Article |
Address correspondence to Christian Haass, Adolf-Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Ludwig-Maximilians-University, Munich, Schillerstrasse 44, 80336 Munich, Germany. Tel.: 49-89-5996-471/472. Fax: 49-89-5996-415. E-mail: chaass{at}pbm.med.uni-muenchen.de
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Abstract |
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Key Words: Alzheimer's disease; presenilin; GFP; nicastrin; amyloid precursor protein
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Introduction |
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Amyloid ß-peptide (Aß) is generated from the ß-amyloid precursor protein (ßAPP) by proteolytic processing mediated by two distinct secretase activities (Esler and Wolfe, 2001). ß-secretase (ß-site APP cleaving enzyme [BACE]) mediates the NH2-terminal cleavage (Vassar and Citron, 2000) and produces a membrane-bound COOH-terminal fragment of ßAPP (ßAPP CTF). BACE has an acidic pH optimum and is preferentially active within early endosomes (for review see Vassar and Citron, 2000). Consistent with the acidic pH optimum of BACE, reinternalization of ßAPP from the cell surface is required for Aß generation (Perez et al., 1999). The BACE-generated ßAPP CTF can be transported back to the cell surface (Yamazaki et al., 1996), and at or close to the cell surface, the final -secretase cleavage takes place, which results in the liberation of Aß into biological fluids (for review see Selkoe, 1999).
Although ß-secretase has been identified (Vassar and Citron, 2000), the nature of -secretase is still unknown. Clearly, PSs are required for the intramembraneous
-secretase cleavage. Gene deletion of PS1 and PS2 fully inhibits
-secretase activity and results in the accumulation of the immediate precursors for
-secretase activity, the ßAPP CTF, as well as in a complete loss of Aß production (Herreman et al., 2000; Zhang et al., 2000). PS inactivation by the mutagenesis of critical aspartates within TM6 and TM7 of PS1 (Wolfe et al., 1999b) and PS2 (Steiner et al., 1999a; Kimberly et al., 2000) also blocks
-secretase activity. In addition, the critical aspartate in transmembrane domain 7 is located within a conserved domain also found in numerous bacterial aspartyl proteases of the type 4 prepilin peptidase family (Steiner et al., 2000). Therefore, it is tempting to speculate that PSs are aspartyl proteases with
-secretase activity (Wolfe et al., 1999a). This is also consistent with the finding that inactivation of PSs blocks the intramembraneous cleavage of its other known substrates, Notch 14 (De Strooper et al., 1999; Saxena et al., 2001), ErbB4 (Ni et al., 2001), E-cadherin (Marambaud et al., 2002), and LDL receptorrelated protein (May et al., 2002). Although this is the most parsimonious conclusion from the above described results, PSs are probably not active by themselves but require the formation of a proteolytically active high molecular weight complex (Capell et al., 1998; Li et al., 2000a). Moreover, the subcellular localization of PSs may not overlap with cellular compartments thought to be involved in Aß production, i.e., where
-secretase activity resides (Annaert et al., 1999; Cupers et al., 2001). This phenomenon, now known as the "spatial paradox" (Checler, 2001; Cupers et al., 2001), describes the findings that PSs are predominantly located within early compartments such as the ER and the intermediate compartment (Annaert et al., 1999; Cupers et al., 2001). Careful cellular analysis of PS1 distribution in cultured neurons indeed revealed very little, if any, PS1 beyond these early compartments (Annaert et al., 1999; Cupers et al., 2001). Based on these findings, it was concluded that PSs may not be identical to
-secretase (Annaert et al., 1999; Cupers et al., 2001), because this protease is thought to be active at or close to the cell surface (Haass et al., 1993). However, a number of studies have found PSs to be localized in post-Golgi compartments (Takashima et al., 1996; Efthimiopoulos et al., 1998; Georgakopoulos et al., 1999; Ray et al., 1999; Schwarzman et al., 1999; Lah and Levey, 2000; Singh et al., 2001).
