APP Intracellular C-Terminal Domain Function is Related ... - IOS Press

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

A␤PP Intracellular C-Terminal Domain Function is Related to its Degradation Processes Erica Buosoa , Fabrizio Biundoa , Cristina Lannia , Gennaro Schettinib , Stefano Govonia and Marco Racchia,∗ a Department

of Drug Sciences, Pharmacology Unit, Center of Excellence in Applied Biology, University of Pavia, Pavia, Italy b Department 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-amyloidogenic pathway; both pathways lead to release of the A␤PP intracellular C-terminal domain (AICD). AICD involvement in signal transduction within Fe65/Tip60 complex is one of the most discussed mechanisms, and different models have been hypothesized to explain the role of AICD within this complex. The analysis of these models in relation to the degradation processes highlights the discrepancy among AICD localization, function, and degradation, leading to the hypothesis that a signaling mechanism may exist which allows A␤PP proteolysis to generate either a transcriptionally active fragment or an inactive one with different involvement of proteasome and IDE (insulin-degrading enzyme). Our work aimed to analyze the functional role of AICD within the Fe65/Tip60 complex considering the AICD degradation processes. Our data suggest a correlation between the role of AICD in gene regulation and its removal operated by proteasome activity. Moreover, treatments with IDE inhibitor underlined the presence of an alternative mechanism involved in AICD removal when the latter is not exerting nuclear activity, thus providing clearer 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 type I transmembrane protein with a large extracellular portion, a membrane anchoring domain, and a short intracellular C-terminal tail. A␤PP can be processed by two distinct proteolytic pathways. The non-amylodogenic pathway involves cleavage by the enzyme ␣-secretase, which cuts A␤PP within the amyloid-␤ (A␤) sequence thereby preventing the ∗ Correspondence to: Marco Racchi, PhD, Department of Drug Sciences, Pharmacology Unit, University of Pavia, Viale Taramelli 14, 27100 Pavia, Italy. Tel.: +39 0382 987738; Fax: +39 0382 987405; E-mail: [email protected].

formation of A␤ [1]. The amyloidogenic pathway involves cleavage of A␤PP by an enzyme referred to as ␤-secretase which cleaves A␤PP at the N-terminal side of the A␤ sequence. Following cleavage by ␣or ␤-secretase, the ␥-secretase complex cleaves inside the membrane the remaining C-terminal fragments of A␤PP, respectively C83 and C99, via a mechanism referred to as regulated intramembrane proteolysis. C83 and C99 cleavage generates p3 and A␤ respectively together with A␤PP intracellular C-terminal domain (AICD) [2]. AICD levels are detectable in membrane fractions of murine total brain homogenates and increase significantly in mice overexpressing the Swedish mutation of human A␤PP [3]. The potential importance of AICD

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has been emphasized by the recognition of similarities between A␤PP and another type I transmembrane protein called Notch [4]. This analogy of A␤PP processing to Notch receptor signaling suggested a possible function for AICD in nuclear signaling [5–7]. The function of AICD as a signaling protein has been suggested to be dependent to its interaction with several transcriptional co-activators. It has been demonstrated that the central YENPTY motif of AICD binds to the phosphotyrosine binding domain 2 (PTB2) of the adaptor protein Fe65 [8], generating the Fe65/AICD complex which is able to bind the histone acetyltransferase tat-interactive protein (Tip60) [9], forming a transcriptionally active complex referred to as an AICD-Fe65-Tip60 (AFT) complex. Data from literature indicate that the AFT complex is able to regulate at the transcriptional level different genes such as KAI, neprylisin, EGFR, A␤PP itself [10], and p53 expression [11]. Moreover AICD-mediated p53 activation can be associated with cell death in Alzheimer’s disease (AD) [12, 13]. The tumor suppressor p53 is a short-lived transcription factor that is post-translationally regulated by the ubiquitin-proteasome pathway [14, 15]. In response to cellular stress or DNA damage, wild-type p53 protein is activated by phosphorylation and other signals that cause it to dissociate from its inhibitor MDM2. Once activated, p53 is no longer targeted for proteasomal degradation by MDM2 and therefore accumulates and binds to specific sequences in DNA, initiating transcription of genes that induce growth arrest, DNA repair, or apoptosis. In some cancer cells, proteasome inhibitors cause the stabilization and accumulation of p53 protein [15]; an increase of p53 mRNA was not observed in proteasome-inhibited cells [16]. The role of AICD in signal transduction is, to date, one of the most controversially discussed mechanisms and the main differences refer to the cellular compartment where the interaction of the complex components takes place. Considering these conflicting observations, Müller and colleagues [17] summarized different models of interaction of the various components of the complex. In model I, the C-terminal domain of A␤PP recruits at the membrane Fe65 which undergoes a conformational change necessary for its activation. Subsequently, Fe65 translocates into the nucleus and binds to Tip60 [18]; in model II, Fe65 and AICD translocate into the nucleus independently, building up the ternary complex AICD/Fe65/Tip60 in the nucleus [19]. Finally, most authors studying AICD localization reported release of AICD from the membrane and translocation into the nucleus after

