Presenilin-1 Regulates Intracellular Trafficking and Cell Surface Delivery of beta -Amyloid Precursor Protein*

Dongming CaiDagger , Jae Yoon Leem§, Jeffrey P. GreenfieldDagger , Pei Wang||, Benny S. KimDagger , Runsheng Wang||, Kryslaine O. Lopes§, Seong-Hun Kim§, Hui Zheng||, Paul GreengardDagger , Sangram S. Sisodia§, Gopal Thinakaran§, and Huaxi XuDagger **

From the Dagger  Fisher Center for Research on Alzheimer's Disease and Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, the § Department of Neurobiology, Pharmacology and Physiology, the University of Chicago, Chicago, Illinois 60637, and || Huffington Center on Aging, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030

Received for publication, September 5, 2002, and in revised form, October 31, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Presenilins (PS1/PS2) play a critical role in proteolysis of beta -amyloid precursor protein (beta APP) to generate beta -amyloid, a peptide important in the pathogenesis of Alzheimer's disease. Nevertheless, several regulatory functions of PS1 have also been reported. Here we demonstrate, in neuroblastoma cells, that PS1 regulates the biogenesis of beta APP-containing vesicles from the trans-Golgi network and the endoplasmic reticulum. PS1 deficiency or the expression of loss-of-function variants leads to robust vesicle formation, concomitant with increased maturation and/or cell surface accumulation of beta APP. In contrast, release of vesicles containing beta APP is impaired in familial Alzheimer's disease (FAD)-linked PS1 mutant cells, resulting in reduced beta APP delivery to the cell surface. Moreover, diminution of surface beta APP is profound at axonal terminals in neurons expressing a PS1 FAD variant. These results suggest that PS1 regulation of beta APP trafficking may represent an alternative mechanism by which FAD-linked PS1 variants modulate beta APP processing.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease (AD)1 is characterized by the excessive generation and accumulation of beta -amyloid peptides (Abeta ). The amyloidogenic Abeta peptide is proteolytically derived from the beta -amyloid precursor protein (beta APP) within the secretory pathway by distinct enzymatic activities known as beta - and gamma -secretase (1, 2). Full-length beta APP is synthesized in the endoplasmic reticulum (ER) and transported through the Golgi apparatus. The major population of secreted Abeta peptides is generated within the trans-Golgi network (TGN) (3-5), also the major site of beta APP residence in neurons at steady state. beta APP can be transported in TGN-derived secretory vesicles to the cell surface if not first proteolyzed to Abeta or an intermediate metabolite. At the plasma membrane beta APP is either cleaved to produce a soluble molecule, sbeta APP (6) or, alternatively, reinternalized within clathrin-coated vesicles to an endosomal/lysosomal degradation pathway (7, 8). Thus, the distribution of beta APP between the TGN and cell surface has a direct influence upon the relative generation of sbeta APP versus Abeta . This phenomenon makes delineation of the mechanisms responsible for regulating beta APP trafficking from the TGN relevant to understanding the pathogenesis of AD.

Expression of autosomal dominant variants of either beta APP, presenilin 1 (PS1), or presenilin 2 (PS2) results in increased Abeta 42 production and predispose individuals to early onset familial Alzheimer's disease (FAD) (9-11). Presenilins (PSs), multitransmembrane proteins, accumulate as endoproteolyzed heterodimers of N- and C-terminal fragments and associate with other membrane proteins (e.g. nicastrin, APH-1, and PEN-2) to form high molecular weight complexes (9, 12-16). Several lines of evidence suggest that presenilin complexes play a crucial role in intramembranous gamma -secretase cleavage of type I membrane proteins including beta APP and the signaling receptor, Notch-1. For example, genetic ablation of PS1, PS2, or other components of the PS complex dramatically impairs Abeta secretion and production of the Notch derivative, S3/NICD, in cells (14, 15, 17, 18). Mutations of conserved transmembrane aspartate residues in PSs result in loss-of-function leading to reduced Abeta secretion (19, 20). Finally, biochemical fractionation studies closely link PSs with gamma -secretase activity, and gamma -secretase inhibitors can be photo-cross-linked to PS1 and PS2 (21, 22).

Although it has generally been accepted that PSs are essential for gamma -cleavage, it has not been firmly established that PSs are the catalytic component of the enzyme complex. For example, recent studies (23) have shown that the production of Abeta 42 in early compartments of the secretory apparatus is unimpaired in the absence of PSs. Whereas the hypothesis remains attractive that PSs are the gamma -secretase, several reports indicate that PSs mediate additional physiological functions, including roles in calcium homeostasis, neurite outgrowth, apoptosis, and synaptic plasticity (24), and some of these functions are influenced by FAD-linked PS mutations. PS1 has been implicated in regulating intracellular trafficking and maturation of selected transmembrane proteins. PS1 deficiency significantly affects trafficking of the tyrosine kinase receptor TrkB, as well as the PS1-interacting protein ICAM-5/telecephalin (25, 26). Indeed evidence has emerged to support the notion that PS1 may facilitate gamma -secretase cleavage of substrates via regulating the maturation and intracellular trafficking of substrates and/or components of the gamma -secretase complex. For example, expression of the PS1 aspartate variants leads to accumulation of beta APP C-terminal fragments (CTFs) as well as full-length beta APP at the cell surface (20, 27). Several recent studies (28, 29) further demonstrate that PS1 regulates the maturation and cell surface accumulation/trafficking of nicastrin.