Recently, nicastrin (Nct) was shown to be a component of the PS complex that is essential for -secretase activity (Yu et al., 2000; Chung and Struhl, 2001; Levitan et al., 2001; Edbauer et al., 2002; Hu et al., 2002; Lopez-Schier and Johnston, 2002). Nct is a type I transmembrane protein containing multiple glycosylation sites. In mammalian cells, it is present in an immature, endoglycosidase H (endoH)sensitive N-glycosylated form, and a mature, endoH-resistant N-glycosylated form (Edbauer et al., 2002; Leem et al., 2002). In Drosophila, Nct might stabilize PS fragments (Hu et al., 2002; Lopez-Schier and Johnston, 2002) and seems to be involved in transport of PSs to the cell surface (Chung and Struhl, 2001). These results suggested to us that a small, but biologically active, fraction of PS bound to Nct could be released from the ER and targeted to the cell surface, where it interacts with the
-secretase substrates. We therefore investigated the subcellular localization of PS1 and its binding partner Nct. Indeed we found PS1 on the plasma membrane (PM). We also found that PS1 binds to mature, cell surfacelocalized Nct. Moreover, inactivation of PSs by mutagenesis of the critical aspartates or treatment with
-secretase inhibitors affected cell surface reinternalization and PM accumulation of ßAPP. Our data suggest that a PS1Nct complex is released from the ER and transported to late Golgi compartments and the cell surface, where it is biologically active.
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Results |
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Detection of presenilin on the cell surface of living cells
We investigated the subcellular distribution of PE using live cell microscopy. Two different planes of focus are shown in Fig. 2, A and B, respectively, and enlargements of the boxed areas are shown in Fig. 2, CG. PE staining can be clearly seen in the nuclear envelope, vesicular structures, and an ER-like network. Surprisingly, the borders of neighboring cells (Fig. 2 C), marked by staining with fluorescent WGA (TRITCWGA; Fig. 2 D) were clearly labeled, suggesting a PM localization of PE. Another example of surface localization is shown in Fig. 2, EG, where a lamellipodium (indicated by phase contrast in Fig. 2 G) of a cell is labeled with PE (Fig. 2 E) as well as TRITCWGA (Fig. 2 F). The observed PM localization of PE is not an artifact of our detection method, because ER-retained GFPKDEL does not show PM staining under identical imaging conditions (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200201123/DC1).
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Functional inactivation of presenilin affects trafficking of cell surface ßAPP
After demonstrating targeting of a PS1Nct complex to the PM, we wanted to investigate whether inactivation of PS would also affect cellular mechanisms other than Aß production. To this end, we investigated endocytosis of ßAPP in living cells expressing either fully functional PS1 or the nonfunctional PS1 D385N mutant. Cells expressing either PS1 wt or PS1 D385N (Fig. 7) were incubated on ice with antiserum 5313 recognizing the ectodomain of ßAPP, washed, and returned to 37°C. After the indicated time points, cells were fixed and processed for immunofluorescence. In PS1 wtexpressing cells, cell surface ßAPP was completely taken up after 10 min (Fig. 7). In contrast, ßAPP was present much longer on the surface of cells expressing PS1 D385N. After 10 min at 37°C, an unchanged surface staining of ßAPP was observed and there were no endocytic structures containing ßAPP. Only after 2030 min did ßAPP-containing endocytic structures become visible, but no complete uptake was observed at these time points (Fig. 7).
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To further prove that inactivation of a PS-dependent -secretase activity affects reinternalization of ßAPP, we used the highly specific
-secretase inhibitor DAPT (Dovey et al., 2001). Incubation of cells with DAPT leads to biochemical phenotypes very similar to those observed in PS1 D385Nexpressing cells (Dovey et al., 2001; Sastre et al., 2001), i.e., inhibition of Aß and ßAPP intracellular domain production, enrichment of ßAPP CTFs, and inhibition of NICD generation accompanied by a lack of Notch signaling (Geling et al., 2002). Cells expressing PS1 wt were incubated for 4 h with DAPT and processed for ßAPP uptake as above. Endocytosis of ßAPP was delayed, and significant levels of surface ßAPP were still present at the PM after 10 min at 37°C. After 20 and 30 min at 37°C, most of the ßAPP had been endocytosed, however, at all time points, there were cells present with surface-retained ßAPP (Fig. 7). Similar, albeit weaker, effects were observed using a different
-secretase inhibitor (Li et al., 2000b; unpublished data). These data therefore demonstrate that functional inactivation of a PS1-associated
-secretase activity not only affects ßAPP processing but also its trafficking from the cell surface to endosomes.