␥-secretase cleavage of the precursor. In this last model, AICD/Fe65 complex generates at the membrane and subsequently, after ␥-secretase cleavage, translocates into the nucleus. Following translocation, Tip60 enters the complex to generate the transcription factor [20, 21]. We turned our attention to the AICD degradation processes because these mechanisms may help to elucidate some of the controversies outlined above; specifically the two best characterized mechanisms of AICD degradation are the proteasome and the insulin degrading enzyme (IDE). Literature data indicated that proteasome inhibition could increase A␤PP processing specifically at the ␥secretase site [22]. In this regard, it was demonstrated that the proteasome is capable of directly cleaving the cytoplasmic domain of A␤PP at several sites, including a region around the YENPTY sequence which interacts with Fe65. This proteasomal cleavage decreases the availability of A␤PP-CTF for ␥-secretase cleavage and consequently inhibits AICD production [23–25]. On the basis of these results, Kerr and Small proposed that the proteasome is not able to degrade A␤PP-CTF when this fragment is bound by A␤PP-binding protein such as Fe65 [26]. IDE is a 110-kDa thiol zinc-methalloendopeptidase that can cleave A␤ peptides and the AICD [27–30] both in vivo and in vitro [31, 32]. The subcellular localization shows that IDE is abundant in cytosol [33]; this observation is supported by biochemical studies which have revealed the IDE presence in the soluble fractions from human brain which contained both extracellular and cytosolic compartments [34, 35]. The analysis of AICD signal transduction models, in relation to these two putative degradation processes, highlights the contrast among mechanism responsible of AICD localization, function, and degradation [36]. On the basis of this consideration in the models I and III, the AICD degradation is primarily due to proteasome action which can be inhibited by Fe65 binding to A␤PP-CTF as suggested by Kerr and Small [26]. In contrast to model III, in model I the AICD does not translocate to the nucleus but it is released in the cytosol becoming an IDE substrate. The model presented by Nakaya and Suzuki [19] argues that the AICD is released alone at the cytosol level, thus it does not act as an anchoring site but acquires the faculty to regulate gene expression in association with Fe65/Tip60 complex in the nucleus. Considering this one as a putative mechanism of AICD’s action, IDE is the only process involved in AICD degradation [36].


Elucidations about the role of AICD and its degradation can contribute to better understand its involvement in AD pathology. In particular, a putative contribution of these proteolytic fragments has been demonstrated in transgenic mice overexpressing AICD. Increased levels of AICD are responsible for a series of events, including the activation of GSK-3␤ [3], hyperphosphorylation and aggregation of tau protein, microtubule destabilization, and reduction of nuclear ␤-catenin levels, thus causing a loss of cell-cell contact mechanisms that may contribute to neurotoxicity in AD. Subsequent neurodegeneration and working memory deficits were also observed in these transgenic mice [37], as well as abnormal spiking events in their electroencephalograms and susceptibility to kainic acid-induced seizures independent of A␤ [38]. AICD could also alter gene expression and induce neuron-specific apoptosis [39, 40]. Elevated AICD levels have also been reported in AD brains [37]; the reason for their increased levels in AD brain is still unknown, though it could be related to an impairment of proteasome degradation pathway [41]. Taking into account this background, our work aimed to analyze the role of AICD within Fe65/Tip60 complex integrating the putative degradation mechanisms. In order to verify the pathway-specific activity of proteasome in AICD removal as previously suggested, we used two proteasome inhibitors, MG-132 and ALLN. MG132 is a non selective protease inhibitor because it is also able to inhibit ␥-secretase; it causes a large accumulation of A␤PP-CTF, by inhibiting both ␥-secretase and the proteasome. ALLN, a proteasome specific inhibitor, does not act on ␥-secretase and thus causes an increase in A␤PP-CTF providing more substrate available for ␥-secretase cleavage [23, 24, 42]. Our data highlight a correlation between AICDmediated proteasome removal and gene expression regulation supporting the existence of at least two distinct AICD degradation processes.