Based on these observations, we investigated the potential role of PS1 in regulating intracellular trafficking of full-length beta APP through the secretory pathway. We previously established a cell-free system in which we can reconstitute both the formation of Abeta and the trafficking of beta APP/Abeta from the ER or the TGN (3, 5, 30-32). By utilizing this system, we directly examined individual cellular processes involved in PS1 regulation of beta APP trafficking. We report here that PS1 deficiency or expression of loss-of-function variants led to robust formation of beta APP-containing vesicles, concomitant with increased maturation and/or cell surface accumulation of beta APP. In contrast, vesicle formation from the TGN and the ER was impaired in cells expressing FAD-linked PS1 mutants, resulting in a reduction of beta APP delivery to the cell surface. We also observed a profound reduction of surface beta APP at axonal terminals in neurons harboring an FAD PS1 mutation (A246E), compared with neurons expressing wild type (wt) PS1. Taken together, these results suggest that PS1 can modulate metabolism of beta APP via regulating beta APP trafficking within the secretory pathway and thus affect Abeta generation by controlling the availability of substrate beta APP to appropriate secretases.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines-- Mouse N2a neuroblastoma cells doubly transfected with cDNAs encoding human beta APP harboring the "Swedish" double mutant (beta APPswe) and human wt PS1 or various PS1 mutants (11, 30) were maintained in medium containing 50% Dulbecco's modified Eagle's medium, 50% Opti-MEM, supplemented with 5% fetal bovine serum, antibiotics, and 200 mg/ml G418 (Invitrogen). Immortalized PS1-/- fibroblasts (33) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. To circumvent potential variability between fibroblasts established from PS1+/+ and PS1-/- embryos, immortalized PS1-/- fibroblasts were transfected with wt PS1 expression plasmid, and stable transfectants were isolated in 400 mg/ml Zeocin (Invitrogen). To express beta APPswe, PS1-/- fibroblasts and wt PS1 transfectants were infected with beta APPswe retroviruses produced from the mouse NIH3T3-based PT67 packaging cell line, and stable expressors (designated PS1-/- and wt) were isolated in media containing 2 mg/ml puromycin (Calbiochem), and ~150-200 colonies were pooled for further studies. Blastocyst-derived wt and PS1-/-/PS2-/- cells have been described previously (28).

Neuronal Culture-- Human wt PS1 (line 17-3) or FAD-linked PS1 A246E mutant transgenic mice (line 16-4) (34) were mated with heterozygous PS1 null mice (PS1+/-) (35). Double heterozygous F1s were then intercrossed to generate offspring that express human wt or A246E PS1 in the PS1-/- background. Genotypes were determined by PCR amplification as described previously (34). Primary neuronal cultures were derived from the cerebral cortices of embryonic day 17 (E17) mouse embryos from the above matings. Dissociated neurons were plated (~5 × 104 cells per chamber) on poly-L-lysine (Sigma)-precoated 8-chamber slides (Lab-Tek) in serum-free neurobasal medium with N2 supplement as well as B27 supplement (Invitrogen) and cultured for 7 days. Media were replaced every 2 days with the addition of 16.5 mg/ml uridine and 6.7 mg/ml 5-fluoro-2'-deoxyuridine to prevent proliferation of glial cells.

Preparation of Permeabilized N2a Cells-- It has been well established that incubation of cells at 15 (36) or 20 °C (30) leads to an accumulation of membrane and secretory proteins in the ER and TGN, respectively. To assay beta APP trafficking from TGN, cells were pulse-labeled with [35S]methionine (500 µCi/ml) for 15 min at 37 °C, washed with phosphate-buffered saline (prewarmed to 20 °C), and chased for 2 h at 20 °C in prewarmed complete media. To assay beta APP trafficking from the ER, cells were pulse-labeled for 4 h at 15 °C. For both types of preparations, cells were permeabilized at the termination of incubation as follows. Cells were first incubated at 4 °C in "swelling buffer" (10 mM KCl, 10 mM HEPES, pH 7.2) for 10 min. The buffer was discarded and replaced with 1 ml of "breaking buffer" (90 mM KCl, 10 mM HEPES, pH 7.2), after which the cells were broken by scraping with a rubber policeman. Cells were centrifuged at 800 × g for 5 min, washed in 5 ml of breaking buffer, and resuspended in 5 volumes of breaking buffer. This results in >95% cell breakage as evaluated by trypan blue staining. Broken cells (cell-free system) were incubated in a final volume of 300 µl containing 2.5 mM MgCl2, 0.5 mM CaCl2, 110 mM KCl, cytosol (30 µg protein) prepared from N2a cells (32, 37), and an energy-regenerating system consisting of 1 mM ATP, 0.02 mM GTP, 10 mM creatine phosphate, 80 µg/ml creatine phosphokinase, and a protease inhibitor mixture. Incubations were carried out at 37 °C for various times (15-120 min) to observe the kinetics of protein trafficking.