The delay in endocytosis was also observed in cells expressing functionally inactive PS2 D366A, demonstrating similar activities of nonfunctional PS1 and PS2 (Fig. 8). Importantly, inactivation of endogenous PSs with DAPT resulted in a delayed endocytosis of ßAPP, very similar to the results observed with PS-transfected cells (Fig. 8). ßAPP-expressing HEK293 and COS7 cells were incubated in the presence or absence of DAPT and processed for ßAPP uptake as above (Fig. 8). In both cell lines, the inactivation of endogenous PSs resulted in delayed endocytosis of ßAPP, fully reproducing the findings observed in transfected cells.
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Loss of PS function, but not FAD mutants or uncleavable PS mutants, affects reinternalization of ßAPP
The above-described results suggest that a loss or reduction of PS function is responsible for the observed reduction of ßAPP reinternalization. To prove if a gain of misfunction, which apparently is caused by all FAD mutations, affects surface metabolism of ßAPP, we analyzed two FAD-associated PS mutations. We chose the PS1 G384A mutation, because this mutant shows an exceptional 5.5-fold increase of Aß42 generation (Steiner et al., 2000). In addition, we also included the PS1 E9 mutation, because that produces high Aß42 levels, but in addition accumulates as an uncleaved holoprotein (like the PS1 D385N and PS2 D366A mutants) (Thinakaran et al., 1996). Because the PS1
E9 may mimic a cleaved PS derivative (Ratovitski et al., 1997; Capell et al., 1998; Steiner et al., 1999b), we also investigated a previously characterized PS1 M292D point mutation, which inhibits endoproteolysis (Steiner et al., 1999c). Expression of any of these PS variants allowed normal uptake of ßAPP, suggesting that a loss or reduction of PS function, but not a gain of pathological function, is responsible for the observed defects in endocytosis (Table I).
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Discussion |
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Considering these two contradictory models, we first reevaluated the cellular distribution of PS1. Endogenously as well as exogenously expressed PS1 was biochemically detected on the PM. Moreover, we found that -secretase activity and PS1 codistributed in a post trans-Golgi compartment in MDCK cells (unpublished data). Furthermore, we could clearly demonstrate that an EGFP-tagged PS1 localizes on the PM in living cells. It is highly unlikely that the fusion of the EGFP domain changed trafficking of PS1, because we could demonstrate that such PS derivatives are fully functional and can be inactivated by the introduction of the D385N mutation as expected. Independent support for a PM localization of PS1 comes from the coimmunoprecipitation of Nct with PS1. Preferentially mature, fully glycosylated Nct associates with PS1, indicating that a PS1Nct complex is targeted to a post-Golgi compartment. Like PS1, a fraction of Nct can be biotinylated at the PM. Moreover, biotinylated endogenous Nct could be coimmunoprecipitated with endogenous PS1, strongly suggesting that a PS1Nct complex is located at the PM.
In addition to our findings on PS localization in a late Golgi compartment and at the cell surface, we found that inactivation of both endogenous and exogenous PS function affects the endocytosis of ßAPP from the PM. Therefore, it appears likely that PSs have a dual function in trafficking and processing. Based on a quantitative analysis, we calculated that 1/30 of total PS is located on the PM. From these data, we conclude that rather small amounts of PSs could indeed exert a biological activity at or very close to the PM. It is not known how much active PS complex is needed for functional
-secretase activity. However, assuming a catalytic activity of the complex, the amounts of PS1 we found on the PM could well account for the cleavage of ßAPP, Notch, ErbB4, E-cadherin, and LDL receptor-like protein and other so far unidentified
-secretase substrates. Our data on the surface localization of PS1 are therefore in full accordance with PSs being part of the proteolytically active
-secretase complex.