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purchased from Sigma-Aldrich (St. Louis, MO, USA). Host specific peroxidase conjugated IgG secondary antibodies were obtained from Pierce (Rockford, IL, USA). Electrophoresis reagents were from Bio-Rad (Richmond, CA, USA). MG-132 and ALLN proteasome inhibitors were obtained from VWR (Milan, Italy) while N-Ethylmaleimide (NEM), an IDE inhibitor, was purchased by Sigma (St Louis, MO, USA). ALLN was solubilized in DMSO at concentration of 100 mM, frozen in stock aliquots which were diluted at the concentration of use in medium without FBS and antibiotic, supplemented with 1% L-glutamine. Cell cultures and treatments

MATERIALS AND METHODS

Human embryonic kidney (HEK)293 cells (ECACC) were cultured in Eagle’s minimum essential medium containing 10% fetal bovine serum (FBS), glutamine (2 mM), and penicillin/streptomycin (2 mM). Cells were maintained at 37◦ C in a humidified 5% CO2 atmosphere. HEK293 cells, stably transfected with the vector of wild type A␤PP isoform of 751 amino acids (A␤PP751 ), were prepared as follows: 5 × 105 HEK293 cells, seeded in a 6-well plate, were transfected with 2.5 ␮g of A␤PP751 vector according to the LipofectamineTM LTX Reagent’s protocol (Invitrogen, Milan, Italy). The antibiotic G418 (Sigma, Milan, Italy) was added at a concentration of 600 ␮g/ml and drug resistant cells were collected after four weeks. G418-resistant clones were picked and analyzed by western blotting for expression of recombinant proteins. Stably transfected cells (HEK293-A␤PP751 ) were maintained in medium supplemented with G418 at a final concentration of 400 ␮g/ml. To study a possible proteasome implication, cells were treated for 6 h either with 20 ␮M MG-132 and 20 ␮M ALLN, both diluted in medium, or with DMSO as vehicle control (0.1% final concentration); to evaluate IDE’s role in HEK293-A␤PP751 cells, they were treated for 2 h with 10 mM NEM [29] prepared for use according to data-sheet instruction.

Chemicals

Construction of the vectors

All reagents for cell cultures were supplied by EuroClone (Milan, Italy). Rabbit anti-A␤PP C terminal domain (AB5352) and mouse anti-Fe65 were purchased from Millipore (Billerica, MA, USA). While rabbit anti-Tip60 from EMD (Gibbstown, NJ, USA). Mouse monoclonal anti ␣-tubulin was

To express the gene of the wild type A␤PP isoform of 751 amino acids (A␤PP751 ), HEK293 cells were transfected with pIRES-GWc-A␤PP751 -EGFP vector which was engineered as previously described [43]. To obtain AICD57 and AICD59 vectors, PCR products 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 rev 5 -CTAGTTCTGCATCTGCT CAA-3 and AICD59 for 5 -ATGACAGTGATCGTCATCACC-3 , AICD59 rev 5 -CTAGTTCTG CATCTGCTCAA-3 . The template for PCR was the cDNA cloned into the vector pIRES-GWc-A␤PP751 -EGFP. The amplified PCR product was cloned into the pCR® 8/GW/TOPO® entry vector (Gateway® Entry vector, Invitrogen) in order to generate the entry clones. The resulting vectors were recombined with the destination empty vector, pIRES-GWc-EGFP thus generating pIRESGWc-C57-EGFP and pIRES-GWc-C59-EGFP. The destination empty vector pIRES-GWc-EGFP was obtained by the Gateway® Vector Conversion System developed by Invitrogen which allows the conversion of any vector of choice to a Gateway® Destination vector. Consequently we inserted the GWc cassette (Invitrogen) in the multiple cloning site of the pIRES2-EGFP vector (BD Biosciences, Clontech), which is between the promoter and the IRES (internal ribosome entry site) sequence, thus our gene of interest is between the IRES sequence and the EGFP protein. This vector allows the expression of EGFP and, separately, the protein of interest. The plasmids were purified with the HiSpeed® Plasmid Midi Kit (Qiagen, Valencia, CA); DNA was quantified and assayed for purity using a DUR24 530 UV/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 in a 60 mm2 Petri plate. Total RNA was extracted using RNeasy Plus Mini Kit (Qiagen, Valencia, CA) following manufacturer’s instructions. QuantiTect reversion transcription kit and QuantiTect Syber Green PCR kit (Qiagen, Valencia, CA) were used for cDNA synthesis and gene expression analysis following manufacturer’s specifications. QuantiTect primer assay for RpL6 were provided by Qiagen, while for p53 amplification we used the following primers: p53for 5 ATGTGCTGTGACTGCTTGTAGA-3 and p53rev 5 TCAACAAGATGTTTTGCCAACT-3 . RpL6 RNA transcription was used as endogenous reference [44] and the quantification of the transcripts was performed by the CT method.

