Interactome of the Amyloid Precursor Protein APP ... - ACS Publications

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Interactome of the Amyloid Precursor Protein APP in Brain Reveals a Protein Network Involved in Synaptic Vesicle Turnover and a Close Association with Synaptotagmin‑1 Bernhard M. Kohli,⊥,† Delphine Pflieger,⊥,‡,§ Lukas N. Mueller,‡ Giovanni Carbonetti,† Ruedi Aebersold,‡,∥ Roger M. Nitsch,† and Uwe Konietzko*,⊥,† †

Institute of Psychiatry Research and Psychogeriatric Medicine, University Zurich, Switzerland Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Switzerland ∥ Faculty of Science, University of Zurich, Switzerland ‡

S Supporting Information *

ABSTRACT: Knowledge of the protein networks interacting with the amyloid precursor protein (APP) in vivo can shed light on the physiological function of APP. To date, most proteins interacting with the APP intracellular domain (AICD) have been identified by Yeast Two Hybrid screens which only detect direct interaction partners. We used a proteomics-based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity-purified complex to mass spectrometric (MS) analysis. Using two different quantitative MS approaches, we compared the protein composition of affinity-purified samples isolated from wild-type mice versus transgenic mice expressing tagged APP. This enabled us to assess truly enriched proteins in the transgenic sample and yielded an overlapping set of proteins containing the major proteins involved in synaptic vesicle endo- and exocytosis. Confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle proteins in vesicular structures throughout the neurites. We analyzed the interaction of APP with these proteins using pulldown experiments from transgenic mice or cotransfected cells followed by Western blotting. Synaptotagmin-1 (Stg1), a resident synaptic vesicle protein, was found to directly bind to APP. We fused Citrine and Cerulean to APP and the candidate proteins and measured fluorescence resonance energy transfer (FRET) in differentiated SH-SY5Y cells. Differentially tagged APPs showed clear sensitized FRET emission, in line with the described dimerization of APP. Among the candidate APP-interacting proteins, again only Stg1 was in close proximity to APP. Our results strongly argue for a function of APP in synaptic vesicle turnover in vivo. Thus, in addition to the APP cleavage product Aβ, which influences synaptic transmission at the postsynapse, APP interacts with the calcium sensor of synaptic vesicles and might thus play a role in the regulation of synaptic vesicle exocytosis. KEYWORDS: Amyloid precursor protein, APP, synaptotagmin, synaptic vesicle, Alzheimer, TAP tag

1. INTRODUCTION

extracellular domain, creating a neurotrophic molecule in the case of α-cleavage. The remaining membrane-anchored stub undergoes further intramembraneous cleavage by the γ-secretase complex.14 Cleavage of β-stubs (generated by β-secretase) generates Aβ peptides which aggregate into oligomers that influence synaptic transmission.15 Further aggregation of Aβ oligomers results in the deposition of amyloid plaques in the brain that are a characteristic of AD. Intramembraneous cleavage further generates the APP intracellular domain (AICD) that translocates to the nucleus to regulate transcription.16 APP undergoes constant proteolytic turnover with one of the shortest reported protein half-lives.17

The amyloid precursor protein (APP) is central to the pathogenesis of Alzheimer’s disease (AD).1 The essential physiological function of this single-pass transmembrane protein has been difficult to pinpoint. Although APP is expressed throughout the body,2 the quest to reveal its functions has concentrated on the nervous system due to the fact that this is the site of AD pathology. APP has been shown to function in cell adhesion and neurite outgrowth,3,4 exert trophic and proliferative activities,5−7 influence synaptic transmission,8−10 and signaling to the nucleus to regulate transcription.11,12 APP is synthesized in the secretory pathway and anterogradely transported in vesicles by kinesin-mediated fast transport. APP is sequentially cleaved by several proteases termed secretases.13 Cleavage by α- or β-secretase leads to shedding of the © 2012 American Chemical Society

Received: February 7, 2012 Published: June 25, 2012 4075

dx.doi.org/10.1021/pr300123g | J. Proteome Res. 2012, 11, 4075−4090

Journal of Proteome Research

Article

streptavidin Washing Buffer (SWB: SBB without protease inhibitor and only 0.4% TX-100). The supernatants (SN) from the preceding preclearing steps were incubated with the preequilibrated cold resin for 2 h at 4 °C. The resins were washed 3 times with 12 mL of SWB. Finally, 1 mL of streptavidin Elution Buffer (SEB: 2 mM biotin in 0.4% TX-100, 150 mM KCl, 50 mM NH4HCO3) was added to each resin and the mixtures were incubated for an elution duration of 1 h, resulting in the eluate (EL) used for mass spectrometry analyses.

Information on APP function gained from APP knockout animals has been hampered by the existence of two homologues, the APP-like proteins APLP1 and APLP2,18 but novel knock-in strategies have shown important functions for the extracellular domain19 as well as essential functions for AICD on synaptic function.20 AICD is highly conserved across vertebrate species, and the identification of cytosolic proteins interacting with AICD can thus shed light on the cellular functions of APP. Among the proteins identified are G0,21 Fe65,22,23 MINT/X11,22 Dab,24 JunN-terminal Kinase interacting protein Jip,25 Abl,26 Shc,27 and numb.28 Most proteins binding to AICD were found through Yeast Two Hybrid screens, which identify direct interactions only. In order to get a more differentiated picture of the APP interactome in brain, we generated transgenic mice expressing APP with a tandem affinity purification (TAP) tag and used affinity purification combined with mass spectrometry (MS) to identify proteins that interact with APP in the brain. We identified many of the main components of synaptic vesicle exoand endocytosis. We further analyzed these interactions by pulldown, colocalization, and fluorescence resonance energy transfer (FRET) studies. Our results show that APP is localized in synaptic vesicles in close association with the calcium-sensor Synaptotagmin-1.

Streptavidin Pull-down with Dynabeads

Dynabeads-Streptavidin M280 (Invitrogen) were washed with 150 mM KCl, 20 mM HEPES pH 7.2, 0.05% BSA before application of the samples. Cotransfected HEK 293 or Neuro2a cells were washed with PBS and scraped off the plate in 150 mM KCl, 20 mM HEPES pH 7.2, 10 mM NaCl, 1% TritonX-100, 2 mM DTT, 5% Glycerol, 5 mM phenathrolene, Protease inhibitor cocktail (Roche, #1 697 498). Cells were disrupted by a 26G syringe and nuclei and debris removed by a 10 min centrifugation at 800g. Supernatants were mixed with the equilibrated streptavidin beads and incubated overnight at 4 °C. Dynabeads were separated with a magnet and the resulting supernatant termed F (flowthrough). The beads were washed 5 times with homogenization buffer. 700 nmol of biotin was added for 30 min to the Dynabeads to disrupt the binding of SBP-tagged APP to streptavidin. Dynabeads were removed with a magnet and the resulting supernatant termed E (eluate). APP-TAP transgenic and wild-type mice were killed by cervical translocation. The brains were removed into ice-cold PBS and cortices and hippocampi prepared. Brain tissue was homogenized with 15 strokes at 800 rpm in a buffer containing 10 mM KCl, 40 mM HEPES pH 7.2, 150 mM NaCl, 1% NP40, 2 mM DTT, 5% Glycerol, 5 mM phenathrolene, Protease inhibitor cocktail (Roche, #1 697 498). Debris was removed by a 10 min centrifugation at 800g. Supernatants were mixed with the equilibrated streptavidin beads and incubated at room temperature for 2 h. Washing and elution was identical to the Dynabeads isolation from cells.

2. MATERIALS AND METHODS Transgenic Mice

APP was mutagenized to contain BsrgGI and NcoI restriction sites at K650 and H657, respectively, without changing the amino acid composition (APP695 numbering). As the tandem affinity purification (TAP) cassette inserted (Stratagene, #240104) has an internal NcoI restriction site, overhanging end PCR cloning was used to prepare the TAP cassette for entry between these two amino acids, yielding full length APP that contains the TAP tag juxtamembraneously (Supporting Information Figure 1A). APP-TAP-AICD was inserted by blunt end cloning behind a PrP promoter in the pMoPrP.Xhovector.29 After removal of the vector sequence, the linear construct was injected into pronuclei of fertilized zygotes of B6D2F1 mice. Founders were screened for transgene expression by tail PCR and Western blot analysis using 6E10 human Aβspecific antibody, and the line used in this study was expanded by pairing littermates. All MS results shown were derived from hemizygous mice.