Formation of Nascent Secretory Vesicles in Permeabilized Cells and Immunoprecipitation-- Following incubation of cell-free systems, vesicle and membrane fractions were separated by centrifugation at 11,000 rpm for 30 s at 4 °C in a Brinkmann centrifuge (Brinkmann Instruments). Vesicle (supernatant) and membrane (pellet) fractions were diluted with IP buffer (50 mM Tris-HCl, pH 8.8, 150 mM NaCl, 6 mM EDTA, 2.5% Triton X-100, 5 mM methionine and cysteine, and 1 mg/ml bovine serum albumin), immunoprecipitated using beta APP C-terminal antibody 369 (7, 38) or anti-NCAM and anti-FGFR1 antibodies (Santa Cruz Biotechnology), and analyzed by SDS-PAGE. Each experiment was performed at least three times. Band intensities were analyzed and quantified using NIH ImageQuant software, version 1.52.

Biotinylation and Detection of Cell Surface beta APP-- Stably transfected N2a cells were labeled with [35S]methionine (500 µCi/ml) for 10 min at 37 °C and chased for 2 h at 20 °C in complete medium to accumulate labeled proteins in the TGN. To restore vesicle trafficking from TGN, cells were transferred to 37 °C for various time intervals. Cells were then incubated at 4 °C with 0.5 mg ml-1 sulfo-N-hydroxysuccinimide biotin (Pierce) to biotinylate cell surface proteins. Biotinylated and non-biotinylated proteins were first separated into two fractions by binding to streptavidin-agarose beads (Pierce), and beta APP from each fraction was immunoprecipitated with antibody 369 (7, 38) and analyzed by SDS-PAGE (39).

Immunofluorescence Confocal Microscopy-- For staining of full-length beta APP, N2a cells grown in chamber slides were incubated at 4 °C with primary antibody 6E10 (diluted 1:100 in growth medium, Senetek PLC) for 1 h without fixation or permeabilization. Following incubation with secondary antibodies and FITC-conjugated Vicia villosa agglutinin (1:100, Vector Laboratories Inc.), cells were fixed with 4% formaldehyde at room temperature for 15 min. Cultured neurons were fixed twice with 4% formaldehyde for 15 min twice. For surface staining of full-length beta APP, cells were directly incubated with primary antibody mAb348 (1:100 dilution, Roche Molecular Biochemicals) at 4 °C overnight. In some experiments, neurons were permeabilized by ice-cold methanol for 2 min prior to overnight incubation with mAb348 or anti-GAP43 antibody (1:4000). Immunofluorescence staining was examined by confocal microscopy (LSM510, Zeiss).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PS1 Deficiency Leads to Increased beta APP Transport from TGN to Plasma Membrane and from ER to Golgi-- It has been well established that PS1-deficient neurons fail to secrete Abeta but accumulate intracellular beta APP C-terminal fragments (25, 40). In addition to affecting gamma -secretase activity, recent observations (25, 28, 29) that PS1 deficiency or loss-of-function alters the maturation and cell surface accumulation of certain membrane proteins, such as beta APP, nicastrin, and Notch-1, suggest a potential role for PS1 in intracellular protein trafficking. To support further the notion that wt PS1 may have a direct regulatory effect on beta APP trafficking through secretory compartments, we assessed the formation of beta APP-containing vesicles from the TGN and from the ER in PS1-/- fibroblasts using a cell-free reconstitution system.

