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Keywords:

  • adapter protein;
  • amyloid precursor protein;
  • Fe65;
  • FRET, gamma cleavage;
  • intracellular domain

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References

Amyloid-β, the peptide that deposits as senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by a gamma secretase-mediated intramembranous cleavage. In addition to amyloid-β, this cleavage produces a carboxyl-terminal intracellular fragment which has an unknown function. The carboxyl-terminal domain of APP interacts in the cytoplasm with an adapter protein, Fe65. We demonstrate by laser scanning confocal microscopy that a gamma secretase generated APP carboxyl-terminal domain, tagged with green fluorescent protein (GFP), translocates to the nucleus in a manner dependent upon stabilization by the adapter protein Fe65; APP which has been mutated to block interactions with Fe65 cannot be detected in the nucleus. The APP-CT domain continues to interact with Fe65 in the nucleus, as determined by both colocalization and fluorescence resonance energy transfer (FRET). Visualization of the APP-CT-Fe65 complex in the nucleus may serve as a readout for processes that modify gamma secretase release of APP-CT.

Abbreviations used

amyloid-β

APP

amyloid precursor protein

APP-CT

APP carboxyl-terminal domain

CT

carboxy terminal

FRET

fluorescence resonance energy transfer

GFP

green fluorescent protein

HRP

Horseradish peroxidase

PBS

phosphate-buffered saline

PVDF

polyvinylidene difluoride

SDS

sodium dodecyl sulfate.

The amyloid precursor protein (APP) is metabolized to give rise to amyloid-β (Aβ), a 40–42 amino acid peptide that is the major constituent of senile plaques in Alzheimer's disease. APP undergoes intramembrane proteolysis by a presenilin-dependent gamma secretase activity, releasing the Aβ peptide from the APP transmembrane domain (Wolfe et al. 1999). The same presenilin-dependent gamma secretase activity is responsible for release of the intracellular signaling domain of Notch (De Strooper et al. 1999; Ray et al. 1999; Song et al. 1999; Steiner et al. 1999; Struhl and Greenwald 1999; Ye and Fortini 1999; Berezovska et al. 2000). The intracellular carboxyl-terminal domain of Notch is released from the membrane and, either directly or indirectly, initiates a signal transduction cascade important in cell differentiation (Artavanis-Tsakonas et al. 1995).

Within the last year, several observations have highlighted a potential biological role for the small cytoplasmic carboxyl domain that is generated by gamma secretase cleavage of APP (APP-CT). Cao and Sudhof (2001) used a heterologous signal transduction assay to suggest that APP-CT interacts with Fe65 and histone acetyltransferase Tip60, leading to transactivation of a reporter construct. Gao and Pimplikar (2001) demonstrated that APP-CT fragment interacts with PAT1 in the nucleus and causes selective degradation of PAT1 and repressed retinoic acid-responsive gene expression. The biochemical identification of the APP-CT has been accomplished along with the observation that it has a very rapid half life in cultured cells (Cupers et al. 2001). Kimberly et al. (2001) demonstrated that Fe65 stabilizes and markedly enhances the half-life of APP-CT, and that it is relatively enriched in the nucleus.

We now extend these observations and demonstrate directly that the APP-CT fragment forms from full length APP (APP770) in a gamma secretase dependent fashion, and, using a green fluorescent protein (GFP)-tagged construct and laser scanning confocal microscopy, directly visualize its translocation to the nucleus. APP-CT appears to be stabilized by collaboration with the adapter protein Fe65, with which it interacts in the cytoplasm (Kinoshita et al. 2001), and APP-CT continues to be closely associated with Fe65 in the nucleus.