Sample Digestion and Quantitative Mass Spectrometry Analyses

In summary (Figure 1), aliquots of digested affinity purified samples were first analyzed by LC-MS/MS on a LTQ instrument (Thermofisher scientific), to obtain a rough estimate of the sample’s relative total protein amounts: the ionic current measured for each digested sample was integrated and used to equalize the total protein amounts before performing further comparative analyses. The remaining samples were then split into two halves either to be analyzed by label-free relative quantification using an LTQ-FT instrument (Thermofisher Scientific), following a procedure developed in our lab,30 or to be differentially labeled with iTRAQ reagents for relative quantification by MALDI-TOF/TOF analysis (MALDI-TOF/ TOF 4800, Applied Biosystems). The cysteine reducing reagent used, tris(2-carboxyethyl)phosphine (TCEP), cysteine alkylating reagent methyl methanethiosulfonate (MMTS), and iTRAQ dissolution buffer (0.5 M triethyl ammonium bicarbonate buffer at pH 8.5) were part of the iTRAQ kit (Applied Biosystems, #4352135). Modified porcine trypsin was purchased from Promega (#V5113), and endoproteinase Lys-C was obtained from Roche (#11 420 429 001).

Protein Purification for Mass Spectrometry

APP-TAP transgenic and wild-type mice were killed by cervical translocation. Each mouse brain (from transgenic or normal animals), excluding the cerebellum, was homogenized using a Potter homogenizer in 4 mL of streptavidin binding lysis buffer (SBB: 1% Triton X-100 (TX-100); 40 mM Tris-HCl, pH 7.4, 150 mM KCl, 5% glycerol, Complete Protease Inhibitor Cocktail (Roche, #11873580001), 5 mM β-mercaptoethanol). The resulting lysate was then centrifuged at 4 °C for 20 min at 20,800g to separate cytosolic and dissolved membrane components from insoluble membrane components and nuclei. All subsequent purification steps were performed at 4 °C. For each sample, two slurries containing 1 mL of Sepharose CL4B (Sigma #CL4B200) per mouse brain were preequilibrated twice with SBB (10 mL). Equal protein amounts from each sample were then incubated with the washed resin for 30 min. This preclearing step was repeated once. 600 μL of streptavidin Sepharose CL4B resin (Novagen, #69203) per mouse brain were pre-equilibrated twice with 12 mL of 4076



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acid, at a flow rate of 1.2 μL/min. The data acquisition mode was set in positive ion mode to obtain FTMS scans at a resolution of 100,000 fwhm (at m/z 400) over the mass range m/z 300−2000, where each FTMS scan was followed by three MS2 scans in the linear ion trap (overall cycle time of approximately 1 s), for precursor ions exceeding 200 ion counts in the FTMS scan. The charge state screening mode was used to exclude unassigned or singly charged ions. The Tune Page parameters were set as follows: AGC in FTMS 200,000; AGC in ITMS2 20,000; maximum injection time 300 ms in FTMS (1 μscan) and 100 ms in ITMS2 (2 μscans). Acquired MS2 scans were searched against the mouse IPI protein database (v.3.15) using the SORCERER-SEQUEST (TM) v3.0.3 search algorithm, which was run on the SageN Sorcerer (Thermo Electron, San Jose, CA, USA). IPI references were automatically matched to SwissProt/Trembl entries when existing. In silico trypsin digestion was performed after lysine and arginine (unless followed by proline), tolerating two missed cleavages in fully tryptic peptides. Database search parameters were set to allow modification of cysteine residues with methyl methanethiosulfonate as a fixed modification and loss of water/ ammoniac from N-terminal E/Q residues as variable modification. The fragment mass tolerance was set to 0.5 Da, and precursor mass tolerance to 10 ppm. Search results were evaluated on the Trans Proteomic Pipeline (TPP) using Peptide Prophet (v3.0).31,32 For relative quantification of ion signals between the four LCMS/MS analyses, data processing was performed by the open source program SuperHirn (Mueller, LN, Proteomics, 2007), which can be downloaded from http://tools.proteomecenter. org/software.php. SuperHirn extracts MS1 peptide signals, combines these with MS/MS peptide identifications, and assembles them by multidimensional LC-MS alignment into a MasterMap. The MasterMap is then subjected to K-means clustering analysis and subsequent peptide and protein profiling. Applied to our samples, the program allowed obtaining a list of proteins which were significantly over-represented in the transgenic mice sample by a high similarity of their profile to the expected dilution curve, as indicated by the protein profile probability (p > 0.9, see Table 1).

Figure 1. Schematic overview of the workflow leading to the data presented in Table 1 (for details, see Materials and Methods). Brain samples from APP-TAP-AICD transgenic and wild-type mice were purified via streptavidin beads. A first estimate of relative peptide amounts was performed by comparing silver stained 1D gels of the two samples before tryptic digestion. Proteins were reduced and alkylated, and then digested with trypsin. More precise peptide concentration normalization was performed based on the LC-MS analysis of aliquots of the samples on a linear ion trap (LTQ). Then the samples were split and two kinds of semiquantitative MS analyses were performed. On the one hand, a dilution series of the samples to compare was mixed and measured on a LTQ-FT instrument, and the data was analyzed by the program SuperHirn.30 On the other hand, the two samples to compare were differentially iTRAQ-labeled and analyzed on a MALDI-TOF/ TOF instrument, for MS/MS data to be finally interpreted by Mascot.

Samples from affinity purifications were precipitated by adding 6 volumes of prechilled (−20°) acetone, incubating at −20 °C for 4 h, and pelleting by centrifugation at 20,000g at 4 °C for 10 min. Pellets were resuspended by vortexing in 50 μL of dissolution buffer composed of 0.1% Rapigest (Waters, #186001861) in 100 mM NH4HCO3 buffer, pH 8.3. Samples were reduced by addition of 5 mM TCEP and incubation for 1 h at 37 °C. MMTS was added at a final concentration of 10 mM, and samples were incubated for 10 min at RT to alkylate cysteine residues. For digestion, we first applied Lys-C at a concentration of 1:200 (LysC: total protein, w:w) for 1 h 30 min at 37 °C. Then trypsin was applied at a 1:100 concentration for overnight incubation at 37 °C. Acid-labile Rapigest detergent was degraded by sample acidification to pH = 1.0 with HCl and incubation at 37 °C for 30 min. The samples were finally purified using C18 micro spin columns (Harvard Apparatus, #74-7206).

MALDI-TOF/TOF

The remaining 50% of the affinity-purified samples were dried in the speedvac and resuspended in 25 μL of iTRAQ dissolution buffer. The negative control sample (obtained from WT mice) was labeled with the 114 iTRAQ reagent, and the transgenic mouse sample was labeled with the 116 iTRAQ reagent according to the manufacturer’s protocol (Applied Biosystems, #4352135). Excess iTRAQ reagents were hydrolyzed by addition of 1 volume of H2O and a 1-h incubation on the bench, and samples from control and transgenic mice were finally mixed in a 1:1 ratio. Samples were desalted using consecutively three ZipTipC18 (Millipore, #ZTC18S096), to enhance peptide recovery. MALDI-TOF/TOF samples were reverse-phase separated and measured on a 4800 MALDI-TOF/TOF from Applied Biosystems and analyzed and quantified using the manufacturer’s GPS Global Explorer software suite. LC separation and MS-MS/MS acquisitions were performed as described previously.33 Briefly, 5 μL of peptide samples were injected and loaded directly onto a C18 capillary column (PepMap100 C18, 75 μm i.d., 15 cm length, 3-μm particle, 100 Å porosity, Dionex) fitted on the injection valve of a Famos system (Dionex-LC Packings, Sunnyvale, CA, USA). Chromatographic

LTQ-FT

For label-free relative quantification of affinity purified samples (AP), the following dilutions of wt-AP/transgenic-AP were prepared with peptides from the above C18 micro spin column elution: (1) 100%/0%, (2) 90%/10%, (3) 80%/20%, and (4) 0%/100%. Each mixture was individually analyzed by LC-MS/ MS coupling an Agilent chromatographic separation system 1100 (Agilent Technologies, Waldbronn, Germany) and a LTQFT instrument (Thermofisher Scientific) equipped with an electrospray ionizer (ESI). LC separations and MS acquisitions were performed as described.30 Briefly, a 10.5-cm-long fused silica emitter of 150 μm inner diameter (BGB Analytik, Böckten, Switzerland) packed in-house with Magic C18 AQ 5 μm resin (Michrom BioResources, Auburn, CA, USA) was used for LC separations. Peptides were separated with a linear gradient of 70 min, from 5% to 40% acetonitrile in water containing 0.1% formic 4077