The formation of beta APP-containing vesicles from the TGN was examined using a cell-free system that has been used extensively to study beta APP trafficking and Abeta generation (see "Experimental Procedures" and Refs. 3, 5, and 32). This cell-free system has been used to investigate nascent secretory vesicle budding from a variety of cells (30, 31), and the integrity of TGN stacks and derived vesicles has been demonstrated by electron microscopy (37). To study trafficking of beta APP-containing vesicles from the TGN, we labeled wt and PS1-/-/PS2-/- fibroblasts with [35S]methionine and then incubated the cells at 20 °C, a temperature at which transport of proteins, including beta APP, from the ER to the TGN is unimpaired. However, under these conditions, the egress of secretory vesicles from the TGN is blocked, thus allowing labeled beta APP (and other membrane proteins) to accumulate in the TGN (3, 5, 32). This experimental design ensures that TGN-specific vesicle biogenesis is measured. In our cell-free trafficking assays, following permeabilization and incubation at 37 °C, budding of beta APP-containing vesicles from the TGN was greatly increased at all time points examined in preparations that lacked PS1 when compared with preparations from cells that express wt PS1 (Fig. 1, a and b). After 2 h of incubation, the amount of beta APP transported out of the TGN was 54.2% higher in PS1-/- cell preparations compared with preparations from PS1 wt cells (i.e. 21.9 versus 14.2% of the total labeled beta APP in the TGN).


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Fig. 1.   PS1 deficiency accelerates beta APP transport from the TGN and from the ER. a-d, PS1-/- fibroblasts expressing human beta APPswe alone (PS1-/-) or coexpressing beta APPswe and wt human PS1 (PS1WT) were labeled for 15 min with [35S]methionine at 37 °C and chased for 2 h at 20 °C to accumulate labeled beta APP in the TGN. Alternatively, cells were labeled for 4 h at 15 °C to accumulate labeled beta APP within the ER. Permeabilized cells were prepared and incubated at 37 °C for various times to allow the formation of post-TGN (a and b) or post-ER vesicles (c and d). Labeled beta APP was immunoprecipitated from nascent vesicles or the donor compartments and analyzed by SDS-PAGE and autoradiography (a and c). The kinetics of beta APP-containing vesicle formation is presented as percent of vesicle budding (b and d); data represent mean ± S.E. from three independent experiments. e, the kinetics of ER budding of FGFR1-containing vesicles was analyzed as above. Data represent mean ± S.E. of three experiments. f, samples of cell lysates with equal amount of protein (50 µg protein/lane) prepared from wt and PS1-/-/PS2-/- cells were analyzed by Western blotting using C-terminal beta APP antibody CT15.

We next tested whether PS1 deficiency might affect beta APP vesicle transport from the ER to Golgi utilizing a modified cell-free system in which beta APP-containing post-ER vesicles were reconstituted. In this case, cells were labeled with [35S]methionine at 15 °C to accumulate beta APP within the ER (see "Experimental Procedures" and Ref. 5), followed by permeabilization and incubation at 37 °C to initiate vesicle release. The budding of beta APP-containing ER vesicles was accelerated (~2-fold) in PS1-/- cells compared with wt cells (Fig. 1, c and d). To assess whether PS1 deficiency selectively affects beta APP trafficking, budding of fibroblast growth factor receptor (FGFR1)-containing vesicles from the ER was determined in parallel and remained unchanged by PS1 deficiency (Fig. 1e). Collectively these results suggest that PS1 selectively regulates beta APP trafficking from ER and TGN compartments.

To support further the notion that beta APP is transported more efficiently out of the ER and Golgi compartments in the absence of PS, we examined the patterns of beta APP maturation in detergent lysates prepared from PS1-/-/PS2-/- cells and wt fibroblasts. As expected from the PS1-/--permeabilized cell studies, the amount of mature (glycosylated/sialylated) beta APP in PS1-/-/PS2-/- cells was significantly higher than that in PS1 wt cells (Fig. 1f), indicative of increased residence and transit of beta APP through the late secretory compartments. However, alternation in beta APP glycosylation/maturation was much less dramatic in PS1-/- cells than in PS1-/-/PS2-/- cells (data not shown). Although the underlying mechanism is not clear, this may be attributed to differential roles of PS1 and PS2 in APP trafficking versus maturation.

Loss-of-Function PS1 Mutants Accelerate beta APP Trafficking from the ER without Altering TGN Vesicle Budding-- To confirm further that loss of PS1 activities results in accelerated beta APP trafficking through the secretory pathway, N2a cells expressing loss-of-function PS1 variants were examined to assess their effects on intracellular transport of beta APP. Previous studies demonstrated that a single amino acid substitution in PS1 transmembrane domain 7 (D385A) (19, 20) or deletion of the first two PS1 transmembranes (Delta 1,2) (41) lowers Abeta secretion and leads to accumulation of beta APP beta CTFs. Unexpectedly, beta APP-containing vesicle budding from the TGN was not changed by the expression of PS1 loss of function mutations (D385A or Delta 1,2) (~35% of maximal beta APP vesicle budding in both D385A and Delta 1,2 versus ~38% in PS1 wt cells) (Fig. 2, a and b). However, the trafficking of beta APP from the ER to Golgi was significantly increased in these loss-of-function PS1 mutants (Fig. 2, c and d). The maximal level of vesicle budding was increased by ~1.5- (for D385A) to 2-fold (for Delta 1,2) when compared with PS1 wt; this increase in budding is similar to that observed in PS1-deficient cells. As a result of accelerated ER trafficking, the total amount of beta APP residing in TGN membrane in loss-of-function PS1 mutants was increased compared with that in wt TGN membrane (Fig. 2a), although the rate of budding of beta APP-containing vesicles from the TGN was unchanged (Fig. 2b).