Generation of expression constructs for human APP and Fe65

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References

Human APP770 cDNA lacking the termination codon was ligated into the SmaI and ScaII sites of pEGFP-N2 vector (Clontech, Pal Alto, CA, USA) after blunting all sticky ends to generate APP770-GFP. To make the carboxyl-terminal myc-tagged constructs of APP770 (APP770-myc), APP770 cDNA lacking the termination codon was generated by PCR and inserted into the HindIII site of pcDNA3.1B (Invitrogen, Carlsbad, CA, USA) in frame. APP751 without any tags in pcDNA3.1 (Invitrogen) was also used to avoid the artificial effect of GFP or myc tag (Nostrand et al. 2001).

The truncated APP constructs, APP-C99 and APP-C58, encoding various lengths of the APP carboxyl terminus were made. C58 construct without any tags was made as follows; the APP770 cDNA template was used to PCR the carboxyl terminal 58 amino acid coding region with a stop codon. A set of primers which add a Kozak sequence to the 5′ end were used: 5′-GCCTCGAGGCCACCATGGCGACAGTGATCGTCATCACCTTG-3′ and 5′- GCGAATTCCTAGTTCTGCATCTGCTCAAAGAA-3′. The fragment amplified by PCR was digested and ligated into the XhoI and EcoRI site of the pcDNA3.1(–)B. The APP-C99 construct without a stop codon was first generated by PCR with a set of primers, 5′-GCAAGCTTGCAGAATTCCGACATGACTCAGGA-3′ and 5′-GCCTCGAGCGTTCTGCATCTGCTCAAAGAACTT-3′, then ligated into pSecTag 2B which contains an ATG (Invitrogen) to generate a carboxyl-terminal myc-tagged APP-C99 (C99-myc) vector.

To inhibit the interaction with Fe65, mutation of tyrosine residues at codons 682 and 687 of YxxNPxY motif (of the 695 numbering) to alanine (Y682/687 A) of the APP770-GFP construct was accomplished using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The sequence of the mutant APP770-GFP [APP770 (Y682/687 A)-GFP] was confirmed by DNA sequencing.

The generation of carboxyl-terminal myc-tagged Fe65 (Fe65-myc) has been reported elsewhere (Borg et al. 1996; Kinoshita et al. 2001). Authenticity of all PCR-generated constructs was confirmed by DNA sequencing.

Antibodies and reagents

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References

Monoclonal antibody 8E5 was raised against the extracellular amino acid residues 520–668 of APP770 (a kind gift of Dr P. Seubert, Elan Pharmaceuticals, South San Francisco, CA, USA). Rabbit polyclonal antibody C8 and was raised against the C-terminal 20 amino acid residues of APP770 (kindly provided by D. Selkoe, Brigham and Women's Hospital, Boston, MA, USA) (Kimberly et al. 2001). Mouse monoclonal antibody 4G8 was raised against the amino acid residues 17–24 of Aβ peptide (Signet Laboratories, Dedham, MA, USA). Mouse monoclonal anti-myc antibody was purchased from Invitrogen.

Two gamma secretase inhibitors were used to inhibit the gamma cleavage of APP; MW-111-20 (Wolfe et al. 2000), and WPE-11–72 (Dovey et al. 2001). The inhibitors (MW-111-20, 5 µm; WPE-11-72, 100 nm) were added to the culture media 3 h after transfection.

Cell culture conditions and transient transfection

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References

H4 cells derived from human neuroglioma cells (American Type Culture Collection, Manassas, VA, USA) were cultured in OPTI-MEMI with 10% fetal bovine serum. Transient transfection of H4 cells was performed using a liposome-mediated method (FuGene 6; Roche Molecular Biochemicals, Indianapolis, IN, USA). Cells were plated onto four-well chambers 1 day before the transfection. First a mixture of 1 µg of plasmid DNA and 3 µL of Fugene6 was made in 100 µL of Dulbecco's modified Eagle's medium and left for 15–30 min at room temperature, and then 25 µL of this mixture was added to the medium in each well. The incubation time was from 24 h to 48 h. The same protocol was used for double transfection of APP and Fe65 plasmids.

Immunohistochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References

Immunostaining was performed 24–48 h post-transfection. Cells were fixed in 4% paraformaldehyde for 10 min, washed in Tris-buffered saline (TBS pH 7.3), permeabilized with 0.5% TritonX-100 for 20 min, and blocked with 1.5% normal goat serum for 1 h. Transfected cells were then incubated with appropriate primary antibodies for 1 h at room temperature; mouse anti-myc monoclonal antibody (Invitrogen, 1 : 1000) to label C58-myc or Fe65-myc, mouse monoclonal 8E5 antibody (1 : 1000) to label extracellular domain of APP, rabbit polyclonal C8 antibody (1 : 500) to label C-terminus domain of APP. The cells were then washed three times in TBS, and labeled with Cy3-conjugated secondary IgG antibody (Jackson Immunoresearch Laboratories, West Grove, PA, USA; 10 µg/mL) for 1 h at room temperature. For the fluorescence resonance energy transfer (FRET) experiments, immunostained cells were stored in TBS at 4°C.

Immunoblotting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References

H4 cells were split into 100 mm dishes and transfected with either APP751, APP or no DNA. After 48 h, plates were washed once with phosphate-buffered saline (PBS) then scraped into 200 µL PBS, and pelleted by centrifugation at 10 000 g for 2 min in a microcentrifuge. PBS was removed and 100 µL lysis buffer containing 0.9% sodium dodecyl sulfate (SDS), 15 mm EDTA plus protease inhibitor cocktail (lysis buffer recipe from Erickson and Blobel 1979) was added to each pellet. Samples were vortexed and heated to 100°C for 10 min (Erickson and Blobel 1979) then vortexed again. Samples were heated to 95°C for 5 min with tricine sample buffer (Invitrogen) and 5%β-mercaptoethanol. For each sample, the well's maximum volume was loaded onto a 10–20% gradient gel and run at 125 V, then transferred to a polyvinylidene difluoride (PVDF) membrane at 385 mA for 2 h. The membrane was blocked in Superblock (Pierce, Rockford, IL, USA) overnight at 4°C, then incubated in C8 antibody (1 : 500) in 5% non-fat milk dissolved in TBS-T for 1 h at room temperature. The membrane was washed three times for 10 min, incubated for 1 h in 1 : 2000 Horseradish peroxidase (HRP) conjugated anti-rabbit IgG at room temperature, washed 3 × 15 min and developed by chemiluminescence method (NEN Life Science Products, Boston, MA, USA).

Fluorescence resonance energy transfer (FRET)

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References

FRET measurements were observed using a Bio-Rad 1024 confocal microscope mounted on a Nikon Eclipse TE300 inverted microscope; the krypton-argon laser (emission 488 and 568 nm lines) was used to excite the fluorescein or EGFP and Cy3, respectively.

FRET was measured using a method developed for laser scanning confocal microscopy (Knowles et al. 1999; McLean et al. 2000; Kinoshita et al. 2001). The energy transfer was detected as an increase in donor fluorescence (EGFP) after complete photobleaching of the acceptor molecules (Cy3). The amount of energy transfer was calculated as the percent increase in donor fluorescence after acceptor photobleaching; initial scan was obtained at low laser energy using the 488 line of the krypton-argon laser to record the fluorescein (or EGFP) signal. A second scan was performed with the 568 line, and area of colocalization was noted. A small part of thecells (approximately 5 × 5 µm) was then photobleached with intense 568 nm light (laser power 100%) to destroy the acceptor molecules. The cells were re-scanned using 488 nm light. An increase of the EGFP signal within the photobleached area was used as a measure of the amount of FRET present. Exposing singly EGFP labeled cells to 568 nm light for equivalent times did not alter the amount of fluorescein emission. The ratio FlD2/FlD1 (where FlD2/FlD1 indicates the ratio of donor fluorescence after photobleaching to donor fluorescence before photobleaching) was compared to the null hypothesis value of 1.0 by one-group t-tests.