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Table 1. Enrichment Profiles of the Two Most Prominent Categories of APP-Interacting Proteins in Braina MALDI-TOF/TOF data entry name

accession no.

prot def

APP (Intrinsic Positive Control) A4_MOUSE P12023 amyloid β A4 protein precursor 14-3-3 Proteins 1433B_MOUSE Q9CQV8 14-3-3β/α 1433E_MOUSE P62259 14-3-3ε 1433F_MOUSE P68510 14-3-3η 1433G_MOUSE P61982 14-3-3γ 1433T_MOUSE P68254 14-3-3θ 1433Z_MOUSE P63101 14-3-3ζ/δ Proteins Involved in Synaptic Vesicle Cycling/Trafficking ARF3_MOUSE P61205 ADP-ribosylation factor 3 CLH_MOUSE Q68FD5 Clathrin heavy chain 1 AP180_MOUSE Q61548 Assembly Protein 180 DYN1_MOUSE P39053 Dynamin-1 RAB1A_MOUSE P62821/Q9D1G1 Rab-1A or 1B RAB1B_MOUSE RAB3C_MOUSE P62823/P35276 Rab-3C or 3D RAB3D_MOUSE RAB3A_MOUSE P63011 Rab-3A SH3G2_MOUSE Q62420 Endophilin-1 SYN1_MOUSE O88935 Synapsin-1 SYN2_MOUSE Q64332 Synapsin-2 SV2A_MOUSE Q9JIS5 synaptic vesicle glycoprotein 2A SNP25_MOUSE P60879 SNAP-25 SYT1_MOUSE P46096 Synaptotagmin-1 STX1A_MOUSE O35526 Syntaxin-1A STX1B_MOUSE P61264 Syntaxin-1B2 STXB1_MOUSE O08599 Munc18 SYUA_MOUSE O55042/ α- or β-synuclein SYUB_MOUSE Q91ZZ3 VAMP2_MOUSE P63044 VAMP2 NSF_MOUSE P46460 NSF

no. ident pept

Mascot score

4

LTQ-FT data

iTRAQ ratio

no. uniq pept

no. quant pept

profile prob.

98

6.05 ± 0.82

13

2

>0.999

6 3

332 157

5.13 ± 1.02 3.97 ± 0.50

4 6 5

179 249 227

4.83 ± 1.39 4.99 ± 1.08 4.53 ± 2.40

1 5 2 5 1 7

1 2 2 3 1 4

>0.999 >0.999 >0.999 0.934 >0.999 1

1 3 2 6 1

71 129 89 278 60

7.14 2.66 ± 0.66 3.32 ± 0.41 4.02 ± 1.81 3.93

3 8 3 10 1

1 2 2 2 1

>0.999 0.833 1 1 >0.999

1

60

0.799

87 102

3.69 ± 0.84 3.41 ± 0.56

2 3 15 6 5

1

2 3

8 3 2

>0.999 1 >0.999

1 4

54 213

4.21 3.80 ± 1.90

3

128

5.31 ± 0.64

3 3 2 8 9

1 2 1 1 6

>0.999 >0.999 >0.999 0.900 1

1

69

4.12

3

1

>0.999

4

234

3.87 ± 1.52

3 7

2 6

>0.999 1

5.49

a

MS data for focus on protein subcategories (14-3-3 proteins and vesicular trafficking/cycling; further MS data in Supporting Information Table 1): Purifications shown in Figure 2C were analyzed both by LTQ-FT and as iTRAQ-labeled samples on an ABI 4800 MALDI-TOF/TOF. For iTRAQ samples, all clearly identified proteins with ratios above the average (p = 0.997) are included. For the LTQ-FT measurements (n = 4, combined data from dilution series), the protein enrichment probability threshold was 90%, as determined by SuperHirn.30. Two additional entries with slightly reduced certainty of enrichment are shown due to qualitative or associative criteria: Clathrin Heavy Chain (iTRAQ 116/114 = 2.66 instead of >2.8) due to the clear identification of Clathrin Coat Assembly Protein (AP180) and Ras-related protein Rab 3A (SuperHirn probability of enrichment: ∼80%), as Rab 3D was clearly found to be enriched in the transgenic sample by MALDI-TOF/TOF. Intrinsic positive control APP listed as MOUSE due to mouse protein database used for MS identifications. Headings: accession no. = protein accession number in Swiss-Prot/Trembl; no. ident pept = number of peptides matching the protein; Mascot score = score of the protein hit (only >50 included); iTRAQ ratio = relative abundance of the protein in transgenic/control samples, as calculated by the GPS explorer software based on ratios of 116/114 reporters; no. uniq pept = number of different peptide sequences identifying the protein; no. quant pept = number of peptide ionic signals taken into account to calculate the protein relative abundance transgenic/control using SuperHirn; profile prob. = probability that the identified protein is enriched in the sample of interest, according to SuperHirn. All proteins listed in the LTQ-FT column were identified as having a ProteinProphet probability above 90%. Alternative names: Endophilin, SH3-containing GRB2-like protein 2; Munc18, Syntaxin-binding protein 1, Unc-18 homolog; VAMP2, Synaptobrevin-2; NSF, vesicle-fusing ATPase.

separations were performed at a flow rate of 300 nL/min delivered by a Ultimate pump (Dionex), with a gradient from 100% A (H2O/acetonitrile/trifluoroacetic acid, 98/2/0.1, v/v/v) to 50% B (acetonitrile/water/trifluoroacetic acid, 80/20/0.1, v/ v/v) in 82 min, followed by a 10 min flush at 100% B. The column eluate was mixed with a matrix solution delivered at a flow rate of 0.8 μL/min: the matrix was α-cyano-4-hydroxycinnamic acid (CHCA, reference 70990 from Fluka), prepared at 2.5 mg/mL in H2O/acetonitrile/trifluoroacetic acid, 30/70/ 0.03, v/v/v. The resulting solution was deposited on a stainless steel MALDI target plate: each spot of a 2000-well plate was

made of 10 s of eluate. The MALDI plate was analyzed in automatic mode by a 4800 MALDI-TOF/TOF instrument (Applied Biosystems). Each spot was first analyzed in MS mode, by accumulating signal with up to 1000 laser shots (20 subspectra of 50 shots) over the mass range 700−4000 Da; MS acquisition was stopped after summing 10 subspectra, when the accumulated spectrum contained at least 5 peaks with S/N > 500. Up to 12 ions giving an MS signal with S/N > 40 were then candidates for further MS/MS analysis, performed in order of increasing precursor intensity. The acquisition of an MS/MS spectrum was obtained by accumulating 1500 laser shots (30 subspectra of 50 4078



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mented with B27 and L-glutamine. Transfections were preformed after 6 days in vitro. For DNA transfection in HEK 293, SH-SY5Y, Neuro2A, or primary cells, Lipofectamine 2000 (Invitrogen, #11668-019) was used according to the manufacturer’s protocol. Transfection medium was replaced after 2 h with fresh medium. Cells were fixed or homogenized 18−22 h later.

shots); acquisition was stopped after summing 20 subspectra, when the spectrum contained at least 4 peaks with S/N > 100. The source air pressure was set to 2.5 × 10−6 Torr for MS/MS analysis; it was 5 × 10−7 Torr for MS analysis. Acquired MS/MS spectra were interpreted using Mascot software version 2.1 embedded in the GPS explorer software version 3.5 (Applied Biosystems). Each MS/MS spectrum was processed as follows: no smoothing or background subtraction was performed; only peaks of S/N ratio above 6 and of masses between 60 and 20 Da below the precursor mass were taken into account, and a maximum of 65 peaks were selected to build the mass list representative of the MS/MS spectrum. An error tolerance on mass measurement in MS mode of 150 ppm was specified; the error tolerance was 0.3 Da in MS/MS mode. The use of trypsin was specified, and one missed cleavage was allowed. iTRAQ labeling of peptide N-termini and Lys residues, as well as alkylation of cysteines by MMTS, were considered to be complete modifications; methionine oxidation was additionally considered as a possible modification. Searches were run using the database SwissProt, restricted to the Mus musculus taxonomy, which corresponded to 12307 protein entries. The relative protein amount between the transgenic sample and the control sample was obtained by averaging the ratios of iTRAQ reporter group areas A(116)/A(114) measured on tryptic peptides identifying each considered protein.