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Fig. 2.   beta APP trafficking from the ER and the TGN in cells expressing loss-of-function mutations of PS1. Stable N2a cells coexpressing beta APPswe and wt PS1 (WT) or PS1 harboring either D385A or Delta 1,2 mutations were used. Budding assays were performed as described in Fig. 1. Quantitative data represent mean ± S.E. from three independent experiments.

Together, these results suggest that the loss of function for PS1 in gamma -cleavage of beta APP and the complete absence of PS1 protein differentially regulate vesicle biogenesis from the TGN, although the efflux of beta APP molecules from the ER is enhanced under both conditions.

Loss-of-Function PS1 Mutations Increase the Amount of Full-length beta APP Delivered to the Plasma Membrane-- Despite the lack of marked differences in TGN vesicle biogenesis, increased ER to Golgi trafficking of beta APP observed in the loss-of-function mutant cells may be sufficient to elevate the steady-state levels of beta APP at the cell surface in intact cells. In addition, as recently reported (42), a delay in the internalization of surface-bound beta APP may further facilitate increased surface accumulation of beta APP. Indeed, live staining of Delta 1,2 cells using monoclonal antibody 6E10 revealed an obvious increase in the amount of surface-bound beta APP compared with PS1 wt cells. The amount of total surface glycoproteins is identical in the two types of cells as judged by staining for V. villosa agglutinin, a lectin that binds to N-acetyl-D-galactosamine linked to serine or threonine residues in glycoproteins (43) (Fig. 3a). The amount of newly synthesized beta APP delivered to the cell surface was measured quantitatively in these cells by pulse-chase labeling in combination with cell-surface biotinylation (39) in intact cells. Cells were pulse-labeled for 10 min with [35S]methionine at 37 °C and chased for 2 h at 20 °C to accumulate labeled beta APP in the TGN. Subsequent incubation at 37 °C allows trafficking of beta APP from TGN to cell surface in a synchronized fashion (39). Newly arrived [35S]methionine-labeled cell surface beta APP molecules were then labeled with biotin at 4 °C for 15 min and separated from TGN-associated beta APP by streptavidin bead precipitation. Up to 14.3% of nascent beta APP was transported to the plasma membrane after 120 min of chase, a value that is 48.9% greater than that in PS1 wt cells (Fig. 3, b and c). The findings from loss-of-function PS1 mutants together with studies using PS1-deficient cells strongly suggest that PS1 plays a role in regulating the delivery of nascent beta APP molecules to the cell surface.


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Fig. 3.   Delivery of full-length beta APP to the plasma membrane in cells harboring loss-of-function PS1 mutations. a, live N2a cells were incubated with primary antibody 6E10 (1:100) at 4 °C for 1 h to label cell surface beta APP (red) and FITC-conjugated V. villosa agglutinin to stain all surface glycoproteins (green). Cells were then fixed and visualized by confocal microscopy. b and c, cells were labeled with [35S]methionine at 37 °C for 10 min and chased at 20 °C for 2 h to accumulate labeled beta APP in the TGN (total cell beta APP). Cells were then incubated at 37 °C for various times to allow transport of beta APP to the plasma membrane. Cell surface proteins were then biotinylated at 4 °C for 15 min. Biotinylated and non-biotinylated proteins were separated into two fractions by binding to streptavidin beads. beta APP was immunoprecipitated from each fraction and analyzed. Quantitative data represent mean ± S.E. from three independent experiments.

FAD-linked PS1 Mutants Delay beta APP Transport from the TGN and the ER-- We next investigated whether FAD-linked PS1 variants might influence beta APP trafficking. The kinetics of beta APP-containing vesicle budding from the TGN of N2a cells expressing wt PS1 was compared with that of cells expressing FAD-linked PS1 variants. The budding of TGN-derived beta APP vesicles was dramatically impaired in FAD-linked PS1 mutants (Delta E9, A246E, and M146L) at all time points examined, compared with PS1 wt cells. Among the three FAD-linked PS1 mutations examined, PS1 A246E demonstrated the highest degree of impairment in beta APP vesicle budding from the TGN, with maximal beta APP vesicle budding of 21.8% at 90 min compared with 38% in PS1 wt cells (Fig. 4, a and b).


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Fig. 4.   FAD-linked PS1 mutants delay beta APP transport from the TGN and the ER. a-d, stable N2a cells coexpressing beta APPswe and either wt PS1 (WT) or PS1 harboring one of the indicated FAD-linked mutations were used in these experiments. Vesicle budding experiments were performed as described in the legend to Fig. 1. Quantitative data represent mean ± S.E. from three independent experiments. e, budding of NCAM-containing vesicles was examined in parallel from WT and mutant cells and quantified.