Subcellular fractionation 5 × 106 HEK293 wild-type and transfected cells were seeded in 100 mm2 dishes and treated with ALLN, MG-132, or NEM; afterwards the medium was removed, and cells were washed with PBS. These cells were subsequently homogenized 15 times using a Teflon glass homogenizer in 0.32 M sucrose buffered with 20 mM Tris-HCl (pH 7.4) containing 2 mM EDTA, 10 mM EGTA, 50 mM ␤-mercaptoethanol, 0.3 mM phenylmethylsulfonyl fluoride, and 20 ␮g/ml leupeptin. The homogenate was centrifuged at 3600 × g for 5 min to obtain the nuclear fraction. The supernatant was centrifuged at 100,000 × g for 30 min; the supernatant obtained represented the cytosolic fraction. The pellet was sonicated in the same homogenization buffer supplemented with 0.2% (vol/vol) Triton X-100. The sample was incubated at 4◦ C for 45 min and centrifuged at 100,000 × g for 30 min. The supernatant was separated and represents the membrane fraction. Aliquots of the fractions were used for protein assay by the Bradford method and the remaining was boiled for 5 min after dilution with sample buffer and subjected to polyacrylamide gel electrophoresis and immunoblotting as described. Immunoprecipitation To analyze AICD, Fe65, and Tip60 interaction, 100 ␮g of the appropriate subcellular fraction was diluted in a volume of 500 ␮l of immunoprecipitation buffer (10 mM Tris, pH 7.6; 140 mM NaCl; 0.5% NP40 including protease inhibitors). Before immunoprecipitation experiments, an aliquot of 20 ␮g of protein extracts from each individual sample were processed for western blotting analysis and probed with anti ␣-tubulin antibody to validate protein content measurements. To prevent non-specific binding, the supernatant of immunoprecipitated samples was pre-cleared with 10% (w/v) protein A/G (50 ␮l) for 20 min on ice, followed by centrifugation. For immunoprecipitation of the protein of interest (AICD, Fe65, or Tip60), 1 ␮g of the specific antibody was added to the samples overnight at 4◦ C. Immuno complexes were collected by using protein A/G suspension and washed five times with immunoprecipitation buffer. Immunoprecipitated complex were recovered by resuspending the pellets in Laemmli sample buffer. The formation of different complexes was