Western Blotting

10−20% Tricine precast gradient gels were used for protein separation (Invitrogen, #EC66252). Western blotting was performed with 1:1000 dilution of anti-HA antibody (Roche, #11867423001), 1:2000 dilution of APP-C-terminal antibody (Sigma, #A8717), and 6E10 (Signet, 1:500). Most recent blots using Neuro2A cells were probed with an APP-C-terminal antibody from Epitomics (Y188, 1:3000), as the currently available antibody from Sigma lacks specificity and detects APP in APP knockout brain homogenates in our hands (probably detects APLP2; also see ref 34). All antibodies against synaptic proteins were from Synaptic Systems (SYSY) and used at dilutions from 1:1000 to 1:2000. For visualization of bands, commercial ECL reagents were used (Pierce, #34095). Immunocytochemistry

Cells were washed with PBS and fixed for 20 min with 4% paraformaldehyde (PFA). They were then washed three times for 10 min each with TBS containing 0.05% TX-100, and blocked with TBS containing 0.02% TX-100 and 10% horse serum for a minimum of 2 h. The mouse anti-Myc (Roche, #11667149001) and rat anti-HA (Roche, #1867423) were applied overnight at 1:100 dilution in blocking solution. Cells were subsequently washed and blocked as described above, and Cy2-, Cy3-, or Cy5conjugated secondary antibodies (Jackson laboratories) applied at 1:250 for a minimum of 2 h. Cells were washed and embedded in Mowiol with the addition of 2.5% 1,4-diazobicyclo(2.2.2)octane (DABCO, Sigma-Aldrich, #D-2522).

Calculation of Probability of Enrichment

The probability that a protein with the iTRAQ ratio D belongs to the enriched population of proteins within the data sample can be denoted as p(+|D) and calculated using the following formula, where the probability of a protein having the iTRAQ ratio D, given it is an enriched protein, is denoted as p(D|+), the probability of a protein belonging to the enriched proteins is denoted as p(+), the probability of a protein having the iTRAQ ratio D, given it is not an enriched protein, is denoted as p(D|−), and the probability of a protein belonging to the nonenriched proteins is denoted as p(−). p( +|D) =

Confocal Microscopy

Images were acquired on a Leica TCS/SP2 confocal microscope (Leica, Wetzlar, Germany) with a 63× water immersion objective. The argon laser line of 458 nm was used to excite CFP (PMT window: 465−485 nm) and the 514 nm line to excite Citrine (PMT window: 525−545 nm). A 543 nm HeNe laser was used to excite Cy3 (PMT window: 553−600 nm). A 633 nm HeNe laser was used to excite Cy5 (PMT window: 655−710 nm). Antibody staining with Cy2 is always color-coded in green, Cy3 in red, and Cy5-staining in blue.

p(D|+) ·p( +) p(D|−) ·p( −) + p(D|+) ·p( +)

Cell Culture

Human embryonic kidney cells (HEK 293, DSMZ, ACC 305) and SH-SY5Y neuroblastoma cells were cultivated at 37 °C, 5% CO2, 95% humidity in Dulbeccos modified eagle medium (DMEM, Invitrogen #52100-039) supplemented with 10% fetal calf serum (FCS) and Penicillin/Streptomycin (PS, Invitrogen #10378-016). Neuro2a cells were grown in DMEM with high glucose (Gibco), 10% FCS, 1% PS, and 1× nonessential amino acids (Gibco). SH-SY5Y cells were differentiated with 5 μM retinoic acid for 5 days followed by 20 ng/mL BDNF for 5 days in DMEM containing 2% FCS. Primary neurons were prepared from E14-16 C57Bl/6 mice. Meninges were removed, and cortices were dissected in ice-cold CSS (120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 25 mM TrisHCl, 15 mM D-glucose). The cortices of 5−7 embryos were treated with 7.2 U Dispase II (Roche, #10295825001) for 10 min at room temperature and triturated in DMEM (InvitrogenGibco, #52100-021) with 10% horse serum (HS), 1 mM sodium pyruvate, 44 mM sodium hydrogen carbonate, 1% BSA, and PenStrep. After dissociation, the cells were plated at a density of 9000 cells/mm2 on poly-L-lysine-coated culture slides. Neurons were grown for 10−14 days in Neurobasal medium supple-

FRET

Fluorescence resonance energy transfer experiments were performed as described.16 Briefly, singly transfected CFP and Citrine fluorescence was measured with identical settings as in FRET measurements to determine CFP-bleachthrough and Citrine-cross-excitation values that were subtracted from measured sensitized emission values in cotransfection experiments. pUKBK Vectors

We designed a series of expression vectors with different promoters and tags that efficiently transfect cells due to their minimized size (Supporting Information Figure 1B). The vector backbone was assembled with the help of oligonucleotides coding for multiple restriction sites enabling the insertion of all necessary plasmid elements without superfluous sequences. Besides a ColEI origin of replication, the backbone includes a 4079



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Figure 2. (A) Schematic of the APP-TAP-AICD construct. Phospholipid icons denote the transmembrane region. (B) The known AICD-interacting protein Fe65 is isolated by our purification protocol with APP-TAP-AICD. HEK 293 cells were transfected with APP-TAP-AICD or a myc-tagged APP (APP-2Myc), as well as with Fe65 containing an N-terminal HA-tag (HA-Fe65). Cells were lysed and streptavidin affinity-purified. Lysates (L), unbound flowthrough (F) and eluates (E) were separated on gels and blots stained for HA-Fe65. (C) Comparison of transgenic mouse lines shows APPTAP-AICD mice to express similar amounts of APP as in wild-type mice. Mouse brains were homogenized and proteins analyzed by Western blot using 22C11 antibody (detects mouse and human APP) and 6E10 (detects only the human transgene). Three different APP-TAP-AICD transgenic lines are shown (L2, L10, L12) together with their transgene-negative littermates ((+) and (-) denote presence and absence of transgenic APP, respectively). For comparison wild-type (wt) mouse brain homogenate and TX-100 (T)- and SDS (S)-extracted brains of arcAβ transgenic mice are shown. This transgenic line overexpresses human APP in contrast to APP-TAP-AICD mice. (D) Streptavidin-affinity purification from the brains of APP-TAP-AICD transgenic mice and negative littermates. Aliquots from purified eluates (E) of two age- and sex-matched mice from founder line 10 were analyzed by antibody and silver staining. Western blot (left side) with an APP C-terminal antibody revealed full-length APP as well as processed fragments only in the eluate (E) of transgene-positive mice. In the silver stained gel (right side), green lines denote proteins that are enriched in the transgenic sample and the red lines those enriched in the nontransgenic sample.

known APP-Interaction35,36 gave us the confidence to continue with this construct into transgenic animals. APP-TAP-AICD transgenic mice were generated by pronucleus injection with the construct under control of the ubiquitously active Prion promoter. Three founder lines were generated. Each showed very similar levels of transgene expression, which also closely matched the levels of endogenous APP. This was determined by WB comparing 22C11 antibody staining, which detects APP of both mouse and human origins, to 6E10 antibody staining, which detects exclusively human APP (Figure 2C). High overexpression is undesired, as this leads to excessive copurification of chaperones during TAP experiments (Brian Raught, ISB, Seattle, personal communication). Initial experiments entailing the entire TAP procedure resulted in very low amounts of protein in the final eluates and, more importantly, in no significant differences in band patterns between samples from transgenic versus nontransgenic animals, as judged by silver staining of 1D gels (data not shown). However, by performing rigorous preclearing prior to streptavidin-based affinity purification, we obtained samples after this first purification step that showed no traces of contaminating endogenous APP, as judged by WB of nontransgenic littermates using the A8717 APP C-terminal antibody (Figure 2D). The staining of APP-TAP-AICD transgenic brains further indicated that we had purified full-length APP as well as αand β-CTFs (verified by 6E10 antibody staining, not shown) and AICD. When silver staining gels, we obtained far more intense bands with a single purification step than after the whole TAP procedure and observed clear differences in intensities in several bands between the APP-TAP-AICD and the wild-type sample (Figure 2D). The obtained samples were therefore amenable for quantitative MS.

