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From: Methods in Molecular Medicine, Vol. 32: Alzheimer’s Disease: Methods and ProtocolsEdited by: N. M. Hooper © Humana Press Inc., Totowa, NJ

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Interaction of the Presenilinswith the Amyloid Precursor Protein (APP)

Andreas Weidemann, Krzysztof Paliga, Ulrike Dürrwang,Friedrich Reinhard, Dai Zhang, Rupert Sandbrink,Geneviève Evin, Colin L. Masters, and Konrad Beyreuther

1. IntroductionThe genes encoding presenilin-1 (PS1) and presenilin-2 (PS2) were identi-

fied as the genes that harbour mutations that cause more than 60% of earlyonset familial Alzheimer’s disease cases (FAD) (1–3). So far, more than 40missense mutations have been described for presenilin-1 and two have beenfound in the gene coding for presenilin-2 (reviewed in refs. 4 and 5). Carriersof mutated presenilin genes develop in their brain neuropathological changescharacteristic of Alzheimer’s disease including the deposition of amyloid Aβpeptide. The latter is released from its cognate amyloid precursor protein(APP) by a two-step proteolytic conversion: first, proteolysis of APP byβ-secretase, which releases the N-terminus of Aβ, and second, conversion ofthe remaining fragment by γ-secretase, which cleaves within the predictedtransmembrane region of APP. This releases the C-terminus of Aβ, which mayend either at position 40 or, to a lesser extent, at position 42 (reviewed inref. 6). The latter species, Aβ1–42, is more prone to aggregation and depositionthan Aβ1–40 and is produced at higher levels in the brains and primaryfibroblasts of FAD patients carrying PS missense mutations (7). The sameresult was obtained when cultured cells transfected with mutated PS1 or PS2, ortransgenic mice harboring missense PS1 were analyzed for the production ofAβ1–42: in every case increased amounts of the longer Aβ1–42 species wereobserved (8–10). The mechanisms by which mutations in the PS genes affectthe proteolytic processing of APP by γ-secretase have not been resolved indetail. There are two possibilities by which the normal processing of APP may

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be disturbed: either mutations in the presenilins affect APP metabolism in anindirect way by modulation of proteases or interaction with proteins involvedin APP intracellular routing, or presenilins may modulate APP processingdirectly through physical interactions with APP. Such a direct interactionbetween presenilins and APP was first demonstrated by us for PS2 (11). Lateron, formation of stable complexes with APP was reported not only for PS2 butalso for PS1 (12,13,13a).

In COS7 cells, transiently transfected with PS cDNA, presenilins weremainly detected as full-length species in the range of 40–55 kDa (Fig. 1).Compared with the amounts of full-length PS, the proteolytically processedderivatives of PS were only poorly observed in these cells, a finding that may

Fig. 1. Identification of PS1 and PS2 in transfected cells. COS7 cells weretransfected with the PS1 (lanes 1 and 4) and PS2 (lanes 2 and 5) encoding pCEP4plasmids and empty vector (lanes 3 and 6). Total cell homogenates were separated bySDS-PAGE and immunoblotted with polyclonal anti-PS1 antiserum (A) or anti-PS2antiserum (B). Both PS1 and PS2 are detected as full-length species migrating in therange of 40–55 kDa.

Interaction of Presenilins with APP 335

be explained by an incomplete proteolytic conversion of highly overexpressedpresenilins (14). To prove the feasibility of a direct interaction between PS andAPP, cells were cotransfected with expression plasmids encoding APP and PS.Analysis of complex formation between APP and PS was performed by twomethods. (1) After immunoprecipitation of PS with polyclonal anti-PS antis-era, the complexes were separated by gel electrophoresis followed by Westernblotting with monoclonal anti-APP antibody (Subheadings 3.2.1. and 3.2.2.;also Fig. 2). (2) PS immunocomplexes derived from lysates of [35S]methionine-labeled cells were first dissociated in the presence of sodium dodecyl sulfate(SDS) and urea and then subjected to a second immunoprecipitation withpolyclonal anti-APP antiserum and successive gel electrophoresis (Sub-headings 3.2.1. and 3.2.3.; also Fig. 3). The species observed in the noncovalentAPP-PS complexes is mainly composed of immature, ER-resident APP(Figs. 2 and 3), in agreement with the predominant localization of presenilinsto the ER, intermediate compartment, and early Golgi of cells (15–17). Nodifference in complex formation was observed for mutant PS as compared with