In all settings, Cy3 signal was photobleached to see changes of GFP signal. Negative controls in which the acceptor fluorophore was omitted did not show any enhancement of donor signal after photobleaching at the acceptor wavelength. A complete description of these techniques is provided in Kinoshita et al. (2001).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References

We hypothesized that gamma secretase action on APP also released a biologically active carboxyl-terminal (CT) fragment which, by analogy to Notch, would translocate to the nucleus. We first attempted to identify APP-CT in H4 neuroglioma cells transiently transfected with vectors containing full-length (APP770) or truncated APP. Initial attempts at demonstrating APP770 derived CT cleavage product by western blot or immunoprecipitation; western were unsuccessful. However, by analogy to other RIP nuclear domain proteins, we used a hot SDS protocol (Erickson and Blobel 1979). Overexposure of the blot allowed clear visualization of an APP 751 derived ∼6 kDa product that comigrated with authentic C58 from C58 (C58-stop) transfected cells (Fig. 1a), analogous to the bands observed by Kimberly et al. (Kimberly et al. 2001). Endogenous or overexpressed APP-C83 which is a product of α-cleavage is detected above the band of C58 in APP C58-transfected and APP751 transfected cells, respectively.

image

Figure 1. (a) Western blot analysis of amyloid precursor protein (APP)-C58-transfected H4 cells (left lane) and APP751-transfected H4 cells (right lane). The lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and detected by C8 antibody, visualized by chemiluminescence. Arrows on the gel indicate the two kinds of APP fragments – APP-C58 and the larger fragment, APP-C83. Overexpressed C58 is clearly detected in both APP-C58 (left) and APP751 (right)-transfected cells with a longer exposure (15 s). (b–d) Localization of APP770 and Fe65 in single or double-transfected H4 cells. APP is localized in the cytoplasm in APP770-GFP transfected cells (b). In H4 cells cotransfected with both APP770-GFP and Fe65, however, the GFP signal is readily observed both in the cytoplasm and in the nucleus (c). Fe65 was immunostained by anti-myc antibody visualized with Cy3 (d) in the same cells as (c). Fe65 was colocalized with APP770-GFP in the nucleus. Bar = 20 µm.

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We next attempted to determine the subcellular localization of APP-CT, which has been inferred to be in the nucleus because it causes transactivation of a reporter construct (Cao and Sudhof 2001). However, confocal analysis of H4 cells transfected with C-terminal GFP-tagged full-length APP770 or membrane-tethered beta cleavage product APP-C99 demonstrated protein expression in the membrane and cytoplasm, but no (or very faint) signal within the nucleus (Fig. 1b and data not shown).

We reasoned that if a C-terminal fragment was generated by gamma secretase activity on APP, it might be short-lived. We therefore coexpressed Fe65, an adapter protein known to bind to APP-CT (Fiore et al. 1995; Guenette et al. 1996; Cao and Sudhof 2001; Kinoshita et al. 2001) with APP770-GFP. Singly transfected APP770-GFP is localized primarily in the ER/Golgi, and is excluded from the nucleus (Fig. 1b), whereas singly transfected Fe65 has strong nuclear localization. We examined the possibility that coexpression of Fe65 altered APP770-GFP cellular localization. When cotransfected, the fluorescent GFP moiety from APP770-GFP colocalizes with Fe65 in the nucleus (Figs 1c and d). Similar results were observed with APP770-myc, detected by immunostaining for myc epitope, or C8, an antibody that recognizes the carboxyl terminal of APP (Kimberly et al. 2001) (data not shown). After cotransfection of APP770-GFP and Fe65, the GFP signal detected in the cytoplasm and in the nucleus colocalized completely with immunostaining using the anti-APP-CT antibody C8 (Figs 2a and b). Likewise, we transfected H4 cells with APP751 (without any tags) with Fe65myc and immunostained the cells with anti-C8 antibody. The signal was clearly detected in the nucleus. Negative controls without Fe65 cotransfection or omitting the primary antibody (C8) did not show any nuclear signals. Consistent with the idea that only the C-terminus of APP enters the nucleus, immunostaining with antibody 8E5, which is directed against a domain near the N-terminal, reveals colocalization with GFP signal in the cytoplasm but not in the nucleus (Figs 2c and d). To clearly identify that this APP-CT signal is derived from gamma cleavage, we immunostained APP-GFP and Fe65 cotransfected cells with 4G8, which is against amino acid residues 17–24 of Aβ peptide, thus recognizes the C83 fragment (alpha cleavage product) but not C58. As shown in Fig. 2(e and f) 4G8 did not stain the nuclear region (Fig. 2e) although the cytoplasmic signal was identical to that of APP-GFP (Fig. 2f).

