possible func-tion for AICD in nuclear signaling [5–7].

The function of AICD as a signaling protein has beensuggested to be dependent to its interaction with severaltranscriptional co-activators. It has been demonstratedthat the central YENPTY motif of AICD binds tothe phosphotyrosine binding domain 2 (PTB2) of theadaptor protein Fe65 [8], generating the Fe65/AICDcomplex which is able to bind the histone acetyl-transferase tat-interactive protein (Tip60) [9], forminga transcriptionally active complex referred to as anAICD-Fe65-Tip60 (AFT) complex. Data from litera-ture indicate that the AFT complex is able to regulateat the transcriptional level different genes such as KAI,neprylisin, EGFR, A�PP itself [10], and p53 expres-sion [11]. Moreover AICD-mediated p53 activationcan be associated with cell death in Alzheimer’s dis-ease (AD) [12, 13].

The tumor suppressor p53 is a short-lived transcrip-tion factor that is post-translationally regulated by theubiquitin-proteasome pathway [14, 15]. In response tocellular stress or DNA damage, wild-type p53 proteinis activated by phosphorylation and other signals thatcause it to dissociate from its inhibitor MDM2. Onceactivated, p53 is no longer targeted for proteasomaldegradation by MDM2 and therefore accumulates andbinds to specific sequences in DNA, initiating tran-scription of genes that induce growth arrest, DNArepair, or apoptosis. In some cancer cells, proteasomeinhibitors cause the stabilization and accumulation ofp53 protein [15]; an increase of p53 mRNA was notobserved in proteasome-inhibited cells [16].

The role of AICD in signal transduction is, todate, one of the most controversially discussed mech-anisms and the main differences refer to the cellularcompartment where the interaction of the complexcomponents takes place. Considering these conflictingobservations, Muller and colleagues [17] summarizeddifferent models of interaction of the various com-ponents of the complex. In model I, the C-terminaldomain of A�PP recruits at the membrane Fe65 whichundergoes a conformational change necessary for itsactivation. Subsequently, Fe65 translocates into thenucleus and binds to Tip60 [18]; in model II, Fe65and AICD translocate into the nucleus independently,building up the ternary complex AICD/Fe65/Tip60in the nucleus [19]. Finally, most authors studyingAICD localization reported release of AICD from themembrane and translocation into the nucleus after

�-secretase cleavage of the precursor. In this lastmodel, AICD/Fe65 complex generates at the mem-brane and subsequently, after �-secretase cleavage,translocates into the nucleus. Following translocation,Tip60 enters the complex to generate the transcriptionfactor [20, 21].

We turned our attention to the AICD degradationprocesses because these mechanisms may help toelucidate some of the controversies outlined above;specifically the two best characterized mechanisms ofAICD degradation are the proteasome and the insulindegrading enzyme (IDE).

Literature data indicated that proteasome inhibitioncould increase A�PP processing specifically at the �-secretase site [22]. In this regard, it was demonstratedthat the proteasome is capable of directly cleaving thecytoplasmic domain of A�PP at several sites, includinga region around the YENPTY sequence which interactswith Fe65. This proteasomal cleavage decreases theavailability of A�PP-CTF for �-secretase cleavage andconsequently inhibits AICD production [23–25]. Onthe basis of these results, Kerr and Small proposed thatthe proteasome is not able to degrade A�PP-CTF whenthis fragment is bound by A�PP-binding protein suchas Fe65 [26].

IDE is a 110-kDa thiol zinc-methalloendopeptidasethat can cleave A� peptides and the AICD [27–30] bothin vivo and in vitro [31, 32]. The subcellular localiza-tion shows that IDE is abundant in cytosol [33]; thisobservation is supported by biochemical studies whichhave revealed the IDE presence in the soluble fractionsfrom human brain which contained both extracellularand cytosolic compartments [34, 35].

The analysis of AICD signal transduction models,in relation to these two putative degradation processes,highlights the contrast among mechanism responsi-ble of AICD localization, function, and degradation[36]. On the basis of this consideration in the mod-els I and III, the AICD degradation is primarily dueto proteasome action which can be inhibited by Fe65binding to A�PP-CTF as suggested by Kerr and Small[26]. In contrast to model III, in model I the AICDdoes not translocate to the nucleus but it is releasedin the cytosol becoming an IDE substrate. The modelpresented by Nakaya and Suzuki [19] argues that theAICD is released alone at the cytosol level, thus itdoes not act as an anchoring site but acquires the fac-ulty to regulate gene expression in association withFe65/Tip60 complex in the nucleus. Considering thisone as a putative mechanism of AICD’s action, IDEis the only process involved in AICD degradation[36].