Kanamycin/Neomycin resistance gene driven by a eukaryotic PGK promoter and a prokaryotic EM-7 promoter. The strong viral CMV promoter and the eukaryotic GAPDH promoter are used to drive the expression of genes bracketed by SfiI and PmeI restriction sites. Multiple tags can be added to the C-terminus via the in-frame AscI site: 3HA, 2myc, Citrine, or Cerulean. Candidate proteins were amplified via PCR from a human fetal brain Pro Quest cDNA Library (Invitrogen) or cDNA derived from SH-SY5Y cells and inserted into pUKBK vectors via SfiI (5′) and AscI (3′) restriction sites. All clones were verified by Sanger sequencing. To control for unspecific binding of proteins to the TAP tag itself, we cloned a construct where the TAP tag is located at a similar position as in the APP-TAP-AICD construct, namely at the cytosolic side of the plasma membrane. This construct consists of the APP signal peptide that triggers cotranslational insertion into the endoplasmic reticulum, followed by an SBP domain, a myc tag, a PreCission cleavage site, the transmembrane domain of the EGF receptor, and finally the TAP tag cassette.

3. RESULTS Optimization of the Affinity Enrichment of APP-Containing Complexes

We inserted the tandem affinity purification (TAP) tag from Stratagene, consisting of Calmodulin binding peptide, followed by streptavidin binding peptide (SBP), into full length human APP, proximal to the cytoplasmic side of the transmembrane domain, yielding APP-TAP-AICD (Figure 2 and Supporting Information Figure 1A). We verified its correct subcellular localization by confocal microscopy of transfected HEK 293 cells (data not shown). Cotransfection of HEK 293 cells with APPTAP-AICD and HA-Fe65 was performed to test the purification efficacy of the construct. In contrast to the case of APP-2myc, the transfection of APP-TAP-AICD enabled the copurification of Fe65 bound to APP (Figure 2B). This reproduction of a well4080



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LC-MS/MS Analyses of the Affinity-Purified Protein Samples (LTQ-FT, and MALDI-TOF-TOF)

The samples generated by streptavidin-based affinity purification were digested in solution by trypsin and split into two sample analysis paths. One part of each sample remained unlabeled and was measured in the form of a dilution series on an LTQ-FT instrument: four peptidic samples, consisting of the mixtures transgenic/control in the proportions 0/100, 10/90, 20/80, and 100/0, were analyzed consecutively. The changes in peptide ion currents, i.e. MS1 signal intensities across the dilution series, were automatically analyzed using specialized software (SuperHirn;37), resulting in relative quantification of proteins in the samples from wild-type and transgenic mice, as depicted in Table 1 (LT-FTQ-data column). Briefly, proteins showing nearly constant abundances in the four dilutions corresponded to contaminants interacting nonspecifically with the affinity resin; in contrast, proteins showing an increase of abundance close to the theoretical trend 0:1:2:10 were enriched in the transgenic mouse sample and were thus potential interaction partners of APP. Only peptides identified by the LTQ-FT analyses of the different samples with a PeptideProphet probability >0.931 were considered for the calculation of protein abundance profiles. The second part of each sample was labeled with the isobaric reagents iTRAQ, which yields relative quantitative information for each peptide during its MS/MS fragmentation. Labeled samples from transgenic (iTRAQ 116) and wild-type (iTRAQ 114) mice were mixed and analyzed by LC-MALDI-TOF/TOF.33 The list of proteins identified by Mascot from the MALDI-MS/MS data were manually curated according to the following criteria: a protein was considered to be reliably identified when it was detected by at least two tryptic peptides, scores of which were above 25 (average identity score threshold calculated by Mascot), or by at least one peptide with a score above 50. Given the different ionization sources used, the two analytical paths could be expected to provide complementary peptide and protein identifications and relative quantification. To decide which of the proteins identified from MALDITOF/TOF analyses were actually enriched and thus overrepresented in the TAP sample from the transgenic mouse, we only regarded those proteins as enriched in the transgenic sample that had an iTRAQ ratio that was above the average of all proteins. To ensure that this threshold is not arbitrary, we applied standard Bayes-statistics to the data to validate the use of the overall mean as a reasonable threshold. We split all certified proteins into two best-fit normal distributions (enriched and nonenriched) based on their iTRAQ ratio and derived an iTRAQ ratio at which the probability of a protein belonging to the nonenriched population would be smaller than 5% (based on the relationship described in the Materials and Methods section). Due to the larger population of enriched proteins, the Bayesderived threshold (2.8) is lower than the average iTRAQ ratio (average = 3.23; p ∼ 0.997). Therefore, it is conservative to apply the average iTRAQ ratio as a threshold (Figure 3A). In a second step, all reliably identified proteins from MALDITOF/TOF analyses were assigned to 1 of 8 protein categories based on general protein function (Figure 3B). The two protein categories most notably overrepresented among the enriched proteins were the 14-3-3 protein class and proteins involved in vesicular trafficking. The percentage of enriched proteins was 100% and 87% for 14-3-3 proteins and vesicular trafficking proteins, respectively, higher than for any other group. Furthermore, the enrichment percentage of these proteins remained constant when switching from using the less stringent

Figure 3. (A) Threshold and categorization of enriched proteins isolated from APP-TAP transgenic mice. For the 132 proteins identified from the MALDI-TOF-TOF analysis, we calculated the distribution of iTRAQ ratios and, in analogy to the Bayes statistics underlying protein identification algorithms such as PeptideProphet, assumed there were two protein populations in the sample. Those that were not enriched (chaperones, etc.) with a low enrichment factor and genuinely enriched proteins (interacting with APP) with a higher factor. The two populations were modeled by fitting to normal distributions, and the iTRAQ enrichment factor at which the probability of a protein belonging to the enriched population is 95% was identified to be 2.8. Due to the larger population of enriched proteins, the average iTRAQ ratio (3.23) is higher than the Bayes-derived threshold. (B) 132 proteins identified and quantified by MALDI-TOF-TOF were manually curated and assigned to different classes of protein function, with the 14-3-3 family grouped into a unique class. 14-3-3 proteins were exclusively and vesicular trafficking proteins nearly exclusively found to be enriched in the transgenic samples. Furthermore, the percentage of enrichment of these proteins remained constant when switching from using the less stringent Bayes p = 0.95 based threshold to the more stringent mean (p = 0.997), while the percentage of enrichment went down for all other categories.

Bayes threshold (p = 0.95) to the more stringent average (p = 0.997), while the enrichment percentage went down for all other categories. The data derived from LTQ-FT analysis allowed consolidating the list of proteins that were clearly over-represented in the affinity-purified samples obtained from the transgenic mice, compared to the control purifications from nontransgenic littermates, by comparing the data from the two mass spectrometry methods. As has been described in published experiments, the peptides that can be successfully identified by MALDI-TOF/TOF versus ESI-LTQ-FT are partly complementary, due to the largely differing properties of the ionization sources and to the different nature of the analyzers.38 Similarly, our LTQ-FT-derived data showed a big overlap with the MALDI-TOF/TOF analyses (Table 1), but some proteins (i.e., 14-3-3-eta, Rab-3A, Synapsin-2, and Syntaxin-1A and 1B2) were only detectable by LTQ-FT analysis. Due to the high sequence homology between, for instance, 14-3-3 or Rab-3 proteins, 4081



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tryptic digestion generates a set of common peptides that may be attributed to any one of the homologous proteins. Furthermore, automated LC-MS/MS analysis usually does not allow fragmenting all the peptides present in a complex sample, and to be detected, the specific peptide(s) of a given protein need to ionize and fragment efficiently on the MS system used. Nevertheless, every single protein identified as enriched by MALDI-TOF/TOF analyses was at least also identified by LTQFT analysis. Moreover, when available, protein semiquantification from LTQ-FT data correlated perfectly with results from MALDI-TOF/TOF data. In summary, mass spectrometry identified the two most prominent categories of putative APP-interacting proteins to belong to the 14-3-3 family and proteins involved in the synaptic vesicle cycle (Table 1 and Figure 4).

