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J Biol Chem, Vol. 274, Issue 45, 32295-32300, November 5, 1999

Expression of -Amyloid Precursor Protein-CD3 Chimeras to Demonstrate the Selective Generation of Amyloid _1-40 and Amyloid _1-42 Peptides within Secretory and Endocytic Compartments^*

Salvador Soriano, Abraham S. C. Chyung^§, Xiaohua Chen, Gorazd B. Stokin, Virginia M.-Y. Lee^§, and Edward H. Koo^¶

From the  Department of Neurosciences 0691, University of California, San Diego, La Jolla, California 92093-0691 and the ^§ Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Amyloid -protein (A) is the main constituent of amyloid fibrils found in senile plaques and cerebral vessels in Alzheimer's disease (AD) and is derived by proteolysis from the -amyloid precursor protein (APP). We have analyzed the amyloidogenic processing of APP using chimeric proteins stably transfected in Chinese hamster ovary cells. The extracellular and transmembrane domains of APP were fused to the cytoplasmic region derived from the CD3  chain of the T cell antigen receptor (CD3). CD3 contains an endoplasmic reticulum (ER) retention motif (RKK), in the absence of which the protein is targeted to lysosomes without going through the cell surface (Letourneur, F., and Klausner, R.D. (1992) Cell 69, 1143-1157). We used the wild-type sequence of CD3 to create an APP chimera predicted to remain in the ER (APP_ER). Deletion of the RKK motif at the C terminus directed the protein directly to the lysosomes (APP_LYS). A third chimera was created by removing both lysosomal targeting signals in addition to RKK (APP). This last construct does not contain known targeting signals and consequently accumulates at the cell surface. We show by immunofluorescence and by biochemical methods that all three APP chimeras localize to the predicted compartments within the cell, thus providing a useful model to study the processing of APP. We found that A_1-40 is generated in the early secretory and endocytic pathways, whereas A_1-42 is made mainly in the secretory pathway. More importantly, we provide evidence that, unlike in neuronal models, both ER/intermediate compartment- and endocytic-derived A forms can enter the secretable pool. Finally, we directly demonstrate that lysosomal processing is not involved in the generation or secretion of either A_1-40 or A_1-42.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

One of the major features of Alzheimer's disease (AD)¹ neuropathology is the deposition of amyloid -peptide (A) in brain parenchyma and cerebral vessels. A can be produced as a 40-amino acid peptide (A_1-40) or, occasionally, as a more amyloidogenic form of 42-43 amino acids (A_1-42). Both forms are generated by the activity of two unknown proteases termed - and -secretase from a larger amyloid precursor protein (APP), a ubiquitously expressed type I membrane glycoprotein (1). A peptide sequence begins at the extracellular domain of APP and ends within its transmembrane domain. In an alternative pathway, APP may be cleaved within the A sequence by another protease termed -secretase to generate a soluble ~100-kDa N-terminal fragment (APPs) and a membrane-retained ~10-kDa C-terminal fragment (2, 3). Because -secretase activity takes place within the A sequence, generation of intact A and APPs are mutually exclusive events.

The identities of , , and  secretases are not known, and the subcellular location of their activities is currently unclear. It is generally thought that -secretase cleavage occurs at the trans-Golgi network (TGN) or at a late compartment in the constitutive secretory pathway, as well as from the cell surface (4-7). Less clear however is the mechanism and intracellular compartments involved in the production of A_1-40 and A_1-42. Full-length APP at the plasma membrane may be internalized to generate A in an unidentified intracellular compartment. APP can also be sorted to the endosomal/lysosomal compartments where several A containing C-terminal APP fragments accumulate (8-11). It is not known whether these potentially amyloidogenic fragments are indeed A intermediates or simply undergo lysosomal degradation. Recently it was shown that in transfected COS cells both A_1-40 and A_1-42 are produced at the cell surface, although the actual sites of production were not identified (12). In contrast, in transfected neurons, A_1-40 appears to be produced at the TGN (6) and A_1-42 at the endoplasmic reticulum (12-14).

To better understand the role of different subcellular compartments in the production of A_1-40 and A_1-42 we constructed several APP chimeric proteins. We fused the extracellular and transmembrane domains of APP to the cytoplasmic region derived from the CD3 chain of the T cell antigen receptor (CD3). CD3 contains an ER retention motif (RKK), in the absence of which the protein is targeted directly from the TGN to the lysosomes without going through the cell surface by virtue of two different lysosomal signals, LL and YQ (15). We used the wild-type sequence of CD3 to create an APP chimera predicted to remain in the ER (APP_ER). A second APP chimera was constructed by removing the RKK motif, thus directing the protein directly to the lysosomes. Finally, a third chimera was created by removing both targeting signals in addition to RKK (APP). This LLYQ double mutation renders the cytoplasmic tail to be devoid of known sorting signals in its sequence, and the resulting mutant protein accumulates at the cell surface at high levels due to impaired internalization (15). This approach therefore provides the opportunity to target APP to multiple intracellular compartments.