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Elucidations about the role of AICD and itsdegradation can contribute to better understand itsinvolvement in AD pathology. In particular, a puta-tive contribution of these proteolytic fragments hasbeen demonstrated in transgenic mice overexpress-ing AICD. Increased levels of AICD are responsiblefor a series of events, including the activation ofGSK-3� [3], hyperphosphorylation and aggregationof tau protein, microtubule destabilization, and reduc-tion of nuclear �-catenin levels, thus causing a lossof cell-cell contact mechanisms that may contributeto neurotoxicity in AD. Subsequent neurodegenera-tion and working memory deficits were also observedin these transgenic mice [37], as well as abnormalspiking events in their electroencephalograms and sus-ceptibility to kainic acid-induced seizures independentof A� [38]. AICD could also alter gene expressionand induce neuron-specific apoptosis [39, 40]. Ele-vated AICD levels have also been reported in ADbrains [37]; the reason for their increased levels inAD brain is still unknown, though it could be relatedto an impairment of proteasome degradation pathway[41].

Taking into account this background, our workaimed to analyze the role of AICD within Fe65/Tip60complex integrating the putative degradation mecha-nisms. In order to verify the pathway-specific activityof proteasome in AICD removal as previously sug-gested, we used two proteasome inhibitors, MG-132and ALLN. MG132 is a non selective protease inhibitorbecause it is also able to inhibit �-secretase; it causes alarge accumulation of A�PP-CTF, by inhibiting both�-secretase and the proteasome. ALLN, a proteasomespecific inhibitor, does not act on �-secretase andthus causes an increase in A�PP-CTF providing moresubstrate available for �-secretase cleavage [23, 24,42].

Our data highlight a correlation between AICD-mediated proteasome removal and gene expressionregulation supporting the existence of at least two dis-tinct AICD degradation processes.

MATERIALS AND METHODS

Chemicals

All reagents for cell cultures were supplied byEuroClone (Milan, Italy). Rabbit anti-A�PP C ter-minal domain (AB5352) and mouse anti-Fe65 werepurchased from Millipore (Billerica, MA, USA).While rabbit anti-Tip60 from EMD (Gibbstown,NJ, USA). Mouse monoclonal anti �-tubulin was

purchased from Sigma-Aldrich (St. Louis, MO, USA).Host specific peroxidase conjugated IgG secondaryantibodies were obtained from Pierce (Rockford, IL,USA). Electrophoresis reagents were from Bio-Rad(Richmond, CA, USA).

MG-132 and ALLN proteasome inhibitorswere obtained from VWR (Milan, Italy) whileN-Ethylmaleimide (NEM), an IDE inhibitor, waspurchased by Sigma (St Louis, MO, USA). ALLN wassolubilized in DMSO at concentration of 100 mM,frozen in stock aliquots which were diluted at theconcentration of use in medium without FBS andantibiotic, supplemented with 1% L-glutamine.

Cell cultures and treatments

Human embryonic kidney (HEK)293 cells(ECACC) were cultured in Eagle’s minimum essen-tial medium containing 10% fetal bovine serum(FBS), glutamine (2 mM), and penicillin/streptomycin(2 mM). Cells were maintained at 37◦C in a humidified5% CO2 atmosphere.

HEK293 cells, stably transfected with the vec-tor of wild type A�PP isoform of 751 aminoacids (A�PP751), were prepared as follows: 5 × 105

HEK293 cells, seeded in a 6-well plate, were trans-fected with 2.5 �g of A�PP751 vector according tothe LipofectamineTM LTX Reagent’s protocol (Invitro-gen, Milan, Italy). The antibiotic G418 (Sigma, Milan,Italy) was added at a concentration of 600 �g/ml anddrug resistant cells were collected after four weeks.G418-resistant clones were picked and analyzed bywestern blotting for expression of recombinant pro-teins. Stably transfected cells (HEK293-A�PP751)were maintained in medium supplemented with G418at a final concentration of 400 �g/ml.

To study a possible proteasome implication, cellswere treated for 6 h either with 20 �M MG-132 and20 �M ALLN, both diluted in medium, or with DMSOas vehicle control (0.1% final concentration); to eval-uate IDE’s role in HEK293-A�PP751 cells, they weretreated for 2 h with 10 mM NEM [29] prepared for useaccording to data-sheet instruction.

Construction of the vectors

To express the gene of the wild type A�PP isoformof 751 amino acids (A�PP751), HEK293 cells weretransfected with pIRES-GWc-A�PP751-EGFP vectorwhich was engineered as previously described [43].