Figure 4. Schematic representation of a synaptic bouton showing the synaptic vesicle cycle with the identified APP-interactome described in Table 1. Synaptic vesicle glycoprotein 2A (SV2A), a regulator of presynaptic calcium, the SNARE protein VAMP2, the calcium-sensor Synaptotagmin-1, and APP are transmembrane synaptic vesicle proteins. Synapsin-1 is localized to the cytoplasmic surface of synaptic vesicles and holds them together in the reserve pool. It also tethers synaptic vesicles to the actin cytoskeleton and is a phosphorylation-state dependent regulator of vesicle mobilization. Rab3 associates with the surface of synaptic vesicles and is a member of a large GTPase family that delivers vesicles to acceptor membranes. SNAP-25 and Syntaxin-1 are plasma membrane resident SNARE proteins. Munc18 binds Syntaxin-1 and keeps it in a locked conformation. α-Synuclein attaches to membranes, binds VAMP2, and promotes SNARE-complex assembly. SNARE-complex formation between Syntaxin-1, SNAP-25, and VAMP2 docks the synaptic vesicles close to the plasma membrane. Influx of calcium triggers rapid exocytosis of neurotransmitters via binding to Synaptotagmin-1. With exocytosis, APP gets exposed to the extracellular space and can be cleaved by α-secretase. After fusion, hexamers of the ATPase NSF disassemble the cis-SNARE complex for retrieval of VAMP2 into endocytosed vesicles. For endocytosis, AP180, among other adaptor proteins, recruits the Clathrin triskelion to the membrane. The BAR domain protein Endophilin-1 stabilizes membrane curvature, has a prominent function in synaptic vesicle endocytosis, and interacts with Dynamin-1. After formation of Clathrin-coated vesicles, they are pinched off the plasma membrane by the GTPase Dynamin-1. Recycling of synaptic vesicle components occurs via endosomes, the place where APP is cleaved by β- and γ-secretase to generate Aβ peptides. ARF3 is a GTPase of the Ras superfamily that functions in vesicular transport, recruiting coat complexes to budding vesicles. 14-3-3 proteins are phospho-Ser/Thr binding proteins with diverse functions. Their highest concentration is found in brain, and binding of 14-3-3 dimers to APP and Fe65 simultaneously has been shown to increase gene transactivation by AICD. Except for the secretases that cleave APP, all molecules depicted have been identified in the proteomic screen for APP-interacting proteins. For references to the individual proteins, please refer to the Discussion.

Verification of MS-Identified APP Candidate Partners

We focused on proteins involved in synaptic vesicle cycling for verification of the APP-associated proteins identified by MS. We amplified the cDNA of the candidates from RNA isolated from human SH-SY5Y cells or a Pro Quest cDNA Library and cloned them into pUKBK vectors. The pUKBK vector system (Supporting Information Figure 1B) was designed to achieve higher transfection efficiency, to be able to switch between different promoters and to attach various tags onto the cDNA (see Materials and Methods). Genes of candidate APPinteracting proteins tagged with 3HA were cotransfected with APP-2myc into HEK 293 cells, and subcellular colocalization was analyzed by confocal microscopy (Figure 5). APP showed a substantial overlap with Stg1 and VAMP2, two synaptic vesicle proteins. Colocalization was seen in ER/Golgi and vesicular structures. SNAP-25 was targeted to the plasma membrane and only overlapped with APP vesicles that were close to the plasma membrane. NSF labeling was mainly absent from APP-positive ER/Golgi structures and overlapped with APP only in processes and at the plasma membrane. Dynamin-1 labeled many vesicular structures not containing APP, but some of the APP-stained vesicles also stained for Dynamin. 14-3-3ε was colocalized with APP in cytosolic structures and showed an additional nuclear staining. Finally, the known APP-binding protein Fe65 had a completely overlapping staining with APP. For pull-down experiments, we cotransfected HEK 293 cells with APP-TAP-AICD or APP-2myc as a control together with the 3HA-tagged candidates. Streptavidin-pulldown was followed by Western blot analysis using HA-tag staining. The APPinteracting protein Fe65 was consistently isolated together with APP-TAP-AICD and used as a positive control for affinity isolations performed on different days. None of the candidate genes tested (Dynamin-1, NSF, Synaptotagmin-1, SNAP-25, VAMP2, and 14-3-3ε) were specifically isolated via APP-TAPAICD. When proteins were found in the isolate, they also appeared after affinity purification with the control APP-2myc, due to unspecific binding (Supporting Information Figure 2). Thus, the association of APP with presynaptic proteins cannot be validated in HEK293 cells which might relate to the indirect nature of the interactions or the lack of bona f ide presynaptic structures in these cells. We next analyzed the subcellular distribution of APP and its associated protein-complex components in primary neurons by cotransfection of APP-2myc and candidate 3HA-tagged interaction partners. Expression was driven by an endogenous GAPDH promoter that is less strong than the viral CMV promoter and shows good expression in neurons and only weak

expression in glial cells. Confocal microscopy revealed that 14-33ε proteins showed substantial overlap with APP in vesicles and in spines (Figure 6; see enlargement of neurite segment). When we cotransfected Fe65, it was completely associated with the APP-14-3-3ε staining. We further analyzed four of the candidates directly involved in the synaptic vesicle cycle. Nearly all of the vesicles in neuronal processes containing APP also stained for Stg1 and VAMP2, two integral synaptic vesicle membrane proteins. Furthermore, most Stg1 or VAMP2-positive vesicles also stained for APP. NSF and SNAP-25 also colocalized with many of the APP-labeled vesicles but additionally localized to other vesicular structures devoid of APP. To resolve subcellular localization at the molecular level, we used fluorescent resonance energy transfer (FRET) techniques 4082



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Figure 5. Subcellular colocalization of APP and candidate binding proteins in HEK 293 cells. HA-tagged candidates and myc-tagged APP were cotransfected into HEK 293 cells and detected by immunofluorescence using a confocal microscope. APP (green) localizes to ER/Golgi structures as well as vesicular structures throughout the cell. APP was colocalized with Synaptotagmin-1 (Stg1) and VAMP2, two proteins that reside in synaptic vesicles. SNAP-25 is targeted to the plasma membrane and overlapped only with APP-labeled vesicles that are close to the membrane. NSF and Dynamin-1 showed only partial overlap with selected APP vesicles. 14-3-3ε shows a strong overlap, and the Fe65 staining has a 100% overlap with APP. The scale bar represents 13 μm; for the VAMP2 and 14-3-3ε panels, it represents 20 μm. The boxed region is zoomed 4-fold. 4083



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Figure 6. Subcellular colocalization of APP and candidate binding proteins in primary neurons. HA-tagged candidates and myc-tagged APP were cotransfected and detected by immunofluorescence using a confocal microscope. (A) 14-3-3ε proteins showed substantial overlap with APP in vesicular structures throughout the processes and in dendritic spines (see enlarged insets). (B) When we cotransfected mCherry-Fe65, it completely colocalized with the APP-14-3-3ε staining. (C−F) We further analyzed candidates directly involved in the synaptic vesicle cycle. Nearly all of the vesicles in neuronal processes containing APP also stained for Stg1 and VAMP2 and vice versa. NSF and SNAP-25 also colocalized with many of the APP-labeled vesicles but additionally localized to other vesicular structures devoid of APP. The scale bar represents 40 μm, for the overviews of Stg1, NSF, SNAP-25, and VAMP2, the bar represents 120 μm. The neurites shown in the right column are zoomed 4-fold.

by fusing improved cyan and yellow fluorescent proteins (Cerulean and Citrine) to APP and the candidate associated proteins. Sensitized emission from Citrine after exciting Cerulean is only detected when the labeled molecules are within nanometer distance of each other. Cotransfecting APP labeled with either of both fluorophores into SH-SY5Y cells showed a

colocalization in vesicular structures. After correction of bleachthrough and cross-excitation, APP-containing vesicles revealed a clear FRET signal (Figure 7A). This is likely due to the dimerization of APP that has been described previously.39,40 VAMP2 again colocalized with APP in vesicles, but we could not detect a FRET signal. The absence of FRET was also seen with 4084



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SNAP-25, which was distributed more homogenously in the plasma membrane. APP colocalized with Stg1 in vesicles, as seen in primary neurons, and additionally showed a strong FRET signal, revealing the close molecular proximity of these proteins. We therefore repeated streptavidin-mediated affinity-isolations from transgenic mouse brain homogenates and analyzed the resulting eluates not by MS but with standard Western blot techniques (Figure 7B). In APP-TAP-AICD transgenic brains, we could elute full-length and cleaved fragments of the tagged APP whereas eluates from wild type brains had no APP in the eluate (left blot). We next probed the blots with antibodies against Stg1, Syntaxin-1B, and VAMP2. Only Stg1 was specifically eluted together with APP from brain homogenates of APP-TAP-AICD transgenic mice but not their wild type littermates. To control for unspecific binding to the TAP tag cassette, we used a control transmembrane construct that expresses a TAP tag attached to the membrane as in APP-TAPAICD, but lacking the APP/AICD sequence. Neuro2A cells were cotransfected with APP-TAP-AICD or the TAP-control construct, together with HA-tagged Stg1 or VAMP2. Streptavidin-Dynabeads-mediated affinity-isolation was followed by Western blot analysis. Again, we could coelute Stg1 together with APP-TAP-AICD but not VAMP2. In contrast, the TAPcontrol construct was bound to the Dynabeads and could be eluted with biotin, but we did not detect Stg1 in the eluate (Figure 7C). The TAP-control construct was absent in the flowthrough (F), in contrast to the case of APP-TAP-AICD. This is likely due to the existence of two SBP domains in the construct that can bind to streptavidin. These experiments show that the TAP tag itself does not interact with Stg1 and that the coelution of Stg1 with APP-TAP-AICD is due to an interaction between APP and Stg1.