detected by western blotting using the specific primary antibodies. Immunoblotting Western blotting samples were prepared mixing the cell lysate with sample buffer (125 mM Tris-HCl pH 6.8, 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 blocked in TBS-Tween 5% non-fat dry milk, and, subsequently incubated with rabbit anti-A␤PP C-terminal domain (1 : 500 in TBS-Tween 5% non-fat dry milk), rabbit anti-Tip60 (1 : 500 in TBS-Tween 5% non-fat dry milk), or mouse anti-Fe65 (1 : 500 in TBS-Tween 5% non-fat dry milk). Mouse monoclonal anti ␣-tubulin primary antibody was diluted 1 : 1000 and used as control for protein loading to normalize data. In all experiments, immunoreactivity was measured using host specific secondary IgG peroxidase conjugated antibodies (1 : 5000 diluted) and ECL (Pierce, USA) as substrate. Plasmid DNA preparation, transient transfections, and luciferase assays The p21 luciferase vector was kindly supplied by B. Volgestain, Johns Hopkins University School of Medicine, Baltimore, MD, USA [14]. Transient transfections were performed in 6 multi well culture plates; for each well 7 × 105 cells were seeded in medium without FBS or antibiotics and with 1% L-glutamine. Transfections were carried out using Lipofectamine 2000 (Invitrogen Carlsbad, CA) following manufacturer’s instructions. p21 luciferasereporter construct plasmid DNA was co-transfected with pRL-TK Renilla luciferase expressing vector to measure transfection efficiency (Promega, Madison, WI). During transfection time HEK293 wild-type and HEK293-A␤PP751 were incubated at 37◦ C in 5% CO2 and then they were lysed with Passive Lysis Buffer 1X provided by Dual-Luciferase Reporter Assay System following manufacturer’s specifications (Promega, Madison, WI). Luminescence was measured using a 20/20 n Luminometer with 10 s of integration (Turner BioSystems, Sunnyvale, CA). Subsequently HEK293A␤PP751 transfected with p21 were also treated for 6 h with ALLN in order to confirm real-time PCR data.

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Densitometry and statistics Following acquisition of the western blotting image through an AGFA scanner and analysis by means of the NIH IMAGE 1.47 program (Wayne Rasband, NIH, Research Services Branch, NIMH, Bethesda, MD, USA), the relative densities of the bands were expressed as arbitrary units and normalized to data obtained from control sample run under the same conditions. Data were analyzed using the analysis of variance test followed, when significant, by an appropriate post hoc comparison such as the Dunnett’s or Student’s t-test; a p value < 0.05 was considered significant. The data reported are expressed as mean ± SD of at least three independent experiments. RESULTS Analysis of HEK293 cells engineered with pIRES-EGFP constructs HEK293 cells were transfected with pIRESGWc-A␤PP751 -EGFP, pIRES-GWc-C57-EGFP, and pIRES-GWc-C59-EGFP in order to overexpress and study the AICD domain. Since each vector possesses the EGFP protein, transfected cells were identified through their ability to emit a green fluorescence as shown in Fig. 1A. These transfected cells were subsequently analyzed by western blotting to evaluate overexpression (Fig. 1B). Cells with pIRESGWc-A␤PP751 -EGFP construct showed considerable overexpression of both A␤PP751 and AICD, whereas cells transfected with the other two constructs (pIRESGWc-C57-EGFP and pIRES-GWc-C59-EGFP) and with the empty vector pIRES-GWc-EGFP did not overexpress AICD, remaining comparable to the untransfected control cells. AICD localization in HEK293 cells overexpressing AβPP751 On the basis of these previous results, we decided to study the role of AICD employing HEK293 cells overexpressing A␤PP751 (HEK293-A␤PP751 ); this cell line, which shows a visible amount of AICD physiologically generated in membrane, represents a good model to carry out AICD’s functions. Since literature presents different opinions concerning AICD cellular localization, we performed a fractionation protocol to evaluate in which cellular compartment these domains are present. Our results

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

Fig. 2. Western blotting analysis of nuclear, cytosolic, and membrane fractions in HEK293 and A␤PP751 overexpressing cells. The figure shows the 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 of A␤PP, and ␣-tubulin antibody for extracts normalization were used. Graph reports densitometric analysis of nuclear, cytosolic, and membrane fraction 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 significant manner at the nuclear level while we were not able to detect them in the cytosol. The AICD nuclear presence correlated with a significant increase in the membrane of CTF fragments which are AICD precursors (Fig. 2).

p53 expression is upregulated compared to HEK293 wild-type cells (Fig. 3A). Since p21 is the first target of activated p53, we further evaluated p21 promoter activation. Our observations indicated that p53 overexpression was correlated to p21 promoter activation (Fig. 3B).

p53 expression in HEK293-AβPP751 cells Proteasome inhibitor treatments Considering that AICD were detected in the nucleus, we analyzed p53 mRNA expression level in HEK293A␤PP751 . Our data showed that, in this cell line,