To obtain AICD57 and AICD59 vectors, PCRproducts were amplified by the PfuUltra High

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Fidelity DNA polymerase (Promega, Milan, Italy)using the following primers: AICD57 for 5′-ATGATAGCGACAGTGATCGTC-3′, AICD57 rev5′-CTAGTTCTGCATCTGCT CAA-3′ and AICD59for 5′-ATGACAGTGATCGTCATCACC-3′, AICD59rev 5′-CTAGTTCTG CATCTGCTCAA-3′. The tem-plate for PCR was the cDNA cloned into thevector pIRES-GWc-A�PP751-EGFP. The amplifiedPCR product was cloned into the pCR®8/GW/TOPO®

entry vector (Gateway® Entry vector, Invitrogen) inorder to generate the entry clones. The resulting vec-tors were recombined with the destination emptyvector, pIRES-GWc-EGFP thus generating pIRES-GWc-C57-EGFP and pIRES-GWc-C59-EGFP.

The destination empty vector pIRES-GWc-EGFPwas obtained by the Gateway® Vector ConversionSystem developed by Invitrogen which allows theconversion of any vector of choice to a Gateway®

Destination vector. Consequently we inserted theGWc cassette (Invitrogen) in the multiple cloningsite of the pIRES2-EGFP vector (BD Biosciences,Clontech), which is between the promoter and theIRES (internal ribosome entry site) sequence, thusour gene of interest is between the IRES sequenceand the EGFP protein. This vector allows theexpression of EGFP and, separately, the protein ofinterest.

The plasmids were purified with the HiSpeed®

Plasmid Midi Kit (Qiagen, Valencia, CA); DNA wasquantified and assayed for purity using a DUR24 530UV/Vis Spectrophotometer (Beckman Coulter Inc.,Fullerton, CA).

All constructs were sequenced by BMR Genomics(Padova, Italy) to check their correctness.

Real-time PCR

For mRNA extraction, 2 × 106 cells were used ina 60 mm2 Petri plate. Total RNA was extracted usingRNeasy Plus Mini Kit (Qiagen, Valencia, CA) follow-ing manufacturer’s instructions. QuantiTect reversiontranscription kit and QuantiTect Syber Green PCRkit (Qiagen, Valencia, CA) were used for cDNAsynthesis and gene expression analysis following man-ufacturer’s specifications. QuantiTect primer assayfor RpL6 were provided by Qiagen, while for p53amplification we used the following primers: p53for 5′-ATGTGCTGTGACTGCTTGTAGA-3′ and p53rev 5′-TCAACAAGATGTTTTGCCAACT-3′. RpL6 RNAtranscription was used as endogenous reference [44]and the quantification of the transcripts was performedby the ��CT method.

Subcellular fractionation

5 × 106 HEK293 wild-type and transfected cellswere seeded in 100 mm2 dishes and treated withALLN, MG-132, or NEM; afterwards the mediumwas removed, and cells were washed with PBS. Thesecells were subsequently homogenized 15 times usinga Teflon glass homogenizer in 0.32 M sucrose bufferedwith 20 mM Tris-HCl (pH 7.4) containing 2 mMEDTA, 10 mM EGTA, 50 mM �-mercaptoethanol,0.3 mM phenylmethylsulfonyl fluoride, and 20 �g/mlleupeptin.

The homogenate was centrifuged at 3600 × g for5 min to obtain the nuclear fraction. The super-natant was centrifuged at 100,000 × g for 30 min;the supernatant obtained represented the cytosolicfraction. The pellet was sonicated in the same homog-enization buffer supplemented with 0.2% (vol/vol)Triton X-100. The sample was incubated at 4◦C for45 min and centrifuged at 100,000 × g for 30 min.The supernatant was separated and represents themembrane fraction. Aliquots of the fractions wereused for protein assay by the Bradford method andthe remaining was boiled for 5 min after dilutionwith sample buffer and subjected to polyacry-lamide gel electrophoresis and immunoblotting asdescribed.

Immunoprecipitation

To analyze AICD, Fe65, and Tip60 interaction,100 �g of the appropriate subcellular fraction wasdiluted in a volume of 500 �l of immunoprecipi-tation buffer (10 mM Tris, pH 7.6; 140 mM NaCl;0.5% NP40 including protease inhibitors). Beforeimmunoprecipitation experiments, an aliquot of 20 �gof protein extracts from each individual sample wereprocessed for western blotting analysis and probedwith anti �-tubulin antibody to validate protein contentmeasurements.

To prevent non-specific binding, the supernatantof immunoprecipitated samples was pre-cleared with10% (w/v) protein A/G (50 �l) for 20 min on ice, fol-lowed by centrifugation.