Fig. 2. Detection of APP-695 in PS2 immunoprecipitates after Western blotting.COS7 cells were transiently transfected with pCEP encoded cDNAs for either PS2 orPS2–N141I together with pCEP/APP-695 as indicated. Proteins were immunoprecipi-tated with anti-APP antiserum (lanes 1–4) or anti-PS2 antiserum (lanes 5–8), followedby SDS-PAGE and immunoblotting with mAb22C11, which recognizes APP. Notethat for demonstration of APP in PS2 immunocomplexes (lanes 5–8), tenfold morecell lysate was subjected to immunoprecipitation compared to lanes 1–4. N: N-glycosylated, immature forms of APP; N,O: N- and O-glycosylated, mature forms(reproduced from ref. 11).

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wild-type PS (Figs. 2 and 3) (11–13a). Thus, mutations in the presenilins maynot directly affect their ability for complex formation with APP molecules, butinstead influence the fate of the PS2–APP complex, e.g., by modifying its transportor its metabolism. This hypothesis is supported by the finding that the conver-sion of APP by γ-secretase(s) is largely inhibited in PS1-deficient mice,suggesting a role of PS as cofactors in this processing step (18). Additionally,mutant PS1 fully complements the severe phenotypical aberrations observedin PS1 knockout mice, which further validates the idea that the clinicalmutations in PS result in a gain of (mis)function of mutant PS rather than aloss of function (19,20). Further studies on the protein domains involved inthe interaction revealed that for PS2, the N-terminal part is sufficient forcomplex formation, whereas for APP, the Aβ and/or transmembrane domainis implicated in the interaction (13a). Again, this supports the idea that PSact as cofactors in the processing of the transmembrane domain of APPby γ-secretases. Alternatively, based on the abundant localization of both PS

Fig. 3. Formation of stable complexes between APP-695 and PS1. Cells weretransfected with pCEP-APP695 and plasmids encoding either wild-type PS1 (lanes2 and 6) or mutant PS1-M146V (lanes 3 and 7). After radiolabeling the cells with[35S]methionine, PS1 complexes were immunoprecipitated with anti-PS1 antiserum,followed by release of bound proteins by addition of SDS/urea and subjected to asecond immunoprecipitation with anti-APP antiserum (B). APP was recovered in bothwild-type PS1 and mutant PS1-M146V immunocomplexes (lanes 6 and 7). The sameresult was obtained for PS1-P264L or PS1-L286V, indicating that the ability forcomplex formation was not influenced by FAD mutations (data not shown). Forcomparison, APP expression was monitored in parallel immunoprecipitations withanti-APP antiserum (A). N: N-glycosylated, immature forms of APP; N,O: N- andO-glycosylated, mature forms.

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and APP-PS complexes in the ER, and based on the influence of overexpressedPS on APP transport and secretion (11), the function of PS could be similar tothat of calnexin, which transports misfolded proteins retrogradely by recyclingthem between the ER, intermediate compartment, and Golgi apparatus (21).Alternatively, mutations in the PS genes may affect APP processing in a patho-logical manner similar to mutations in ERGIC-53 (ER-Golgi intermediate com-partment protein-53). Here, mutations of ERGIC-53, which is localized to theintermediate compartment, cause a combined deficiency of coagulation factorsV and VIII, two secretory proteins found in the blood plasma (22). However,although the exact function of the presenilins is not known, the demonstrationof physical interactions suggests a direct participation of PS on APP processing.

2. Materials2.1. Calcium Phosphate Coprecipitation

Buffers 2–5 should be filter-sterilized before use:

1. COS7 cells (see Note 1); cell culture media: Dulbecco’s modified Eagle’s medium(DMEM), DMEM + 10% fetal calf serum (FCS).

2. 2× HEPES-buffered saline (2× HBS): 280 mM NaCl, 1.5 mM Na2HPO4, 50 mMHEPES; adjust exactly to pH 7.13 with 1 N NaOH.

3. CaCl2 solution: 2 M CaCl2 in distilled H2O.4. Tris-HCl buffer: 10 mM Tris-HCl, pH 7.5, in distilled H2O.5. Glycerol solution: mix a 30% glycerol solution with 2× HBS in a 1:1 ratio.6. DNA (see Note 2): pCEP4 (vector), pCEP-APP695, pCEP-PS1, pCEP-PS2 (and

mutant derivatives of PS1 and PS2) dissolved at a concentration of 1 μg/μL insterile TE (10 mM Tris-HCl, pH 7.5–8.0, 1 mM EDTA). The DNA should be ofhigh quality (Qiagen, Chatsworth, CA).