It is unclear why conventional immunolabeling techniques did not allow the detection of PSs beyond the intermediate Golgi apparatus. However, GFP fluorescence in our constructs is rather weak, suggesting low expression levels, and thus sensitive detection methods are required to visualize PS. In agreement with previous reports (Walter et al., 1996; De Strooper et al., 1997; Annaert et al., 1999; Cupers et al., 2001), in our hands, most of PS seems to be located in the ER, sometimes masking the weak staining on the PM. It is also important to note that upon fixation, the GFP fluorescence is significantly reduced (unpublished data), which makes the detection of surface PS very difficult.
Our data demonstrate that trafficking of ßAPP is altered in cells expressing nonfunctional PS1, whereas the trafficking of another ßAPP processing enzyme, BACE, is unaffected. The accumulation of ßAPP CTFs on the surface (Capell et al., 2000a; Kim et al., 2001) could simply reflect accumulation of the precursor of -cleavage, because this cleavage is blocked. However, accumulation of full-length ßAPP on the surface indicates an effect on trafficking, independent of the role of PSs in
-secretase function (this study; Kim et al., 2001). The cellular mechanism responsible for the surface accumulation of ßAPP could be the delayed reinternalization due to the saturation by the accumulation of APP and its derivatives on the PM. Indeed, we found that ßAPP endocytosis is slowed down in cells expressing nonfunctional PS1 or PS2, which would lead to an accumulation of surface ßAPP if exocytic transport is unaltered. In line with unaltered exocytosis, it has been shown that maturation of ßAPP is not affected in cells expressing nonfunctional PS1 (Wolfe et al., 1999b).
Taken together, our data demonstrate that a small, but functionally active, fraction of PS1 bound to Nct is released from the ER and targeted to the PM. In agreement with the spatial paradox, the majority of PS1 is retained within the ER, where little -secretase activity is observed. However, in clear contrast to the proposals of the spatial paradox, a fraction of PS1 (bound to Nct) is located within a post-Golgi compartment and at the PM. A function of PS on the cell surface is supported by our finding that inactivation of PSs by two independent methods slows endocytosis of ßAPP from the PM. The loss of PS function may indirectly affect ßAPP uptake due to a toxic gain of misfunction. This is supported by our previous finding that the aspartate mutants of PS cause a massive accumulation of ßAPP CTFs (Capell et al., 2000a), which cannot only be explained by the loss of
-secretase activity, but rather by reduced degradation. The latter may very well be due to reduced endosomal/lysosomal targeting of the ßAPP CTFs, resulting in their accumulation on the cell surface (Capell et al., 2000a; Kim et al., 2001).
Our data suggest a dual function for PSs: they may be involved in the trafficking of ßAPP, via so far unidentified mechanisms, and in the -secretase processing of ßAPP by providing the catalytically active sites within the
-secretase complex. Both functions are apparently related to the formation of a PSNct complex, which may contain other additional subunits, such as aph-1 (Goutte et al., 2002). In fact, absence of Nct (Yu et al., 2000; Chung and Struhl, 2001; Levitan et al., 2001; Edbauer et al., 2002; Hu et al., 2002; Lopez-Schier and Johnston, 2002) and aph-1 (Goutte et al., 2002) severely affects
-secretase activity.
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Materials and methods |
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cDNA constructs, transfections, and screening of stably transfected cell lines
To generate an EGFP-tagged PS, a NotI restriction site was introduced between codon 351 and 352 of the cytoplasmic loop of human PS1, resulting in PS1not. EGFP cDNA was then fused in-frame into the PS sequence using the NotI site. Introduction of the NotI site results in two additional glycine codons at NH2 and COOH termini of EGFP, respectively. Proper orientation of EGFP was checked by transfecting miniprep DNA into COS7 cells and analyzing GFP fluorescence. One clone, PE, was subcloned in pCDNA3.1Zeo (Invitrogen). PS1 D385NEGFP and PS1 L166PEGFP were generated by mutagenizing PE with appropriate oligonucleotides using QuikChange Site-Directed Mutagenesis Kit (Stratagene). The former construct was called PEASP, the latter PE (L166P).