For immunoprecipitation of the protein of interest(AICD, Fe65, or Tip60), 1 �g of the specific antibodywas added to the samples overnight at 4◦C. Immunocomplexes were collected by using protein A/Gsuspension and washed five times with immunopre-cipitation buffer. Immunoprecipitated complex wererecovered by resuspending the pellets in Laemmli sam-ple buffer. The formation of different complexes was

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detected by western blotting using the specific primaryantibodies.

Immunoblotting

Western blotting samples were prepared mixing thecell lysate with sample buffer (125 mM Tris-HCl pH6.8, 4% SDS, 20% glycerol, 6% �-mercaptoethanol,0.1% bromophenol) and denaturing at 95◦C for 5 min.Samples were electrophoresed into a 10% or 15%SDS-polyacrylamide gel under reducing conditions.Proteins were transferred to nitrocellulose membrane(Amersham, Little Chalfont, UK), which was blockedin TBS-Tween 5% non-fat dry milk, and, subsequentlyincubated with rabbit anti-A�PP C-terminal domain(1 : 500 in TBS-Tween 5% non-fat dry milk), rab-bit anti-Tip60 (1 : 500 in TBS-Tween 5% non-fat drymilk), or mouse anti-Fe65 (1 : 500 in TBS-Tween 5%non-fat dry milk). Mouse monoclonal anti �-tubulinprimary antibody was diluted 1 : 1000 and used ascontrol for protein loading to normalize data. In allexperiments, immunoreactivity was measured usinghost specific secondary IgG peroxidase conjugatedantibodies (1 : 5000 diluted) and ECL (Pierce, USA)as substrate.

Plasmid DNA preparation, transient transfections,and luciferase assays

The p21 luciferase vector was kindly supplied byB. Volgestain, Johns Hopkins University School ofMedicine, Baltimore, MD, USA [14].

Transient transfections were performed in 6 multiwell culture plates; for each well 7 × 105 cells wereseeded in medium without FBS or antibiotics andwith 1% L-glutamine. Transfections were carried outusing Lipofectamine 2000 (Invitrogen Carlsbad, CA)following manufacturer’s instructions. p21 luciferase-reporter construct plasmid DNA was co-transfectedwith pRL-TK Renilla luciferase expressing vector tomeasure transfection efficiency (Promega, Madison,WI). During transfection time HEK293 wild-type andHEK293-A�PP751 were incubated at 37◦C in 5% CO2and then they were lysed with Passive Lysis Buffer1X provided by Dual-Luciferase Reporter Assay Sys-tem following manufacturer’s specifications (Promega,Madison, WI). Luminescence was measured using a20/20 n Luminometer with 10 s of integration (TurnerBioSystems, Sunnyvale, CA). Subsequently HEK293-A�PP751 transfected with p21 were also treated for 6 hwith ALLN in order to confirm real-time PCR data.

Densitometry and statistics

Following acquisition of the western blotting imagethrough an AGFA scanner and analysis by meansof the NIH IMAGE 1.47 program (Wayne Rasband,NIH, Research Services Branch, NIMH, Bethesda,MD, USA), the relative densities of the bands wereexpressed as arbitrary units and normalized to dataobtained from control sample run under the sameconditions.

Data were analyzed using the analysis of variancetest followed, when significant, by an appropriate posthoc comparison such as the Dunnett’s or Student’st-test; a p value < 0.05 was considered significant. Thedata reported are expressed as mean ± SD of at leastthree independent experiments.

RESULTS

Analysis of HEK293 cells engineered withpIRES-EGFP constructs

HEK293 cells were transfected with pIRES-GWc-A�PP751-EGFP, pIRES-GWc-C57-EGFP, andpIRES-GWc-C59-EGFP in order to overexpress andstudy the AICD domain. Since each vector pos-sesses the EGFP protein, transfected cells wereidentified through their ability to emit a green fluo-rescence as shown in Fig. 1A. These transfected cellswere subsequently analyzed by western blotting toevaluate overexpression (Fig. 1B). Cells with pIRES-GWc-A�PP751-EGFP construct showed considerableoverexpression of both A�PP751 and AICD, whereascells transfected with the other two constructs (pIRES-GWc-C57-EGFP and pIRES-GWc-C59-EGFP) andwith the empty vector pIRES-GWc-EGFP did notoverexpress AICD, remaining comparable to theuntransfected control cells.

AICD localization in HEK293 cells overexpressingAβPP751

On the basis of these previous results, we decided tostudy the role of AICD employing HEK293 cells over-expressing A�PP751 (HEK293-A�PP751); this cellline, which shows a visible amount of AICD physi-ologically generated in membrane, represents a goodmodel to carry out AICD’s functions.