image

Figure 2. H4 cells cotransfected with amyloid precursor protein (APP)770 and Fe65 show nuclear localization of APP-CT, but not full-length APP. Cells cotransfected with APP770-GFP and Fe65-myc cells are immunostained using anti-APP antibodies directed against either the intracellular (C8) or extracellular (8E5) domains of APP. The signal observed by GFP fluorescence (a) is identical to that detected by C8 (b), indicating that the GFP signal present in the nucleus contains the APP C-terminus. By contrast, a cell expressing APP770-GFP (c and e) shows colocalization in the cytoplasm but not the nucleus with either 8E5 (d) or 4G8 (f) immunostaining. Bar = 20 µm.

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To investigate the effect of interaction with Fe65 on APP-CT translocation to the nucleus, we generated a mutant form of APP770-GFP that contains two alanine amino acids instead of two tyrosines at positions 682 and 687, preventing Fe65-APP binding (Borg et al. 1996). In cells cotransfected with APP770-GFP (Y682/687 A) and Fe65-myc, no GFP signal was detected in the nucleus (Figs 3a and b), suggesting thatinhibition of the APP–Fe65 interaction either prevents translocation of the APP-CT to the nucleus, or diminishes APP-CT levels in the nucleus below our detection threshold.

image

Figure 3. APP-CT translocation to the nucleus is in Fe65 and gamma secretase-dependent manner. When H4 cells are cotransfected with APP770 (Y682/687 A)-GFP mutant and Fe65-myc, Fe65 is localized in the nucleus (b) whereas APP-CT signal is not detected in the nucleus (a). (c) shows the effect of a gamma secretase inhibitor (MW-111-20) (Wolfe et al. 2000), which inhibits the translocation of APP C-terminus to the nucleus in Fe65 and APP770 cotransfected cells, but did not prevent the normal nuclear localization of Fe65 (d). Bar = 20 µm.

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We then investigated whether nuclear translocation of APP-CT was dependent on gamma secretase activity or not, by inhibiting gamma secretase pharmacologically. Fe65 dependent nuclear translocation of APP770-derived CT was blocked by each of two gamma secretase inhibitors (Figs 3c and d), MW111-20 and WPE-11-72, which have been previously shown to block Aβ production (Wolfe et al. 2000; Dovey et al. 2001) and generation of the carboxyl-terminal domain of Notch (Berezovska et al. 2001). Furthermore, we applied the gamma secretase inhibitors to the APP-C58 overexpressing cells, which did not affect the nuclear translocation (data not shown). This result suggests that nuclear translocation of APP-CT occurs only after its cleavage by gamma secretase; the gamma cleavage releases APP-CT from the membrane, causing its accumulation of translocation to the nucleus in a Fe65-dependent manner. The downstream gamma cleavage (S3) did not affect the nuclear translocation of the APP-CT fragment.