Our results suggest that, in transfected Chinese hamster ovary (CHO) cells, A_1-40 is generated in the ER/IC, as well as in the endocytic pathway, whereas, as previously reported for other cell models, A_1-42 is generated in the early secretory pathway, mainly in the ER/IC. More importantly, we provide evidence that, unlike in neuronal models, both ER/IC- and endocytic-derived A forms can enter the secretable pool. Finally, we demonstrate that, in CHO cells, the lysosomes are not involved in the generation or secretion of either A_1-40 or A_1-42.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Construction of APP Chimeras-- cDNAs encoding the chimeric APP/CD3 proteins were generated by oligonucleotide-directed mutagenesis with the expression vector pCI-NEO (Promega) from the parental APP₇₅₁ containing the extracellular and transmembrane but lacking the entire cytoplasmic tail (residues 734-770; APP₇₇₀ numbering) fused to the cytoplasmic domain of the  chain of the CD3 (T cell) receptor (15). Three different APP/CD3 chimeric constructs were used: full-length tail (Gln-116 to Lys-159) was used to generate APP_ER; Gln-116 to Leu-156 (deletion of the C-terminal RKK residues) was used to generate APP_LYS; and APP was obtained by deleting residues L130L131 and Y137QPL140. Both the dileucine- and tyrosine-based targeting motifs were deleted in the last construct. All constructs were verified by DNA sequencing.

Cell Culture-- CHO cell lines were transfected using LipofectAMINE^TM (Life Technologies) reagent and selected by G418 resistance. The stably transfected CHO cell lines expressing wild-type APP and various APP chimeras were chosen with comparable levels of expression of the exogenous gene product. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with G418 (200 µg/ml) and 10% fetal bovine serum at 37 °C, with 5% CO₂. All experiments using these transfected CHO cells were repeated 3-5 times, and results from either representative experiments or the mean (± S.E.) of all experiments are shown.

Antibodies-- Monoclonal antibodies 5A3 and 1G7 and polyclonal antibody 863 directed to the mid-region of APP have been described (16, 17). Anti- tubulin monoclonal antibody was from Amersham Pharmacia Biotech and was used in Western blots at 1:5,000 dilution.

Immunofluorescence Microscopy-- APP-transfected cells grown on coverslips were fixed and permeabilized in methanol for 5 min at 20 °C. Following extensive washing in PBS, cells were blocked with 3% BSA in PBS (PBS/BSA) and then incubated with 5A3/1G7 (10 µg/ml in PBS/BSA) for 20 min at room temperature. Cells were then extensively washed in PBS and anti-mouse IgG conjugated to fluorescein isothiocyanate (Roche Molecular Biochemicals) for 20 min at room temperature. Control samples were incubated with mouse IgG instead of primary antibody. Immunostained cells were visualized by conventional epifluorescence microscopy.

Metabolic Labeling and Immunoprecipitation-- Confluent cultures of stably transfected CHO cells were incubated in methionine-free DMEM for 20 min followed by incubation with methionine-free DMEM supplemented with 250 µCi/ml [³⁵S]methionine for 15 min (pulse labeling) or 8 h with 150 µCi/ml (long labeling). In pulse-chase experiments, cells were lysed immediately after brief pulse-labeling or incubated in DMEM with 1 mM methionine (chase) for the indicated time points. APP was immunoprecipitated with antibodies 5A3/1G7 or 863 (16) and separated by SDS-polyacrylamide gel electrophoresis. Where indicated, cells were incubated for 4 h with 10 µM proteasome inhibitor MG-132 (Calbiochem) or 8 h with 100 µM leupeptin (Sigma) before immunoprecipitation. Gels were analyzed by phosphorimaging (Bio-Rad).

Cell Surface Biotinylation-- Confluent cultures of stably transfected CHO cells were surface-biotinylated on ice using sulfosuccinimidobiotin (sulfo-NHS-biotin, Pierce, IL) as described previously (16). Cells were lysed, and immunoprecipitations were performed using antibody 863 against APP. Biotinylation of precipitated APP was analyzed by phosphorimaging (Bio-Rad) after enhanced chemiluminescence using an antibody against biotin (Jackson Laboratories). Ratios of biotinylated versus total APP were used to calculate relative amounts of cell surface APP.