Since literature presents different opinions con-cerning AICD cellular localization, we performed afractionation protocol to evaluate in which cellularcompartment these domains are present. Our results

398 E. Buoso et al. / AICD Function and its Degradation Processes

Fig. 1. Microscopic and western blotting analysis of HEK293 cells transfected with A�PP751, C57, and C59 expression vectors for AICDoverexpression. A) HEK293 cells transfected with pIRES-GWc-A�PP751-EGFP, pIRES-GWc-C57-EGFP, and pIRES-GWc-C59-EGFP werefixed in 3.7% formaldehyde and observed by fluorescence microscopy Axioskop 40 (Zeiss). Upper panel shows over-expression of specificconstructs as green fluorescence; bottom panel shows the nuclei of HEK293 cells counterstained with Hoechst 33342. The scale bar correspondsto 20 �M. B) Total lysates of transfected and control cells (CTRL) were analyzed to assess both A�PP751 and AICD protein levels. For thisanalysis the AB5352 antibody, which recognizes the C-terminal portion of A�PP, and �-tubulin antibody for extracts normalization wereused.

Fig. 2. Western blotting analysis of nuclear, cytosolic, and membrane fractions in HEK293 and A�PP751 overexpressing cells. The figure showsthe differences in AICD and its precursor (CTFs) expression between HEK293 control cells (CTRL) and HEK293-A�PP751 cells (A�PP751),in nuclear, cytosolic, and membrane fractions. For this western blotting analysis, the AB5352 antibody, which detects the C-terminal portion ofA�PP, and �-tubulin antibody for extracts normalization were used. Graph reports densitometric analysis of nuclear, cytosolic, and membranefraction immunoblots performed. Bar represent the mean values ± S.D. of three independent experiment with **p < 0.01 versus control cells(CTRL); Student’s t- test.

demonstrated that AICD was present in a significantmanner at the nuclear level while we were not able todetect them in the cytosol. The AICD nuclear presencecorrelated with a significant increase in the membraneof CTF fragments which are AICD precursors (Fig. 2).

p53 expression in HEK293-AβPP751 cells

Considering that AICD were detected in the nucleus,we analyzed p53 mRNA expression level in HEK293-A�PP751. Our data showed that, in this cell line,

p53 expression is upregulated compared to HEK293wild-type cells (Fig. 3A). Since p21 is the first targetof activated p53, we further evaluated p21 promoteractivation. Our observations indicated that p53 over-expression was correlated to p21 promoter activation(Fig. 3B).

Proteasome inhibitor treatments

After 6 h exposure to 20 �M MG-132 andALLN, HEK293-A�PP751 subcellular fractions were

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Fig. 3. p53 expression and its p21 target gene in HEK293-A�PP751 cells. A) HEK293-A�PP751 cells (A�PP751) show a significant increasein p53 expression levels compared to control cells (CTRL). Statistical analysis was performed with Student’s t-test; bars represent the meanvalues ± S.D. with **p < 0.01 versus the CTRL values. B) Control (CTRL) and HEK293-A�PP751 (A�PP751) cells were transfected with p21-lucpromoter vector and subsequently luciferase activity, expressed as RLU%, was measured. Statistical analysis was performed with Student’st-test with *p < 0.05 versus the CTRL values; bars represent the mean values ± S.D of three independent experiments.

Fig. 4. Proteasome inhibitors effect on HEK293- A�PP751 subcellular fractions. Representative blot of membrane (A) and nuclear (B) fraction ofHEK293- A�PP751 treated for 6 h with or without 20 �M MG-132 or ALLN. Graphs report densitometric analysis of immunoblots with *p < 0.05and **p < 0.01 versus membrane A�PP751; ***p < 0.001 vs nucleus A�PP751; bars represent the mean values ± S.D. of three independentexperiments; Dunnett’s t-test.

separated. According to the literature [23, 24], bothtreatments were able to induce a considerable increaseof CTFs in the membrane compartment comparedto control cells thus highlighting how proteasomeactivity could be involved in CTFs degradation andconsequently in AICD production (Fig. 4A). How-ever, while ALLN induced a related increase ofAICD in the nucleus, MG-132 did not, becauseof its �-secretase inhibitory activity (Fig. 4B) [23,24, 42]. Both proteasome inhibitors thus providedmore substrate to the �-secretase, but MG-132

impairing �-secretase activity blocked the productionof AICD.