2.2. Analysis of PS Immunocomplexes for Bound APP

1. Phosphate-buffered saline (PBS).2. [35S]methionine (Amersham, Arlington Heights, IL), methionine-free cell

culture medium (Life Technologies, Gaithersburg, MD).3. Antibodies: Polyclonal anti-PS antiserum; polyclonal anti-APP antiserum;

monoclonal anti-APP antibody (22C11, Boehringer Mannheim, Mannheim,Germany, ref. 23); secondary anti-mouse antibody coupled to horseradishperoxidase (Amersham).

4. Protein A-Sepharose as a 1:1 slurry in buffer A (see step 6) or in PBS (Pharmacia,Uppsala, Sweden).

5. Cell lysis buffer: 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1%Nonidet P40 (NP40), 1% Triton X-100, supplemented with protease inhibitors(50 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride).

6. Wash buffers: Buffer A: 150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA,0,2% NP40; Buffer C: 10 mM Tris-HCl, pH 7.5.

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7. Sample buffer (2×): 125 mM Tris-HCl, pH 6.8, 5% SDS, 50 mM dithiothreitol(DTT), 20% glycerol, 0.01% bromophenol blue.

8. Release buffer (2×): 125 mM Tris-HCl, pH 6.8, 1% SDS, 8 M urea, 25 mM DTT.DTT should be stored as 1 M stock solution at –20°C and added freshly.

9. 8% SDS Tris-glycine gels, running buffer.10. Equipment and reagents for immunoblotting; ECL solutions (Amersham).11. HAc/MeOH solution: 30% Methanol, 10% glacial acetic acid in H2O.

3. Methods3.1. Transfection of Cells with PS-and APP-Encoding Plasmids by Calcium Phosphate Coprecipitation

1. Split COS7 cells grown to confluency on a 10-cm dish (see Note 3) the day beforetransfection in a ratio of about 1:4 and seed the cells in 12 × 5 cm dishes. Onthe day of transfection, the confluence of cells should be 70–90% (calciumphosphate coprecipitation protocol from ref. 24).

2. For each transfection, transfer 420 μL Tris-HCl buffer into microtubes; add atotal of 20 μg DNA, i.e., 10 μg pCEP-APP695 and 10 μg pCEP-PS2 or 10 μgpCEP-APP695 and 10 μg pCEP-PS1. Include also control transfections withpCEP4 only, pCEP-APP695/pCEP4, and pCEP-PS/pCEP4.

3. Add 62 μL CaCl2 buffer to the DNA solution and mix the sample by pipeting upand down several times.

4. Prepare 500 μL 2× HBS in 13 mL screw-cap tubes or equivalent.5. Place the tube with the 2× HBS solution on a vortex with moderate agitation and

add dropwise the 500 μL CaCl2/DNA solution.6. Incubate the mixture for 20–30 min at room temperature, thus allowing the

formation of calcium phosphate crystals containing DNA.7. Without previous removal of the conditioned media, distribute dropwise the

solution on the cells. After a few minutes, the crystals are detectable as a fine,uniform precipitate on the cells.

8. Incubate the cells for 3 h in the cell culture incubator.9. Aspirate the conditioned media and add 1.5 mL glycerol solution per dish.

Incubate for 21⁄2 min. Remove the glycerol solution, wash twice with DMEM,and add fresh culture medium containg FCS (see Note 4).

10. Incubate the transfected cells overnight until subsequent analysis.

3.2. Analysis of PS Immunocomplexes for Bound APP

The following protocols describe methods for the detection of APP695 in PSimmunoprecipitates. This can be achieved by analysis of PS immunocomplexesfrom nonlabeled cells by immunoblotting with monoclonal anti-APP antibody.However, the use of radioactive labeled proteins allows the concurrent detectionof PS by phosphorimaging the blots after ECL development. This is especiallyuseful for quantitating of the PS/APP ratio, i.e., for comparing bound APP incomplexes with mutant or wild-type PS. An alternative approach can be

Interaction of Presenilins with APP 339

employed to identify APP in PS immunocomplexes: cells are radiolabeled,followed by a first immunoprecipitation with anti-PS antisera; the releasedimmunocomplexes are analyzed by a second immunoprecipitation withanti-APP antiserum. Again, using radiolabeled cells allows the simultaneousdetection of both PS and APP, and thus, quantitative studies of the correspondingratios can be performed. The latter method of consecutive immunoprecipita-tions can also be used for the reverse experiments to identify PS in APPimmunoprecipitates. The alternative, a direct analysis of PS in APPimmunocomplexes by immunoblotting with anti-PS antisera, is not conclusivebecause the signal of the immunoglobulin heavy chains from the first immuno-precipitation is observed in the range of about 50 kDa, and therefore interfereswith the signals obtained for full-length PS in the same molecular weight range.