Lastly, we tested the hypothesis that Fe65 and APP-CT form a stable complex in the nucleus by performing fluorescence resonance energy transfer (FRET) experiments. FRET is a sensitive biophysical technique that can detect protein–protein interactions within ∼10 nm. As expected from our previous studies (Kinoshita et al. 2001) and from the observations that Fe65 binds the C-terminal domain of APP, APP770 and Fe65 colocalized in the cytoplasm and membrane. In addition, in the cotransfected cells, there was clear translocation of APP-CT from full length APP770 to the nucleus and colocalization of APP-CT with Fe65 in the nucleus (Figs 4a and c). Photobleaching of the Cy 3 signal from Fe65 (Fig. 4b) led to enhanced APP-CT-GFP fluorescence (Fig. 4d). Strong FRET was observed between the APP-CT from full length APP770-GFP and Fe65 within the nucleus [FRET increase = 27.3 ± 4% (mean ± SE), n = 9, p < 0.0001], demonstrating directly a close intermolecular interaction (Fig. 4d).

image

Figure 4. Amyloid precursor protein carboxy terminal (APP-CT) accumulates in the nucleus when cotransfected with Fe65 and forms a heterodimer with Fe65 confirmed by fluorescence resonance energy transfer (FRET). H4 cells were transfected with APP770-GFP and Fe65-myc. APP-CT-GFP is strikingly localized to the nucleus (c), and colocalizes with Cy 3-immunolabeled Fe65 (a). Photobleaching of the Cy 3 label of Fe65 in the nucleus (b) leads to enhanced APP-CT-GFP fluorescent signal within the photobleached area, demonstrating FRET (d). The average increase influorescence was 27.3 ± 4%, n = 9, p < 0.0001. Bar = 20 µm.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References

Cao and Sudhof (2001) recently made the striking observation that a fusion protein of APP containing Gal4- or LexA-DNA binding domains within the carboxyl-terminal region could stimulate transcription using a heterologous signal transduction assay. Reporter gene induction depended on the presence of Fe65 and the histone acetyltransferase Tip60. Although the APP-CT could not be detected directly, these data implied that the carboxyl terminus of APP reached the nucleus to transactivate the reporter construct. APP-CT was detected by Western blot in nuclear fractions (Cupers et al. 2001; Kimberly et al. 2001) and its detection was enhanced by Fe65 in a microsomal preparation (Kimberly et al. 2001). Our current data confirm and extend these observations in several ways.

  • (i)
    We show that native APP-CT is released from full length APP in a gamma secretase dependent fashion in intact cells and that it is translocated to the nucleus.
  • (ii)
    This report is also the first to directly image the translocation of APP-CT to the nucleus in a cellular preparation.
  • (iii)
    Blockade of APP CT–Fe65 interactions prevents APP-CT from being detected in the nucleus, suggesting either that Fe65 stabilizes the APP-CT fragment or that it is important for its translocation to the nucleus.
  • (iv)
    APP-CT colocalizes with Fe65 in the nucleus, and a FRET assay shows a close intermolecular interaction of APP-CT and Fe65 in the nucleus, complementing our earlier studies using the FRET assay which showed that APP-CT interacts closely with Fe65 in the cytoplasm as well (Kinoshita et al. 2001).

These data raise the possibility that Fe65 plays a crucial role in APP-CT trafficking, and also support the hypothesis that the signaling functions of APP-CT may be modulated by interactions with Fe65.

In addition to Fe65, two other phosphotyrosine binding domain containing adapter proteins, X11 (and related molecules) and mammalian disabled, also bind the carboxyl terminus of APP in cells (Borg et al. 1996; McLoughlin and Miller 1996; Howell et al. 1999) and may well be involved in the APP-CT signal transduction machinery; our current assay will allow examination of the roles of these adapter proteins, as well as other molecules that have been demonstrated by biochemical methods to interact with APP-CT such as c-Jun N-terminal kinase (JNK) (Matsuda et al. 2001; Scheinfeld et al. 2002), PAT1 (Zheng et al. 1998), APP-BP1 (Chowet al. 1996) and LRP (Trommsdorff et al. 1998; Kinoshita et al. 2001) in APP-CT translocation to the nucleus.