After 6 h exposure to 20 ␮M MG-132 and ALLN, 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 increase in p53 expression levels compared to control cells (CTRL). Statistical analysis was performed with Student’s t-test; bars represent the mean values ± S.D. with **p < 0.01 versus the CTRL values. B) Control (CTRL) and HEK293-A␤PP751 (A␤PP751 ) cells were transfected with p21-luc promoter vector and subsequently luciferase activity, expressed as RLU%, was measured. Statistical analysis was performed with Student’s t-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 of HEK293- A␤PP751 treated for 6 h with or without 20 ␮M MG-132 or ALLN. Graphs report densitometric analysis of immunoblots with *p < 0.05 and **p < 0.01 versus membrane A␤PP751 ; ***p < 0.001 vs nucleus A␤PP751 ; bars represent the mean values ± S.D. of three independent experiments; Dunnett’s t-test.

separated. According to the literature [23, 24], both treatments were able to induce a considerable increase of CTFs in the membrane compartment compared to control cells thus highlighting how proteasome activity could be involved in CTFs degradation and consequently in AICD production (Fig. 4A). However, while ALLN induced a related increase of AICD in the nucleus, MG-132 did not, because of its ␥-secretase inhibitory activity (Fig. 4B) [23, 24, 42]. Both proteasome inhibitors thus provided more substrate to the ␥-secretase, but MG-132

impairing ␥-secretase activity blocked the production of AICD. IDE involvement in AICD degradation Since IDE is preferentially located in the cytosol and data from the literature suggested its role in AICD removal in this compartment, we evaluated whether IDE exerted a degradative function on this domain. Consistent with a previous study [45], HEK293-A␤PP751 cells were exposed for 2 h

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Fig. 5. AICD presence in the cytosol and nucleus subsequent to NEM treatment. Cellular lysates were fractionated to obtain cytosol and western blotting analysis was performed to evaluate AICD presence in this cellular compartment. The figure shows a representative immunoblot of HEK-A␤PP751 treated with 10 mM NEM for 2 h. Graph reports densitometric analysis of cytosolic (A) and nuclear (B) fraction immunoblots performed. Bar represent the mean values ± S.D. of three independent experiment with ***p < 0.001 versus control cells (A␤PP751 ); Student’s t-test.

to 10 mM NEM, an IDE inhibitor. After treatment, subcellular fractions were isolated and analyzed by western blotting. Unlike proteasome inhibitor treatments, NEM exposure promoted AICD accumulation in the cytosol (Fig. 5A), thus suggesting that IDE is involved in AICD cytosolic removal. No variations were observed in the nucleus, where the presence of 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 increase in the nucleus may affect p53 mRNA expression. For this purpose, HEK293-A␤PP751 cells were exposed to ALLN inhibitor and then RNA was extracted to analyze p53 expression level by real-time PCR. Cells treated with ALLN showed an increase of p53 expression level (Fig. 6), which could be correlated to the AICD nuclear increase observed by western blotting. Furthermore, we evaluated p53 expression after 2 h of 10 mM NEM exposure in HEK293-A␤PP751 cells. Contrary to ALLN, NEM treatment was not able to influence p53 expression, thus supporting the observation, obtained from

Fig. 6. p53 mRNA expression analysis after NEM and ALLN treatment. Real-time PCR analysis of cells after treatment for 2 h with 10 mM NEM and for 6 h with 20 ␮M ALLN and MG-132. Bars represent the mean values ± S.D with **p < 0.01 versus CTRL; Dunnett’s t-test.

western blotting experiments, that AICD levels in the nucleus were not modified by NEM treatment (Fig. 6). In addition, we also analyzed p21 promoter activity data following ALLN treatment. We performed a luciferase assay in HEK293-A␤PP751 cells and found that ALLN treatment promoted a higher p21 promoter activation compared to untreated cells (Fig. 7).


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Fig. 7. ALLN influence on p21 promoter activation. HEK293A␤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 values ± S.D. of three independent experiments. Statistical analysis was performed with Student’s t-test with ***p < 0.001 versus A␤PP751 values.