3.2.1. Immunoprecipitation of APP and PS/APP Complexes

1. Remove media from the cells, wash three times with PBS, and add 1.5 mLmethionine-free cell culture medium Petri-dish (when using nonlabeled cells,omit steps 1–3).

2. Add 100 μCi [35S]methionine per dish.3. Incubate for 4 h in a humidified incubator at 37°C.4. Remove the supernatant; wash once with PBS; scrape off the cells, add 0.5 mL

PBS and transfer the cells into a microtube; and centrifuge for 1 min at 4000g,discard the supernatant (see Note 5).

5. Add 50 μL ice-cold lysis buffer to the cell pellet, resuspend the cells in the bufferby pipeting up and down (see Note 6).

6. Incubate for 10 min on ice.7. Centrifuge for 10 min at 10,000g.8. Collect the clear supernatant in fresh tubes, discard the pellets.9. Dilute the samples with 250 μL buffer A, mix briefly (PS-IP); transfer 30 μL into

a tube with 270 μL buffer A (APP-IP). Add 5 μL anti-PS2 antiserum to theremaining 270 μL PS-IP samples and 3 μL anti-APP antiserum to the 300 μLAPP-IP samples (see Note 7). Add 30 μLProtein A-Sepharose (Pharmacia) to all tubes.

10. Incubate for 2 h at room temperature on a head-over-head roller (see Note 8).11. Wash six times with 1 mL buffer A and once with 1 mL buffer C (see Note 9).

3.2.2. Immunoblotting with Anti-APP Antibodies

The treatment of the precipitates depends on the successive experiments.For immunoblotting with anti-APP antibody, add 30 μL sample buffer andboil the immunoprecipitates for 10 min (see Note 10). The samples areanalyzed by gel electrophoresis on 8% polyacrylamide SDS gel followed byimmunoblotting and ECL detection with monoclonal anti-APP antibody22C11 by using standard conditions.

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3.2.3. Analysis of Radiolabeled Immunoprecipitates for APP

1. Release bound antigens from the Protein A-Sepharose (Pharmacia) by incubationof the samples with 30 μL release buffer for 15 min at 50°C (see Note 10).

2. Add 270 μL buffer A to the samples to diminish the effective concentrations ofSDS to 0.1% and the urea concentration to 0.8%, which is necessary for thesubsequent immunoprecipitations.

3. Centrifuge briefly and transfer the supernatant into a fresh tube.4. Add 5 μL polyclonal anti-APP antiserum, and 30 μL Protein A-Sepharose;

incubate for 2 h on a head-over-head roller at room temperature.5. Centrifuge briefly. The supernatant contains released PS (and released immuno-

globulin heavy and light chains from the first immunoprecipitation), which canbe analyzed separately after concentration of proteins by TCA (trichloroaceticacid) precipitation and subsequent gel electrophoresis. If this analysis is notperformed, discard the supernatant, wash the Protein A-Sepharose twice withbuffer A and once with buffer C.

6. Denature samples in sample buffer and apply to an 8% polyacrylamide SDS gel.7. To immobilize the proteins, incubate the gel in HAc/MeOH solution with moder-

ate agitation for 1 h.8. Dry the gel and visualize proteins by phosphorimaging or autoradiography.