For transient or stable transfection, Fugene (Roche) was used. HEK293 cells stably expressing Swedish ßAPP were transfected with PE or PEASP or PE (L166P) and clones were selected for stable integration. Several clones expressing each construct were analyzed and one representative clone was chosen for further analysis. PE17 was the clone used for analysis of PE, and PEASP1 was the one used for PEASP. For analysis of Notch processing, PE 17 cells were transfected with NotchE/pcDNA3.1/Hygro+, and a pool of stably expressing cells was obtained by selection with hygromycin. Alternatively, PE 17 cells were transfected and processed for immunofluorescence the following day.
As a control for microscopy, HEK293 cells were transiently transfected with pEF/myc/ER/GFP (Invitrogen), coding for a GFPKDEL. For APP uptake experiments, COS7 cells were transiently transfected with ßAPP695.
Quantitation of expression levels of exogenous PS
For quantitating membrane lysates (Capell et al., 1998) and total lysates of HEK293, cells expressing endogenous PSs or stably expressing PS1 wt or PE were separated on 12% urea gels, blotted, and probed with antiserum 3027. Expression levels were quantitated using 125I secondary antibodies and a phosphoimager system. Total exogenous PS (holoprotein and CTF) was overexpressed 10-fold, compared with endogenous PS1 levels. Only a twofold overexpression of exogenous PS CTF was observed. Because the biologically active PS complex contains the PS fragments (Capell et al., 1998; Li et al., 2000a), this demonstrates a rather low level of overexpression.
Surface biotinylation
HEK293 cells grown on poly-L-lysinecoated 10-cm (for PS and Nct detection) or 6-cm dishes (for APP detection) were washed in ice cold PCM (PBS supplemented with 1 mM CaCl2, 0.5 mM MgCl) and incubated for 30 min on ice in PCM containing 1 mg/ml (for PS and Nct) or 0.5 mg/ml (for ßAPP) sulfo-succinimidyl-6-([+]-biotinamido)-hexanoate (Molecular Biosciences). Thereafter, biotinylation was quenched by washing two times in 50 mM NH4ClPBS on ice, followed by a 10-min incubation on ice in 50 mM NH4ClPBS. In some experiments, 20 mM glycinePBS was used for quenching. After two additional washes, cells were lysed in STEN lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% NP-40), and biotinylated proteins were precipitated with streptavidin-Sepharose. Biotinylated proteins and 1/60 of total cell lysates were separated on 12% SDS-urea gels (PS) or 8% SDS gels (ßAPP and Nct) and blotted onto PVDF membranes. PS1 was detected using antibodies 3027 or PS1 N, Nct using anti-Nct antibody, and ßAPP using antiserum 5313. As a control, blots were stripped and reprobed with EEA1 or GSK-3 antibody. Similar blots to those shown in Fig. 5 were incubated with 125I secondary antibodies, and the radioactive signal was detected using a phosphoimager system. The ratio of biotinylated versus total PS was then calculated.
Quantitation of cell surface ßAPP
For quantitation of surface ßAPP, cells were biotinylated as described above. Biotinylated ßAPP was detected using antiserum 5313 and 125I secondary antibody and quantified by phosphoimaging. In each experiment, the values of biotinylated ßAPP divided by total ßAPP of PS1 wtexpressing cells were set to 1, and the values of biotinylated ßAPP divided by total ßAPP of PS1 D385Nexpressing cells was related to 1.
Coimmunoprecipitation and deglycosylation of Nct
Nct was coimmunoprecipitated from membrane fractions extracted in 2% CHAPS with antiserum 3027 against PS CTF (Capell et al., 1998). EndoH and N-glycosidase F digestion was performed according to the supplier's instructions (Roche). For coimmunoprecipitation and recapture of biotinylated Nct CHAPS lysates from the cell surface, biotinylated cells were immunoprecipitated with antiserum 3027 against PS CTF. Bound proteins were eluted from protein ASepharose as described (Bonifacino et al., 2000), and biotinylated proteins were precipitated using streptavidin-Sepharose. Nct was detected using Nct antibody.