AICD/Fe65/Tip60 complex assembly in HEK293-AβPP751 Fe65/AICD/Tip60 complex modulation after proteasome inhibitors treatment HEK293-A␤PP751 cells were exposed for 6 h to 20 ␮M MG-132 and ALLN. The membrane fraction was then immunoprecipitated with anti-Fe65 antibody and western blotting analysis was performed with the antibody which recognizes AICD (Fig. 8). We found that proteasome inhibition increased Fe65 interaction with CTFs in membrane. Since we showed that ALLN enhanced the presence of AICD in the nucleus and some literature data argue that, after Fe65 binding to CTFs in membrane, the AICD/Fe65 complex translocates to the nucleus, we evaluated whether these domains where still associated to Fe65 within the nucleus as detected in the membrane fraction. After expose to 20 ␮M ALLN for 6 h, the nuclear fraction was immunoprecipitated with anti-AICD antibody, and western blotting analysis was conducted with the anti-Fe65 antibody. We found that, in the nucleus, AICD was associated with Fe65 to constitute the AICD/Fe65 complex and that ALLN treatment was able to promote a consistent increase of this complex compared to untreated cells (Fig. 9A). The nuclear fraction was also immunoprecipitated with the anti-Tip60 antibody in order to appraise either the interaction of Fe65 with Tip60 or the presence of the entire AICD/Fe65/Tip60 complex. In the first case, western blotting analysis using the anti-Fe65 antibody showed how the ALLN treatment increases the 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 and ALLN were immunopreciptated with the antibody against Fe65 and western blotting analysis was performed with the antibody which recognized the AICD. Immunoprecipitated antibody was omitted in negative control samples (blank). ␣-tubulin was indicated to appreciate protein content measurements. Graph reports densitometric analysis of membrane fraction immunoblots performed. Bar represent the mean values ± S.D. of three independent experiment with *p < 0.05, **p < 0.01 versus control cells (A␤PP751 ); Dunnett’s t-test.

case, the western blotting examination was conducted with the anti A␤PP C-terminal domain antibody and showed that ALLN treatment favored the increase of AICD/Fe65/Tip60 complex formation (Fig. 9C). Fe65/AICD/Tip60 complex in HEK293-AβPP751 cells treated with IDE inhibitor We immunoprecipitated the nuclear cellular compartment with Tip60 and then we conducted the western blotting analysis using the anti-A␤PP C-terminal antibody. NEM treatment was not able to influence AICD/Fe65/Tip60 complex, unlike ALLN treatment (Fig. 9C). To evaluate whether NEM action may affect the Fe65/AICD complex in the cytosol, HEK293A␤PP751 cells were treated and immunoprecipitated with the anti-A␤PP C terminal antibody and western blotting analysis was then performed with the antiFe65 antibody. We found that Fe65/AICD complex was not influenced by NEM treatment (Fig. 10). These data suggest that AICD nuclear levels were not influenced by NEM treatment and that the AICD removed by IDE was not involved in Fe65/Tip60 complex.

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

Fig. 10. Cytosol and nuclear fraction immunoprecipitation to detect AICD/Fe65/Tip60 variation dependent to IDE inhibition. Cytosol fractions of HEK293-A␤PP751 control (CTRL) and exposed to 20 ␮M ALLN for 6 h (ALLN) were immunoprecipitated with the antibody directed against the AICD domain. Immunoprecipitates were analyzed by western blotting with anti-Fe65 antibody.

DISCUSSION To achieve our goal, we engineered three different vectors for AICD overexpression: pIRES-GWc-

A␤PP751 , pIRES-GWc-C57-EGFP, and pIRES-GWcC59-EGFP. One remarkable difference in the use of these vectors consists of the process of AICD generation. Endogenous AICD is generated from A␤PP by cleavage in the membrane region, whereas exogenous AICD is synthesized in the cytoplasm as a cytoplasmic protein. Since the expression of the entire A␤PP751 protein was necessary to observe a considerable amount of A␤PP-CTFs in membrane with a corresponding AICD presence in the nucleus, we held that AICD generation in the membrane can be essential to obtain the AICD domain as other authors suggested [18, 25]. We demonstrated that proteasome activity influence A␤PP proteolytic processing according to studies previously discussed; proteasome inhibition mediated by ALLN promoted A␤PP-CTF accumulation at the membrane, thus increasing their availability for ␥secretase cleavage. In fact, ALLN treatment induced a significant AICD increase at the nuclear level. ALLN exposure also favored the Fe65 binding increase to