We further tested whether FAD-linked PS1 mutations affect beta APP vesicle transport from the ER to Golgi. Similar to the results obtained for TGN budding, beta APP trafficking from the ER to Golgi, as judged by the release of beta APP-containing vesicles from the ER, was significantly reduced in FAD-linked mutants, with a 50.9 and 57.9% decrease in the maximal levels of vesicle budding in PS1 Delta E9 and A246E cells, respectively, compared with PS1 wt cells. The impairment in ER vesicle biogenesis was relatively mild in the PS1 M146L mutation, with an ~30% reduction compared with PS wt (Fig. 4, c and d).

To ascertain the selectivity of impaired beta APP-containing vesicle budding in cells expressing FAD-linked PS1 variants, we examined the trafficking of neural cell adhesion molecule (NCAM) from the TGN using the permeabilized cell system. In contrast to beta APP, the rate of TGN budding of vesicles containing NCAM isoforms was almost identical in PS1 wt and mutant cells (Fig. 4e), suggesting that FAD-linked PS1 variants specifically delay beta APP transport from the TGN to the cell surface.

FAD-linked PS1 Mutants Lower the Amount of Full-length beta APP Delivered to the Plasma Membrane-- We reasoned that the alteration in beta APP trafficking by PS1 mutations seen in the permeabilized cell system should affect the steady-state level of beta APP on the plasma membrane. To test this hypothesis we stained live N2a cells with monoclonal antibody 6E10 to visualize surface-bound beta APP and beta APP C-terminal fragments and FITC-conjugated V. villosa agglutinin to label all glycoproteins. As shown in Fig. 5a, the intensity of beta APP immunofluorescence on the cell surface of mutant cells was markedly reduced as compared with PS1 wt cells. V. villosa agglutinin staining of surface glycoproteins was comparable between PS1 wt and mutant cells (Fig. 5a). These findings are consistent with the results from permeabilized cell experiments described above and suggest that FAD-linked PS1 mutants impair the trafficking of beta APP to the cell surface.


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Fig. 5.   FAD-linked PS1 mutations reduce the amount of full-length beta APP delivered to the plasma membrane. Surface staining and budding assays were performed as described in Fig. 3, except that N2a cells coexpressing beta APPswe and PS1 harboring one of the indicated FAD-linked mutations were used. These assays were done in parallel to the experiments described in Fig. 3, and the WT data are the same as in Fig. 3. Quantitative data represent mean ± S.E. from three independent experiments.

To examine directly the delivery of newly synthesized full-length beta APP trafficking from the TGN to the plasma membrane, we carried out pulse-chase labeling in combination with cell-surface biotinylation. As shown in Fig. 5, b and c, in PS1 wt cells, 9.6% of nascent beta APP that accumulated in the TGN during incubation at 20 °C left the TGN and traveled to the cell surface after a 2-h chase at 37 °C. However, the amount of newly synthesized beta APP that accumulated on the cell surface at various chase time periods was much lower in PS1 (Delta E9) mutant cells, with only 3-5% of nascent beta APP molecules delivered to the plasma membrane even after 2 h of chase. Therefore, both immunofluorescence and biochemical studies indicate that the levels of full-length beta APP delivered from TGN to plasma membrane are diminished in cells expressing FAD-linked PS1 variants, most likely by delaying the budding of beta APP-containing vesicles from the TGN and the ER.

An FAD-linked PS1 Variant Causes a Profound Reduction of Cell Surface-bound beta APP at Axons and Axonal Terminals in Primary Neurons-- Previous studies (44-48) have demonstrated that beta APP is axonally transported by the kinesin-mediated, fast anterograde component. It has also been reported (49) that both full-length and processed derivatives of beta APP accumulate at presynaptic terminals of cortical neurons. We further assessed whether FAD-linked mutations affect the distribution of full-length beta APP on the surface of primary neurons.

Embryonic cortical neurons from PS1 knockout mouse embryos rescued with comparable levels of expression of either wt human PS1 (line 17-3) or FAD-linked PS1 A246E mutant (line 16-4) (34) were grown in culture, and beta APP molecules on the cell surface at the axonal terminals and in the cell body were visualized by immunofluorescence staining using monoclonal antibody mAb348, which recognizes the ectodomain of beta APP. As shown in Fig. 6a, immunofluorescence of full-length beta APP on the surface of live (non-permeabilized) wt PS1 rescued neurons was evident both in the cell body and at the axonal terminals, whereas the immunofluorescence of surface-bound beta APP in PS1 A246E mutant rescued neurons was limited to the cell body, being absent from axons and axonal terminals (Fig. 6a, top panels). As a control, live cells were also doubly stained with V. villosa agglutinin to confirm that the staining of surface-bound glycoproteins was comparable in wt and mutant PS1 neurons (Fig. 6a, bottom panels). However, after permeabilization, the distribution pattern of intracellular full-length beta APP was identical in PS1 wt and A246E neurons (Fig. 6b, top panels). In addition, intracellular localization of growth-associated protein 43 (GAP43), a protein associated with growth cone membranes, was similar in wt and mutant PS1 neurons (Fig. 6b, bottom panels). These data suggest that the FAD-linked mutants selectively impair the targeting or fusion of beta APP-containing vesicles to the plasma membrane, especially at axons and axonal terminals of neurons.