Cell surface uptake of ßAPP and BACE
Cells plated on poly-L-lysinecoated coverslips were incubated for 4 h in the presence or absence of 250 nM DAPT (provided by Boehringer Ingelheim Pharma KG), washed in ice cold PCM, and incubated on ice in a 1:200 5313 (for ßAPP detection) or 7523 (for BACE detection) antibody dilution in PCM. After 20 min, cells were washed in PCM on ice, and then PCM was replaced by prewarmed culture medium and cells were placed for various time points in a 37°C incubator. After indicated time points, coverslips were transferred to 4% paraformaldehyde, 4% sucrose in PBS, fixed for 20 min, and processed for standard immunofluorescence (Wacker et al., 1997) using Alexa®488- or Alexa®594-coupled secondary antirabbit antibodies (Molecular Probes). Endocytosis of ßAPP or BACE was normally fully completed after 1020 min, depending on the individual experiment. Experiments were performed at least twice, and 50100 cells were scored per cell line and time point. Representative images are shown.
Microscopy
Fixed cells were analyzed on a Leica DMRB microscope equipped with a 100x/1.3 objective and standard FITC and TRITC fluorescence filter sets. Images were obtained using a Spot Camera (RT Monochrome Diagnostics) and the MetaView Imaging software (Universal Imaging Corp.). For analysis of living cells, cells were cultured on poly-L-lysinecoated coverslips and mounted on custom-made aluminum holders. For labeling of the PM, cells were incubated for 20 min on ice in 20 µg/ml TRITCWGA/PCM (Sigma-Aldrich) followed by a wash in ice cold PBS. Imaging was performed on an Olympus IX70 microscope using a 60 x 1.4 Planapo objective and a GFP/Texas red double dicroic emission filter. Images were recorded with a TILL Vision setup consisting of a TILL Imago camera, Polychrome IV monochromator, and Vision software (T.I.L.L. Photonics GmbH). The EGFP fluorescence in general was very low; typical exposure times ranged from 14 s. In cells labeled with TRITCWGA, special care was taken to avoid excitation of TRICTWGA when viewing GFP. Excitation with 480490 nm led to considerable excitation of TRITCWGA, leading to a bleedthrough of the red fluorescence (due to the double dicroic filter used). Control experiments revealed that excitation with 470 nm showed only GFP, but no TRITC fluorescence, even in very long exposures. Images therefore were recorded with an excitation of 470 nm for GFP and 570 nm for TRITCWGA.
TIRM
PE or PEASP cells were grown in poly-L-lysinecoated glass bottom dishes (MatTek Corporation). TIRM was performed on an objective type setup based on an inverted microscope (Olympus IX70) equipped with an oil immersion objective (PlanApo x60, NA 1.45 TIRFM; Olympus). The specimen was illuminated with an argon laser (model 163-All; Spectra Physics). Images were recorded on an Imago CCD sVGA camera using Vision software.
Online supplemental material
The supplemental figures for this article are available at http://www.jcb.org/cgi/content/full/jcb.200201123/DC1. To exclude the possibility that the detection of PSEGFP at the cell surface is not an artifact of overexpression, we analyzed the subcellular distribution of an overexpressed ER-resident protein. For this, a GFPER with a KDEL retention motif was expressed in HEK293 cells and analyzed by conventional fluorescence microscopy (Fig. S1) and TIRM (Fig. S2). No staining of the plasma membrane could be detected.
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Footnotes |
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* Abbreviations used in this paper: Aß, amyloid ß-peptide; Aß42, 42amino acid amyloid ß-peptide; BACE, ß-site APP cleaving enzyme; ßAPP, ß-amyloid precursor protein; CTF, COOH-terminal fragment; EEA1, early endosomal autoantigen 1; endoH, endoglycosidase H; FAD, familial Alzheimer's disease; GSK-3, glycogen synthase kinase 3; HEK, human embryonic kidney; Nct, nicastrin; NICD, Notch intracellular domain; NTF, NH2-terminal fragment; PE, PS1EGFP; PEASP, PS1 D385NEGFP; PM, plasma membrane; PS, presenilin; TIRM, total internal reflection microscopy; wt, wild type.
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Acknowledgments |
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This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) (priority program "Cellular Mechanisms of Alzheimer's Disease") to C. Haass and C. Kaether. S. Lammich is a recipient of a fellowship from the DFG.
Submitted: 29 January 2002
Revised: 11 June 2002
Accepted: 11 June 2002
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