4. DISCUSSION Using a proteomic approach with a novel transgenic mouse line expressing tagged APP, we have identified proteins that associate with APP in brain. The two categories of proteins that most clearly were enriched with APP affinity isolation were 14-3-3 proteins and proteins involved in the synaptic vesicle cycle, implicating a role of APP in synaptic function and signaling. To circumvent the necessity of using APP C-terminal antibodies for purification that could compete with endogenous binding proteins, we inserted an affinity tag preceding the AICD sequence. We generated transgenic mice that express the APPTAP-AICD construct at similar levels as endogenous APP, therefore reducing the probability of incorrect subcellular localization and unspecific binding. We performed affinity purifications from brain and split the resulting samples for analysis using two different semiquantitative proteomic approaches. MALDI-TOF-TOF analysis was performed on iTRAQlabeled samples. Defining a threshold of the iTRAQ ratio above which a protein could be considered to be enriched was important in order to avoid false positives. We used Bayes statistics to define a probabilistic threshold of 2.8 above which a protein would be considered enriched at a p-value smaller than 0.05 (Figure 3A). However, as described in the Results section, we analyzed the data based both on this threshold as well as on the average iTRAQ ratio of 3.2 as a threshold. The latter threshold is more stringent but nevertheless shows that the conclusions derived from the lower threshold remain valid, especially for proteins in the 14-3-3 and synaptic vesicle trafficking categories (Figure 3B). Next, we analyzed the other

Figure 7. Synaptotagmin-1 directly interacts with APP. (A) Fluorescence resonance energy transfer (FRET) measurements were performed to determine molecular proximity. Improved cyan and yellow fluorescent proteins (Cerulean and Citrine) were fused to APP and the candidate associated proteins and transfected into differentiated SHSY5Y cells. The last column (FRET) shows sensitized emission from Citrine after exciting Cerulean with correction of cross-excitation and bleach-through. Only the interaction of APP with itself and with Synaptotagmin-1 resulted in FRET emission whereas VAMP2, although showing a strong colocalization with APP, did not lead to sensitized emission. (B) Streptavidin pull-down from APP-TAP and wild-type brains was analyzed by Western blotting. The first blot is stained with an APP C-terminal antibody, revealing full-length APP and stubs in the lysates (L), the flowthrough (F), and the eluate (E) of APP-TAP mice but not in the eluate of wild-type mice. Of the candidates tested, only Synaptotagmin-1 was specifically pulled down from APP-TAP brains. (C) Neuro2A cells were cotransfected with APP-TAP-AICD or a TAPcontrol construct, together with HA-tagged Stg1 or VAMP2. Streptavidin-mediated pull-down was followed by Western blot analysis with the blots cut horizontally to probe different antibodies without stripping (APP C-term antibody to detect full-length (fl)-APP and cleaved APP stubs, HA tag antibody to detect Stg1 and VAMP2, myc tag antibody to detect the TAP-control construct). Stg1, but not VAMP2 was coeluted with APP. In contrast, the TAP-control construct could be retrieved by Streptavidin-mediated pull-down (marked with arrows), but no Stg1 was seen to coelute, i.e. bind unspecifically to the TAP tag. Due to the similar size of VAMP2 and the TAP-control, construct blots were first probed with HA tag and then with myc-tag antibodies. The asterisk marks the signal from VAMP2. 4085



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and synapses.47−49 Among the APP-associated proteins, we identified many components necessary for synaptic vesicle fusion, including the core SNARE complex proteins VAMP2,50 Syntaxin-1,51 and SNAP-2552 as well as other proteins necessary for vesicle fusion such as Munc1853 and NSF.54 Additionally, we identified other synaptic vesicle proteins such as Synaptotagmin1,55,56 Synapsin-1 and -2,57 and the synaptic vesicle glycoprotein 2A.58 Furthermore, we also identified several of the proteins necessary for the endocytosis of synaptic vesicles from the membrane. Among them are the coat protein Clathrin and its adaptor protein AP180 as well as Dynamin-159,60 and Endophilin-161,62 that are necessary for synaptic vesicle invagination and fission from the plasma membrane. Finally, our proteomic screen identified proteins involved in vesicle fission/fusion such as the ARFs (ADP-ribosylation factors) and Rab proteins, including Rab3, that regulates a late step in synaptic vesicle fusion.63 ARFs showed the highest enrichment with APP. ARFs are small GTPases regulating the assembly of coat complexes such as Clathrin on budding vesicles.64 ARFs have been described to bind directly to MINTs (Munc18-interacting proteins)/X11,65 known APP-binding proteins.22 Together, Arfs and MINTs regulate the post-Golgi trafficking of APP.66 Furthermore, MINTs can couple APP to Munc1867 and are important regulators of neurotransmitter release.68 The APPMINT-Munc18 complex also associates APP with Syntaxin-1, thereby inhibiting amyloidogenic processing of APP.69,70 Many of the proteins we identified to be associated with APP in brain are thus involved in the synaptic vesicle cycle. A recent publication described immuno-isolation of APP from brain membrane vesicles and analyzed the coisolated proteins with an array of antibodies directed against known synaptic components.71 Many proteins were identified that we also found with our unbiased proteomics approach, for instance Synapsin-1, SNAP-25, Syntaxin-1B, VAMP2, and Rab3. In a different approach, synaptic vesicles have been purified, followed by detailed proteomic analysis of vesicle constituents.72 This synaptic vesicle preparation has been reanalyzed, and the presence of APP in these vesicles has been verified recently.73 Furthermore, using elegant optical imaging, these authors showed that synaptic vesicles containing APP undergo activitydependent exocytosis/endocytosis cycles, whereby some of the cleaved extracellular domain gets secreted into the synaptic cleft. Another presynaptic protein enriched in our isolations from transgenic mice was α-synuclein. Mutations in α-synuclein can lead to dopaminergic neuron death, and presynaptic aggregation of α-synuclein generates Lewy bodies that are found in Parkinson’s disease (PD) and AD.74−77 Interestingly, UCH-L1 mutations have been found in familial PD, and transgenic mice expressing mutated UCH-L1 show dopaminergic cell loss.78 UCH-L1 was strongly enriched in our APP purifications (cf. Supporting Information Table 1) and might cooperate with APP to regulate synaptic structure and function.79,80 Indeed, transduction of UCH-L1 in slices restored synaptic deficits in transgenic APP expressing mice.81 Together, these recent publications and the data presented in this paper clearly show that APP is a synaptic vesicle protein with a function at the presynapse. APP has been shown to play a role at the synapse. The protein has been localized to both pre- and postsynaptic sites10,49 and has been postulated to be involved in homotypic trans-synaptic interactions.39 The role of the APP-derived Aβ peptide in inhibiting synaptic plasticity has been thoroughly examined. Synaptic activity has been shown to stimulate the processing of