IDE involvement in AICD degradation

Since IDE is preferentially located in the cytosoland data from the literature suggested its rolein AICD removal in this compartment, we eval-uated whether IDE exerted a degradative functionon this domain. Consistent with a previous study[45], HEK293-A�PP751 cells were exposed for 2 h

400 E. Buoso et al. / AICD Function and its Degradation Processes

Fig. 5. AICD presence in the cytosol and nucleus subsequent to NEM treatment. Cellular lysates were fractionated to obtain cytosol and westernblotting analysis was performed to evaluate AICD presence in this cellular compartment. The figure shows a representative immunoblot ofHEK-A�PP751 treated with 10 mM NEM for 2 h. Graph reports densitometric analysis of cytosolic (A) and nuclear (B) fraction immunoblotsperformed. Bar represent the mean values ± S.D. of three independent experiment with ***p < 0.001 versus control cells (A�PP751); Student’st-test.

to 10 mM NEM, an IDE inhibitor. After treatment,subcellular fractions were isolated and analyzed bywestern blotting. Unlike proteasome inhibitor treat-ments, NEM exposure promoted AICD accumulationin the cytosol (Fig. 5A), thus suggesting that IDE isinvolved in AICD cytosolic removal. No variationswere observed in the nucleus, where the presenceof AICD remained comparable to untreated cells(Fig. 5B).

p53 expression after inhibitors treatment

Based on the results of the ALLN treatment(Fig. 4), we investigated whether the AICD increasein the nucleus may affect p53 mRNA expression. Forthis purpose, HEK293-A�PP751 cells were exposedto ALLN inhibitor and then RNA was extractedto analyze p53 expression level by real-time PCR.Cells treated with ALLN showed an increase ofp53 expression level (Fig. 6), which could be cor-related to the AICD nuclear increase observed bywestern blotting. Furthermore, we evaluated p53expression after 2 h of 10 mM NEM exposure inHEK293-A�PP751 cells. Contrary to ALLN, NEMtreatment was not able to influence p53 expres-sion, thus supporting the observation, obtained from

Fig. 6. p53 mRNA expression analysis after NEM and ALLN treat-ment. Real-time PCR analysis of cells after treatment for 2 h with10 mM NEM and for 6 h with 20 �M ALLN and MG-132. Barsrepresent the mean values ± S.D with **p < 0.01 versus CTRL; Dun-nett’s t-test.

western blotting experiments, that AICD levels inthe nucleus were not modified by NEM treatment(Fig. 6). In addition, we also analyzed p21 promoteractivity data following ALLN treatment. We per-formed a luciferase assay in HEK293-A�PP751 cellsand found that ALLN treatment promoted a higherp21 promoter activation compared to untreated cells(Fig. 7).

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Fig. 7. ALLN influence on p21 promoter activation. HEK293-A�PP751 cells were transfected with p21 luc promoter vector,treated for 6 h with or without 20 �M ALLN and luciferase activity,expressed as RLU%, was measured. Bars represent the mean val-ues ± S.D. of three independent experiments. Statistical analysis wasperformed with Student’s t-test with ***p < 0.001 versus A�PP751values.

AICD/Fe65/Tip60 complex assemblyin HEK293-AβPP751

Fe65/AICD/Tip60 complex modulationafter proteasome inhibitors treatment

HEK293-A�PP751 cells were exposed for 6 h to20 �M MG-132 and ALLN. The membrane fractionwas then immunoprecipitated with anti-Fe65 antibodyand western blotting analysis was performed with theantibody which recognizes AICD (Fig. 8). We foundthat proteasome inhibition increased Fe65 interactionwith CTFs in membrane.

Since we showed that ALLN enhanced the pres-ence of AICD in the nucleus and some literature dataargue that, after Fe65 binding to CTFs in membrane,the AICD/Fe65 complex translocates to the nucleus,we evaluated whether these domains where still asso-ciated to Fe65 within the nucleus as detected in themembrane fraction. After expose to 20 �M ALLN for6 h, the nuclear fraction was immunoprecipitated withanti-AICD antibody, and western blotting analysis wasconducted with the anti-Fe65 antibody. We found that,in the nucleus, AICD was associated with Fe65 toconstitute the AICD/Fe65 complex and that ALLNtreatment was able to promote a consistent increaseof this complex compared to untreated cells (Fig. 9A).

The nuclear fraction was also immunoprecipitatedwith the anti-Tip60 antibody in order to appraise eitherthe interaction of Fe65 with Tip60 or the presenceof the entire AICD/Fe65/Tip60 complex. In the firstcase, western blotting analysis using the anti-Fe65antibody showed how the ALLN treatment increasesthe Fe65/Tip60 association (Fig. 9B). In the second

Fig. 8. Fe65 binding to CTFs improved by proteasome inhibition.HEK293-A�PP751 cells exposed for 6 h to 20 �M MG-132 andALLN were immunopreciptated with the antibody against Fe65 andwestern blotting analysis was performed with the antibody whichrecognized the AICD. Immunoprecipitated antibody was omittedin negative control samples (blank). �-tubulin was indicated toappreciate protein content measurements. Graph reports densito-metric analysis of membrane fraction immunoblots performed. Barrepresent the mean values ± S.D. of three independent experimentwith *p < 0.05, **p < 0.01 versus control cells (A�PP751); Dunnett’st-test.

case, the western blotting examination was conductedwith the anti A�PP C-terminal domain antibody andshowed that ALLN treatment favored the increase ofAICD/Fe65/Tip60 complex formation (Fig. 9C).