3.3. Other Methods

3.3.1 DNA Constructs

Cloning of PS2 cDNA and generation of pCEP4 derivatives encodingwild-type PS2 or mutant PS2–N141I (changing Asn141 into Ile) has beendescribed (11). PS1 was cloned by reverse transcriptase-polymerase chain reac-tion using total RNA isolated from human blood lymphocytes as an alternativesplice variant lacking the four residues VRSQ encoded at the 3' end of exon 3.The design of the oligonucleotide primers was based on the published sequencesof PS1 (1,2) and encoded an additional XhoI site in the 5'-sense primer and anClaI site in the 3'-antisense primer. The sites were used for cloning in pBluescriptSK+ (Stratagene, La Jolla, CA). Sequencing one positive clone revealed com-plete alignment with the cDNAs published. The PS1 FAD mutants PS1-M146V,PS1-P264L, and PS1-L286V were constructed by site-directed mutagenesis ofsingle-stranded DNA (25) and verified by DNA sequencing. For expression ineukaryotic cells, PS1 and mutant PS1 derivatives were cloned into the expres-sion vector pCEP4 by using KpnI and BamHI restriction sites. Cloning and con-struction of eukaryotic APP expression vectors was as described before (26).

3.3.2. Cell Culture

COS7 cells were maintained in DMEM containing penicillin (50 U/mL),streptomycin (40 μg/mL) and 10% FCS (Life Technologies).

Interaction of Presenilins with APP 341

3.3.3. Antisera

Preparation of rabbit polyclonal anti-APP antiserum and mouse monoclonalanti-APP antibody 22C11 has been described (23). Antisera recognizing PS2was obtained by serial immunizations of rabbits with synthetic peptide corre-sponding to PS2 residues 42–58 (11). Production and characterization ofpolyclonal rabbit PS1 antiserum directed against a synthetic peptide encom-passing residues 1–20 of PS1 has been published (17).

4. Notes1. Instead of COS cells, most other cell lines that can be transfected with high

efficiency should be suitable for analysis of PS-APP complex formation includingChinese hamster ovary (CHO), HeLa or human embryo kidney 293 (HEK293) cells.

2. For expression of APP and PS cDNAs, we used the commercially availablepCEP4 vector (Invitrogen, San Diego, CA). Here, the gene expression is drivenby the cytomegalovirus early/late promoter (CMV promoter) which belongs toone of the strongest promotors known and which is active in a wide variety ofdifferent cell lines from different species. Additionally, the pCEP4 vectors encodea marker for selection in eucaryotic cells (hygromycin B resistance gene) and anorigin of replication derived from the Epstein-Barr virus. The latter allows anepisomal propagation of the pCEP4 plasmids in some human and monkey celllines, including COS7 cells or SH-SY5Y cells. Because of this episomalpropagation, all transfected cells should express the cloned gene, which allowsthe use of pools of cells instead of screening individual colonies for establishingstable transfected cell lines. However, most other eucaryotic expression vectorsshould be suitable as well for transient expression of PS and APP genes ineucaryotic cells as described in this chapter.

3. Among the many different protocols, the calcium phosphate coprecipitation isstill one of the common methods to transfect cells. However, most of the othertransfection methods, including liposome-mediated DNA transfer orelectroporation of cells, can be used for transfection of common cell lines likeCHO, COS7, HeLa, or HEK293 cells. Transfection efficiency can be easilymonitored by reporter plasmids encoding proteins like β-galactosidase, luciferase,or green-fluorescent protein.

4. Other cell lines than COS7 cells may require different conditions for hightransfection efficiency. Frequently, incubation of the crystals overnight withoutglycerol treatment also gives high transfection rates.

5. It is recommended not to freeze cells or cell lysates because this results indecreased recoveries of APP from PS-APP complexes.

6. The use of prechilled lysis buffer is required to avoid the rupture of nuclei and therelease of the chromosomal DNA, which would result in viscous solutions.

7. The direct immunoprecipitation of APP from cell lysates should be performedfor the following reasons. First, to prove the expression of APP in the controlexperiments in which cells were only transfected with APP encoding plasmid.

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Second, to control the level of APP expression because if there is no APP detectedin the analysis of PS immunoprecipitates, this could be due to low transfectionrates. Thus, the direct analysis of APP should be helpful to interpret negativeresults. Third, under the conditions described in this chapter, about 10% of totalAPP is found in PS immunoprecipitates. If no signal is obtained for APP by directimmunoprecipitation from the ten percent of the total cell lysates, it means thatthe assay/antibody used is not sensitive enough to detect APP in PSimmunocomplexes.

8. Prolonged incubation (i.e., overnight) is not recommended because of somedissociation of the PS-APP complexes.

9. At this point, the immunoprecipitates may be stored at –20°C.10. Do not boil the samples in the case of concurrent detection of radioactive

presenilins because the latter tend to aggregate heavily at high temperatures.Instead, the immunoprecipitates should be denatured at 37–50°C for 15 min in30 μL sample buffer containing 8 M urea.

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