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Fig. 6.   FAD-linked PS1 mutations cause more profound reduction of surface-bound beta APP in axons and axonal terminals. Primary neurons that express comparable levels of either wt human PS1 or FAD-linked PS1 A246E mutant in PS1-/- background were cultured for 7 days. a, after 15 min of 4% formaldehyde fixation, non-permeabilized cells were incubated with APP N-terminal antibody mAb348 and FITC-conjugated V. villosa agglutinin. b, in some experiments neurons were permeabilized with ice-cold methanol for 2 min and then incubated with mAb348 (red) and anti-GAP43 (green) antibodies. Immunofluorescence staining was visualized by confocal microscopy.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To date, the mechanisms by which PSs exert their effects on beta APP metabolism are not fully understood, although multiple lines of evidence support a direct role of PS1 in facilitating gamma -cleavage of beta APP, Notch, and other substrates (1, 50). On the other hand, it has been reported (24) that PS1s may play multiple physiological roles such as those in calcium homeostasis, neuronal development, neurite outgrowth, apoptosis, synaptic plasticity, and tumorigenesis. Recent evidence indicates a novel function of PS1 in regulating intracellular trafficking of a selected set of proteins including those associated with PS1 and beta APP metabolites (25, 26, 42). In the present report, we demonstrate the following: 1) PS1 deficiency leads to the accelerated trafficking of beta APP from both the TGN and the ER; 2) loss-of-function PS1 mutants with impaired gamma -secretase function increase ER biogenesis of beta APP-containing vesicles without affecting TGN budding and eventually elevate the amount of beta APP transported to the plasma membrane; 3) FAD-linked PS1 mutants impair beta APP trafficking from the TGN to the plasma membrane, as well as from the ER to Golgi, resulting in delayed delivery of beta APP to the cell surface; 4) a profound reduction of surface beta APP at axonal terminals of neurons that express FAD-linked PS1 mutants. Taken together, these results indicate that the role of PS1 in facilitating gamma -secretase processing of APP extends beyond its putative function in the catalytic process.

The above findings are consistent with a model in which PS1 might regulate beta APP metabolism by altering beta APP trafficking (Fig. 7). Our model proposes that PS1 provides a retention signal, which guides beta APP delivery to appropriate compartments where gamma -secretase processing occurs (Fig. 7a). Gain-of-function FAD mutants (Fig. 7c) may direct sustained retention of beta APP-containing vesicles and consequently increase the availability of beta APP to cleavage enzymes resident in the TGN and/or ER, the major sites of intracellular Abeta generation. In contrast, PS1 knockout (Fig. 7b) or loss-of-function mutants (Fig. 7b') abolish or reduce, respectively, the retention property and thereby allow accelerated beta APP trafficking to the cell surface. In addition, based upon our observations and another report (42), it is conceivable that PS1 regulates beta APP trafficking in both the secretory as well as the endocytic pathway where some Abeta may also be generated (51).


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Fig. 7.   Model proposing that PS1 may regulate beta APP metabolism by altering beta APP trafficking through the secretory pathway. a, it is proposed that PS1 provides a retention signal to guide beta APP delivery to appropriate compartments where gamma -secretase processing occurs. b, in the absence of PS1 expression beta APP trafficking is accelerated. As a consequence, Abeta generation is reduced. b', PS1 loss-of-function mutants accelerate beta APP transport from the ER without affecting budding from the TGN. The amount of beta APP molecules in each vesicle derived from TGN membrane is increased without altering the rate of vesicle budding. Therefore, beta APP delivery to cell surface is increased. c, in contrast, FAD-linked PS1 mutants may direct sustained retention of beta APP, and consequently increase the availability of beta APP to cleavage enzymes resident in the TGN and/or ER, the major intracellular sites for Abeta generation. However, the possibilities that PS1 may recruit certain cytosolic trafficking factors (such as phospholipase D1 and/or Rab11) to TGN and/or ER membrane and thereby regulate beta APP transport cannot be excluded.