Regulated intramembranous proteolysis has beensuggested to be a conserved mechanism of cell signaling in which an intramembranous cleavage releases a cytosolic fragment that enters the nucleus to control gene transcription (Brown et al. 2000). Four proteins (SREBP, Notch, Ire1, and ATF6; see Brown et al. 2000 for review) are known to undergo regulated intramembrane proteolysis, and Brown et al. observed that, although the fate of the APP cytosolic domain was not known, the mechanism for intramembrane processing of APP is so similar to the other proteins studied that it should be considered as a potential member of this class (Brown et al. 2000). Our current data confirm this prediction: we observe intranuclear accumulation of the C-terminal fragment of APP derived from gamma secretase processing of APP, noting that it forms a stable heterodimeric complex with a known multifunctional adapter protein. If, as it appears, APP-CT translocates to the nucleus and forms a transcriptionally active complex (Cao and Sudhof 2001), it joins Notch (Artavanis-Tsakonas et al. 1995) and ErbB-4 (Ni et al. 2001), as a subclass of presenilin/gamma secretase dependent regulated intramembranous proteolysis family that functions to facilitate signal transduction through novel cell surface receptor mechanisms (Fig. 5). We suggest that our current assay, demonstrating APP CT translocation to the nucleus, may prove useful for experiments aimed at understanding the factors that regulate gamma secretase activity, including screens for potential ligands that, by analogy to Notch processing, could induce activation of an APP-dependent signaling cascade.

image

Figure 5. Summary illustration of APP–CT and Fe65 interactions. Fe65 binds APP-CT via YxNPxY motif of APP in the cytoplasm. After gamma secretase cleavage, the liberated C-terminal fragment travels to the nucleus where it interacts with Tip60 (Cao and Sudhof 2001) or other transcription factors and influences gene expression.

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While our studies were in progress, two groups reported that APP CT is produced by the gamma secretase at a novel site, corresponding to the S3 cleavage of Notch (between Leu 645 and Val646 of APP 695), several amino acids carboxyl to the cleavage site expected from analysis of the carboxyl cleavage site of Aβ (Sastre et al. 2001; Yu et al. 2001). Whether this shorter fragment derives from direct gamma secretase cleavage of APP or is a result of a secondary cleavage of the predicted C57–C59 carboxyl-terminal product has not been determined with certainty. Our observations, using a novel hot SDS extraction from APP overexpressing cells finding a ∼6–7 kDa band, are comparable to the results from membrane preparations utilizing overexpressed APP (Yu et al. 2001) or APP-CT domains as substrate (Sastre et al. 2001), although the exact composition of the band observed in our preparations has not been determined by sequencing. However, our result applying the gamma secretase inhibitor to APP-C58 overexpressing cells showed that nuclear localization is dependent on the C58-site cleavage by the gamma secretase, and not the S3, cleavage site.

The possible role of a C-terminal fragment in Alzheimer's disease is intriguing. Our and other groups' results raise the possibility that the C-terminal fragment of APP, released during gamma secretase processing, is a bioactive molecule that translocates to the nucleus and modifies the transcription of genes (Cao and Sudhof 2001; Gao and Pimplikar 2001). Thus, presenilin-related gamma secretase cleavage of APP could lead to neurodegeneration both by generation of Aβ leading to deposition in senile plaques and by overproduction of bioactive C-terminal domain fragments. Further investigation is needed to define the exact role of Fe65 and the function of APP-CT as a modulator of transcription.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References

We thank Dr Michael Wolfe (Brigham and Women's Hospital, Boston) for gamma secretase inhibitor compounds and Drs Dennis Selkoe (Brigham and Women's Hospital, Boston) and Rudolph Tanzi (Massachusetts General Hospital, Boston) for helpful discussions. Supported by NIH P01 AG15379 and AG12406.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of expression constructs for human APP and Fe65
  5. Antibodies and reagents
  6. Cell culture conditions and transient transfection
  7. Immunohistochemistry
  8. Immunoblotting
  9. Fluorescence resonance energy transfer (FRET)
  10. Results
  11. Discussion
  12. Acknowledgements
  13. References
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