Fe65/AICD/Tip60 complex in HEK293-AβPP751

cells treated with IDE inhibitorWe immunoprecipitated the nuclear cellular com-

partment with Tip60 and then we conducted thewestern blotting analysis using the anti-A�PPC-terminal antibody. NEM treatment was not able toinfluence AICD/Fe65/Tip60 complex, unlike ALLNtreatment (Fig. 9C).

To evaluate whether NEM action may affectthe Fe65/AICD complex in the cytosol, HEK293-A�PP751 cells were treated and immunoprecipitatedwith the anti-A�PP C terminal antibody and westernblotting analysis was then performed with the anti-Fe65 antibody. We found that Fe65/AICD complexwas not influenced by NEM treatment (Fig. 10).

These data suggest that AICD nuclear levels werenot influenced by NEM treatment and that the AICDremoved by IDE was not involved in Fe65/Tip60complex.

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Fig. 9. Nuclear fraction immunoprecipitation to detect AICD/Fe65/Tip60 variation dependent to proteasome inhibition. A) Protein extractsderived from nuclear fraction of untreated HEK293-A�PP751 cell line (CTRL) and treated for 6 h with 20 �M ALLN (ALLN) were immuno-precipitated with the A�PP C-terminal domain antibody. Immunoprecipitates were analyzed by western blotting with anti-Fe65 antibody.Immunoprecipitated antibodies were omitted in negative control samples (blank). Graph reports densitometric analysis of nuclear fraction. Barrepresent the mean values ± S.D. of three independent experiment with *p < 0.05 versus control cells (A�PP751); Student’s t-test. B) Nuclearfraction of HEK293-A�PP751 cell line control (CTRL) and exposed to 20 �M ALLN for 6 h (ALLN) were immunoprecipitated with the Tip60antibody. Immunoprecipitates were analyzed by western blotting with anti-Fe65 antibody. Graph reports densitometric analysis of nuclear frac-tion. Bar represent the mean values ± S.D. of three independent experiment with *p < 0.05 versus control cells (A�PP751); Student’s t-test. C)Nuclear fraction exposed to 20 �M ALLN for 6 h (ALLN) and 10 mM NEM for 2 h (NEM) were immunoprecipitated with the Tip60 antibodyand also analyzed by western blotting with anti-A�PP C-terminal domain antibody. Immunoprecipitated antibodies were omitted in negativecontrol samples (blank). Graph reports densitometric analysis of nuclear fraction. Bar represent the mean values ± S.D. of three independentexperiment with *p < 0.05 versus control cells (A�PP751); Dunnett’s t- test. Before immunoprecipitation experiments, an aliquot of 20 �g ofprotein extracts from each individual sample was processed for western blot analysis and probed with anti �-tubulin antibody in order to validateprotein content measurements.

Fig. 10. Cytosol and nuclear fraction immunoprecipitation to detectAICD/Fe65/Tip60 variation dependent to IDE inhibition. Cytosolfractions of HEK293-A�PP751 control (CTRL) and exposed to20 �M ALLN for 6 h (ALLN) were immunoprecipitated with theantibody directed against the AICD domain. Immunoprecipitateswere analyzed by western blotting with anti-Fe65 antibody.

DISCUSSION

To achieve our goal, we engineered three differentvectors for AICD overexpression: pIRES-GWc-

A�PP751, pIRES-GWc-C57-EGFP, and pIRES-GWc-C59-EGFP. One remarkable difference in the use ofthese vectors consists of the process of AICD gener-ation. Endogenous AICD is generated from A�PP bycleavage in the membrane region, whereas exogenousAICD is synthesized in the cytoplasm as a cyto-plasmic protein. Since the expression of the entireA�PP751 protein was necessary to observe a consid-erable amount of A�PP-CTFs in membrane with acorresponding AICD presence in the nucleus, we heldthat AICD generation in the membrane can be essentialto obtain the AICD domain as other authors suggested[18, 25].