half of the samples with a LTQ-FT-based method that does not require differential sample labeling to yield estimation for protein relative abundances between the two compared samples.37 The resulting two lists of proteins identified as enriched in the sample from transgenic mice had a good degree of overlap, showed some complementarity, and were without conflicts. In spite of the fact that we were able to show that our purification protocol did not disrupt interactions between AICD and its binding protein Fe65 (Figure 2B and Supporting Information Figure 2), we did not detect Fe65 in our MS experiments. The amount of Fe65 protein may be too limited to allow its identification in LC-MS/MS analyses, as APPassociated complexes are isolated from transgenic brains that express the APP-TAP transgene but no additional Fe65. The identification of APP-associated Fe65 using Western blots was possible using cells that were transiently cotransfected with APPTAP and Fe65. Thus, the amounts of both APP-TAP and Fe65 are much higher in the cell culture experiment. The limited overlap of proteins identified by our MS analyses and previous Yeast Two Hybrid methods is a manifestation of the complementarity of the techniques. However, we identified the previously described guanine nucleotide-binding protein G0 subunit α121 to be 4.3-fold enriched in APP-TAP-AICD eluates. A published proteomics study reported proteins binding to APP in post-mortem brains of healthy control subjects and of AD patients.41 Several APP-interacting proteins were found: 14-3-3ζ, NEM sensitive fusion protein (NSF), Munc18, Dynamin-1, and ubiquitin C-terminal hydrolase L1 (UCH-L1), which were all again identified and quantified as enriched in our MS experiments, mutually confirming the data published by Cottrell et al. and indicating the validity of the data presented in this work. Another recent study used transcardiac perfusion cross-linking to isolate the APP interactome from brain via antibody pull-down.42 The resulting candidate list shows no overlap with our data. In vivo cross-linking can conserve weak interactions throughout the purification procedure, resulting in the identification of proteins that are lost during our purification method, but might also cause cross-linking of proteins not directly interacting with each other. We identified five of the six mammalian 14-3-3 proteins, and all of them showed enrichment in the samples from APP-TAPAICD mouse brains. 14-3-3 proteins are phosphoserine/ threonine binding modules that regulate a variety of signaling processes regulated by phosphorylation as a means of signal propagation.43 14-3-3 proteins function as cup-shaped dimers, and their binding to target proteins can mask subcellular localization signals and phosphorylation sites. The simultaneous interaction of 14-3-3γ dimers with both AICD and Fe65 has been described,44 and it facilitates transactivation of luciferase reporters in a phosphorylation-dependent fashion. 14-3-3γ is one of the identified 14-3-3 proteins of our proteomics approach that was identified in both LTQ-FT and MALDI-TOF/TOF measurements. From the MALDI-TOF/TOF analysis of differentially iTRAQ-labeled samples, 13 enriched proteins are involved in synaptic vesicle endo- or exocytosis (18.3% of all enriched proteins), while only two such proteins (3.3% of all nonenriched proteins) are present at below-average levels. Also, the latter two proteins have iTRAQ ratios of 2.66 and 2.47 and are thus only slightly below our threshold iTRAQ ratio of enrichment. The APP interactome we identified in brain thus revealed many proteins involved in the synaptic vesicle cycle.45,46 This implies a role for APP in synaptic vesicle function in the brain, in concordance with localization of APP to neuronal growth cones 4086



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I restriction enzyme sites via the primers. Size-selected PCR fragments were cloned into our in-house developed pUKBK vectors. These vectors have a minimized backbone necessary for plasmid replication (ColE1) and prokaryotic or eukaryotic antibiotic selection. The expression of inserted cDNAs can be driven by a CMV or the endogenous GAPDH promoter, and the cDNAs are C-terminally tagged with either small epitope tags (3HA and 2myc) or fluorescent proteins (Cerulean and Citrine). Supplementary Figure 2: Fe65 is consistently pulled down from HEK293 expressing APP-TAP. HEK 293 cells were cotransfected with APP-TAP or APP-2myc together with different HAtagged candidate binding proteins. Cell homogenates were affinity-purified with streptavidin-Dynabeads and Western blots probed with APP C-terminal antibodies and anti-HA tag antibodies. Full-length APP and APP stubs from APP-TAP but not from APP-2myc were always detected in the eluate. The APP-binding protein Fe65 was consistently coeluted with APPTAP but not with APP-2myc and was used as a positive control for all transfections performed on different days. None of the candidate APP-binding proteins was found to specifically elute with APP-TAP. L, lysates; F, flowthrough; E, eluate. Supplementary Figure 3: Analysis of the expression levels of candidate APP-interacting proteins in wild type and APP knockout animals. (A) Brain homogenates were analyzed by Western blot using antibodies against the different proteins. (B) Quantification of expression levels analyzed by Western blot in four animals each. None of the putative APP-interacting proteins showed a significant change in expression levels in the APP knockout animals. Supplementary Table 1. Description of all proteins identified by MALDI-TOF-TOF. This table includes all protein categories, not just the 14-3-3 and synaptic vesicle proteins shown in Table 1. This material is available free of charge via the Internet at http://pubs.acs.org.

APP, leading to enhanced Aβ production, which in turn can feed back to inhibit synaptic plasticity.8,9,15,82−85 In addition, several reports show synaptic effects of full-length APP or cleavage products other than Aβ on synaptic function.10 In contrast to transgenic mice expressing human APP harboring familial AD mutations, expression of wild-type APP in mice resulted in enhanced synaptic plasticity and memory performance86 as well as an increased number of synapses containing more vesicles per synapse.87,88 Furthermore, analysis of neurons derived from APP knockout mice revealed dramatic changes in synaptic properties related to the increased number of functional synapses.89 The expression levels of APP have been reported to correlate with synaptogenesis,90 and expression of APP in HEK293 cells promoted synaptogenesis in contacting axons.91 Our proteomic analysis has identified APP as a synaptic vesicle component strongly colocalizing with other vesicle proteins VAMP2 and Stg1. Given the strong FRET signal observed between tagged APP and Stg1 and the lack thereof between APP and VAMP2, we conclude that APP in synaptic vesicles is located in close proximity to Stg1. Our data show that MS analysis identifies many more proteins associated with APP than detected in Western blots. One cause may be the increased sensitivity of MS (high attomole to low femtomole) as compared to Western blot analysis (low nanogram, which for a 50 kDa protein would be high femtomole). Analysis of brain homogenates or cell lysates with Western blots revealed similar amounts of APP in the lysate and flowthrough, meaning that only a minor fraction of the tagged APP, including its binding partners, is bound to the beads during isolation. It has been shown that expression of Stg1 in fibroblasts leads to the generation of long filopodial processes92 and that APP enhances neurite outgrowth in neurons.3,4 We observed a similar effect in HEK293 cells after transfecting Stg1 and APP. This resulted in the formation of a high number of thin filopodia and a strong process outgrowth resulting in a neuron-like morphology of the cells. Thus, APP and Stg1 might interact to together regulate process outgrowth. APP could also influence the calcium-sensing function of Stg1, which is essential for the calcium influx-triggered fast synchronous release pathway of synaptic vesicles. APP knockout animals show decreased paired pulse facilitation (PPF) in the dentate gyrus.93 Because PPF results from the accumulation of calcium in the presynapse, this would imply that interaction with APP increases the calcium sensitivity of Stg1 to trigger vesicle exocytosis. In conclusion, our proteomic analysis of APP-TAP-AICD mouse brains has identified many proteins centrally involved in the synaptic vesicle cycle as well as 14-3-3 proteins that regulate phosphorylation-dependent signaling. The progression of AD has been previously correlated with synaptic pathology. Our data raise the possibility that APP effects on the synaptic vesicle cycle are involved, with an emphasis on the interplay of APP and calcium sensor Stg1.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 0041-786619632. Present Address §

CNRS, LAMBE UMR 8587, Université d’Evry Val d’Essonne, 91025 Evry, France. Author Contributions ⊥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Michelle Meier, Michelle Meyer, Kirsten Kallina, and Diana Bundschuh for excellent technical assistance and to Martin Hubalek for help in acquiring LTQ-FT data. This work was supported by the Transregio SFB (6027) on Structure and Function of Membrane Proteins and the SNF NCCR on Neural Plasticity and Repair.

ASSOCIATED CONTENT

S Supporting Information *

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Supplementary Figure 1: (A) Amino acid alignment showing the insertion site of the tandem affinity purification (TAP) cassette in the APP C-terminal sequence. CBP: Calmodulin binding peptide (red). SBP: streptavidin binding peptide (blue). (B) cDNA of selected putative interaction partners (4 examples depicted) amplified by PCR from mixtures of HEK and SH-SY5Y human cDNA or a Pro Quest cDNA Library, incorporating Sfi I and Asc

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Interactome of the Amyloid Precursor Protein APP ... - ACS Publications

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