A␤PP-CTFs. Since Tip60 has been identified as a Fe65 nuclear binding partner [46], we further evaluated the existence of the entire complex inside the nucleus. According to the literature [10], we demonstrated that the AFT complex was present in transfected control cells and it was positively influenced by ALLN treatment; this event is in agreement with the increase of Fe65 binding to A␤PP-CTFs in membrane where the AICD/Fe65 complex is generated as suggested in the model III. Hence, our findings correlate with the observations that the intramembranous proteolysis of A␤PP could play a signaling role [9, 47]. It was also demonstrated that AICD regulates phosphoinositidemediated calcium signaling through a ␥-secretase dependent signaling pathway [48]. Although some authors found no consistent effects of ␥-secretase inhibitors or of genetic deficiencies in the ␥-secretase complex or the A␤PP family on the expression levels of KAI1, GSK-3␤, A␤PP, and neprylisin genes [49, 50], a recent work revealed by chromatin immunoprecipitation that AICD was associated with the NEP promoter [51]. Concerning the p53 gene, Checler and coworkers [12] demonstrated that AICD controls this gene at a transcriptional level, both in vitro and in vivo. As this purpose, our data demonstrated that the accumulation of AFT complex in the nucleus, induced by ALLN, correlated with p53 overexpression, which in turn induced p21 activation. Altogether these data establish that proteasome can influence AICD generation and consequently its participation in Fe65/Tip60 complex as we previously hypothesized [36]. Aiming to investigate the role of IDE in AICD degradation, HEK293 cells were exposed to NEM following previously described procedures; this treatment determined AICD accumulation at the level of the cytosol, thus suggesting that this enzyme is involved in AICD degradation in this cellular compartment. However, this cytosolic accumulation did not promote any nuclear modification of the AICD level, which remained comparable to untreated cells. Moreover, unlike ALLN treatment, NEM treatment did not modify the presence of the AFT complex in the nucleus. This observation was in agreement with both western blotting and p53 expression data (Fig. 6). Altogether these data indicate that the AICD removed by IDE do not translocate into the nucleus and is not involved in Fe65/Tip60 complex. A recently published work established that the ␤-secretase is the predominant pathway generating the transcriptionally active AICD/Fe65/Tip60 complex [27]. This observation supports the concept that

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AICD function is related to the metabolic pathway through which it is generated, and this aspect can be integrated with our degradation data; since we demonstrated that proteasome activity could prevent nuclear AICD formation, we could suggest that the transcriptionally active AICD deriving from amyloidogenic pathway is influenced by proteasome activity. On the contrary, since ␣-secretase action is predominantly at the cell surface, the subsequent action of ␥-secretase may induce AICD release in the cytosol where they can be degraded by IDE [45, 51]. This consideration is consistent with our NEM data showing that IDE inhibition promoted AICD accumulation in the cytosol without affecting nuclear AICD levels and their transcriptional function. Nevertheless, further investigation is requested to elucidate the functions of AICD degraded by IDE. In conclusion our data help to solve the discrepancy in literature between AICD function and degradation within Fe65/Tip60 complex, suggesting the existence within cells of at least two different AICD degradation mechanisms. In particular, we have demonstrated that there is a correlation between the role of AICD in gene regulation within Fe65/Tip60 complex and its removal dependent by proteasome activity. Evidence suggests that the proteasome system could be impaired in AD [41, 52, 53]. This decrease in proteasome-dependent processing would be expected to cause an increase in A␤PP-CTFs available for ␥-secretase processing, thereby increasing AICD production, which is in turn responsible for an alteration in gene expression and may result in different effects such as modifications in cytoskeletal dynamics [54]. Defective proteasome function could directly contribute to increase A␤ production in cases of sporadic AD [26, 55]. These data highlight that the involvement of the proteasome activity in A␤PP processing has important consequences for the regulation of A␤PP intracellular fragment production and could be involved in the pathogenesis of AD. On the other hand, results obtained by NEM treatments underline the presence of an additional mechanism which is involved in the removal of AICD which does not exert nuclear activity and does not take part in Fe65/Tip60 complex.

ACKNOWLEDGMENTS This work was supported by the contribution of grants from CARIPLO to M.R. and grant PRIN2009B7ASKP to S.G. We are grateful to Giulia

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Stefania Porcari for careful editing and proofreading of the manuscript. Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=1175).

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