Previously it was shown that PS1 deficiency causes missorting of select type I membrane proteins. For example, PS1 is required for the maturation and intracellular trafficking of nicastrin, an integral component of the gamma -secretase complex (14, 28, 29, 52). Moreover, in PS1-deficient neurons, telencephalin/ICAM is translocated from the plasma membrane to large intracellular clusters (26). It is interesting to note that PS1 deficiency causes enhanced localization of beta APP at the cell surface but has the opposite effect on surface accumulation of nicastrin and telencephalin/ICAM.

To fully understand the regulation of beta APP metabolism by PS1, it would be important to determine whether PS1 exerts an effect on trafficking of beta APP CTFs similar to that on full-length beta APP. It has been shown that loss of PS1 results in the accumulation of the alpha -/beta -CTF (25, 40). However, further studies on the subcellular distribution of CTFs in PS1-deficient cells indicate the complexity of intracellular trafficking of CTFs. For example, the generation of CTFs mostly occurs in the late secretory compartments, whereas beta APP CTFs accumulate in the ER, Golgi, and lysosomes (53). It has also been reported (53) that CTFs may accumulate in restricted and unpredicted intracellular compartments in PS1-deficient cells. Although our preliminary data indicate that PS1 may regulate intracellular trafficking of CTFs in a similar manner as its regulation of full-length beta APP (data not shown), vesicle budding assays (pulse-chase and low temperature incubations) have not yet distinguished between trafficking and production of CTFs in the ER and Golgi compartments. More rigorous studies are underway to establish appropriate cell lines (e.g. those overexpressing beta CTFs) and optimal experimental conditions.

Much remains to be learned about the mechanisms by which PS1 regulates intracellular trafficking of beta APP. Based on our permeabilized cell data, budding of vesicles containing FGFR1 and NCAM is not influenced by PS1 function, indicating that PS1 exerts its regulation on select membrane proteins. One attractive model is that PS1 regulates the recruitment or the association of trafficking factors with cytoplasmic sorting signals within beta APP, thereby selectively regulating the sorting of beta APP to the surface. In the absence of PS function, beta APP containing vesicles may be transported to the cell surface via the default constitutive pathway. On the other hand, FAD-linked mutations modify the interaction between beta APP and trafficking factors in a manner that interferes with efficient beta APP trafficking. In this regard, it has been reported that PS1 and PS2 associate with Rab11, a member of the GTP-binding protein family of membrane trafficking regulators implicated in protein transport along the biosynthetic and endocytic pathways (54). In addition, PS1 binds to Rab GDP dissociation inhibitor (RabGDI), a protein that functions in vesicular membrane transport to recycle Rab GTPases, and PS1 deficiency leads to impaired Rab-GDI membrane association (55). Furthermore, our preliminary data suggest that addition of phospholipase D1 to the cell-free budding assays prevents impaired beta APP trafficking from the TGN in cells expressing FAD PS1 mutations, whereas inhibition of phospholipase D1 activity by primary butanol diminishes the accelerated beta APP trafficking from the TGN in cells expressing loss-of-function PS1 mutations.

Finally, our studies demonstrate that FAD-linked PS1 mutants result in a decreased distribution of surface beta APP in axons and axonal terminals of neurons. This finding suggests that PS1 may specifically affect targeting to and/or fusion of beta APP-containing vesicles at the nerve terminus. As reported previously, full-length beta APP has been implicated in a number of physiological functions such as synapse formation, growth cone outgrowth, and axon guidance (55, 56). Furthermore, based on the demonstration that beta APP plays an essential role in axonal trafficking (47, 48), our findings that PS1 may regulate beta APP sorting along the axon have much broader implications as well. For example, impaired delivery of full-length beta APP to the cell surface at axonal terminals by FAD-linked PS1 variants may interfere with neurite initiation, elongation and branching, and synaptic plasticity. It is important to note that in AD presynaptic pathology is more severe than neuronal loss (57). By affecting vesicle transport and surface delivery of full-length beta APP, pathogenic PS1 mutants might directly modulate beta APP metabolism and, in addition, indirectly contribute to the pathogenesis and progression of AD.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants AG09464 (to P. G. and H. X.), AG19070 (to G. T.), AG21495 (to G. T.), AG021494 (to S. S. S.), and NS40039 (to H. Z.), the Alzheimer's Association (to G. T. and H. X.), the Ellison Medical Foundation (to H. X.), and the American Health Assistance Foundation (to G. T. and H. X.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by an Adler Foundation post-doctoral fellowship.

** To whom correspondence should be addressed. Tel.: 212-327-7567; Fax: 212-327-7888; E-mail: xuh@mail.rockefeller.edu.

Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M209065200

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; PS, presenilins; beta APP, beta -amyloid precursor protein; Abeta , beta -amyloid; FAD, familial AD; TGN, trans-Golgi network; ER, endoplasmic reticulum; CTFs, C-terminal fragments; wt, wild type; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; FGFR1, fibroblast growth factor receptor; NCAM, neural cell adhesion molecule.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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