We demonstrated that proteasome activity influ-ence A�PP proteolytic processing according to studiespreviously discussed; proteasome inhibition mediatedby ALLN promoted A�PP-CTF accumulation at themembrane, thus increasing their availability for �-secretase cleavage. In fact, ALLN treatment induced asignificant AICD increase at the nuclear level. ALLNexposure also favored the Fe65 binding increase to

E. Buoso et al. / AICD Function and its Degradation Processes 403

A�PP-CTFs. Since Tip60 has been identified as aFe65 nuclear binding partner [46], we further eval-uated the existence of the entire complex inside thenucleus. According to the literature [10], we demon-strated that the AFT complex was present in transfectedcontrol cells and it was positively influenced by ALLNtreatment; this event is in agreement with the increaseof Fe65 binding to A�PP-CTFs in membrane wherethe AICD/Fe65 complex is generated as suggested inthe model III. Hence, our findings correlate with theobservations that the intramembranous proteolysis ofA�PP could play a signaling role [9, 47]. It was alsodemonstrated that AICD regulates phosphoinositide-mediated calcium signaling through a �-secretasedependent signaling pathway [48]. Although someauthors found no consistent effects of �-secretaseinhibitors or of genetic deficiencies in the �-secretasecomplex or the A�PP family on the expression levels ofKAI1, GSK-3�, A�PP, and neprylisin genes [49, 50], arecent work revealed by chromatin immunoprecipita-tion that AICD was associated with the NEP promoter[51]. Concerning the p53 gene, Checler and coworkers[12] demonstrated that AICD controls this gene at atranscriptional level, both in vitro and in vivo. As thispurpose, our data demonstrated that the accumulationof AFT complex in the nucleus, induced by ALLN, cor-related with p53 overexpression, which in turn inducedp21 activation.

Altogether these data establish that proteasome caninfluence AICD generation and consequently its par-ticipation in Fe65/Tip60 complex as we previouslyhypothesized [36].

Aiming to investigate the role of IDE in AICDdegradation, HEK293 cells were exposed to NEM fol-lowing previously described procedures; this treatmentdetermined AICD accumulation at the level of thecytosol, thus suggesting that this enzyme is involvedin AICD degradation in this cellular compartment.However, this cytosolic accumulation did not promoteany nuclear modification of the AICD level, whichremained comparable to untreated cells. Moreover,unlike ALLN treatment, NEM treatment did not mod-ify the presence of the AFT complex in the nucleus.This observation was in agreement with both westernblotting and p53 expression data (Fig. 6). Altogetherthese data indicate that the AICD removed by IDE donot translocate into the nucleus and is not involved inFe65/Tip60 complex.

A recently published work established that the�-secretase is the predominant pathway generatingthe transcriptionally active AICD/Fe65/Tip60 com-plex [27]. This observation supports the concept that

AICD function is related to the metabolic pathwaythrough which it is generated, and this aspect can beintegrated with our degradation data; since we demon-strated that proteasome activity could prevent nuclearAICD formation, we could suggest that the transcrip-tionally active AICD deriving from amyloidogenicpathway is influenced by proteasome activity. On thecontrary, since �-secretase action is predominantly atthe cell surface, the subsequent action of �-secretasemay induce AICD release in the cytosol where theycan be degraded by IDE [45, 51]. This considera-tion is consistent with our NEM data showing thatIDE inhibition promoted AICD accumulation in thecytosol without affecting nuclear AICD levels and theirtranscriptional function. Nevertheless, further investi-gation is requested to elucidate the functions of AICDdegraded by IDE.

In conclusion our data help to solve the discrepancyin literature between AICD function and degradationwithin Fe65/Tip60 complex, suggesting the existencewithin cells of at least two different AICD degradationmechanisms. In particular, we have demonstrated thatthere is a correlation between the role of AICD in generegulation within Fe65/Tip60 complex and its removaldependent by proteasome activity. Evidence suggeststhat the proteasome system could be impaired in AD[41, 52, 53]. This decrease in proteasome-dependentprocessing would be expected to cause an increasein A�PP-CTFs available for �-secretase processing,thereby increasing AICD production, which is in turnresponsible for an alteration in gene expression andmay result in different effects such as modificationsin cytoskeletal dynamics [54]. Defective proteasomefunction could directly contribute to increase A� pro-duction in cases of sporadic AD [26, 55]. These datahighlight that the involvement of the proteasome activ-ity in A�PP processing has important consequencesfor the regulation of A�PP intracellular fragment pro-duction and could be involved in the pathogenesis ofAD.

On the other hand, results obtained by NEMtreatments underline the presence of an additionalmechanism which is involved in the removal of AICDwhich does not exert nuclear activity and does not takepart in Fe65/Tip60 complex.

ACKNOWLEDGMENTS

This work was supported by the contribution ofgrants from CARIPLO to M.R. and grant PRIN-2009B7ASKP to S.G. We are grateful to Giulia

404 E. Buoso et al. / AICD Function and its Degradation Processes

Stefania Porcari for careful editing and proofreadingof the manuscript.

Authors’ disclosures available online (http://www.j-alz.com/disclosures/view.php?id=1175).

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