Internalization Assay-- Internalization of cell surface APP was performed as described (5). Briefly, iodinated (3-6 µCi/µg) whole 1G7 monoclonal antibody was diluted in binding medium (BM) (RPMI 1640 supplemented with 20 mM Hepes + 0.2% BSA) and added to triplicate cultures of CHO cells grown to confluence in 6-well tissue culture plates. Cells were incubated with radiolabeled 1G7 antibody at 37 °C for 30 min, chilled on ice, and washed once with ice-cold BM. After extensive washing with ice-cold Dulbecco's PBS, 1G7 antibody bound to cell-surface APP was uncoupled by two 5-min washes with ice-cold PBS, pH 2.5 followed by cell lysis with 0.2 M NaOH. Radioactivity from both the acid washes and the cell lysates was determined in a  counter. The ratio of radioactivity of acid-resistant to acid-labile fractions constitutes a measure of internalized versus cell-surface pool of APP.

Measurement of A by Sandwich ELISA-- Sandwich ELISA was performed as described (18, 19) using monoclonal antibodies specific for different species of A. BAN-50, specific for the N-terminal 10 amino acids of A, was used as capturing antibody. Horseradish peroxidase-conjugated BA-27 (A_1-40) and horseradish peroxidase-conjugated BC-05 (A_1-42) were used as secondary antibodies.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression and Localization of APP and APP/CD3 Chimeras-- Stably transfected wild-type APP and APP/CD3 (Fig. 1) CHO lines with comparable levels of expression were analyzed by immunoprecipitations after 8 h of [³⁵S]-methionine labeling. The results are shown in Fig. 2, a and b. Essentially, no differences in the levels of accumulated "mature" and "immature" forms of APP were found between APP, APP_LYS, and APP, indicating that at this level of analysis, all three forms are appropriately post-translationally processed through the secretory pathway. In contrast, very low levels of APP_ER mature forms were detected, suggesting that the majority of the newly synthesized protein is, as expected, retained in the endoplasmic reticulum and does not undergo normal maturation.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Schematic diagram of APP and CD3/APP chimeras containing the cytoplasmic tail of the CD3 chain and the transmembrane and extracellular domain of APP. Panel a, schematic diagram of wild-type APP; panel b, schematic diagram CD3/APP chimeras. APPER contains the two lysosomal sorting motifs, "LL" and "YQ", as well as the "RKK" ER retention motif and is predicted to stay in the ER. APPLYS lacks the motif RKK and is directed to the lysosomes from the TGN without sorting to the cell surface. APP lacks both the lysosomal signals LL and YQ and the ER retention motif RKK. This chimera accumulates at the cell surface and is endocytosis-deficient. The sequence of the cytosolic tail of CD3 (amino acids 116 to 159) is represented at the bottom with the motifs LL and YQ in bold.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Expression of APP and CD3/APP chimeras. Panel a, total APP was immunoprecipitated with antibodies 5A3/1G7 from cell lysates after 8 h [35S]methionine labeling. Note the absence of high molecular weight mature forms (m) from the APPER chimera (i, immature forms). Results from a representative experiment are shown. Panel b, quantitation of the ratio of mature to immature forms of APP shows a significant decrease for APPER, again representative of its lack of maturation. The results are mean ± S.E. from three experiments.

To further investigate the subcellular localization of the APP chimeras, we first performed immunofluorescence on methanol fixed/permeabilized cells. As expected, APP localized mainly to diffuse vesicular structures with a juxtanuclear distribution (Fig. 3), consistent with Golgi localization (20). In contrast, APP_ER-staining pattern showed a strong perinuclear ring extending into fine reticular structures, a distribution characteristic of ER resident proteins. APP_LYS distribution partially overlapped with that of APP at a juxtanuclear region due to the presence of both proteins in the biosynthetic pathway, but also in larger vesicular structures in the periphery. These latter vesicular structures were more intensely stained after leupeptin treatment (not shown), suggesting that APP_LYS does indeed localize to the lysosomes. Finally, APP was found at the cell surface (arrowheads, APP panel) in addition to the juxtanuclear staining also present in both APP and APP_LYS. Double staining experiments also showed that APP_ER co-localized with calnexin, whereas APP_LYS co-localized with the lysosomal markers LAMP-1 and LAMP-2 (not shown).

View larger version (102K): [in this window] [in a new window]   Fig. 3.   Localization of APP and CD3/APP chimeras in CHO cell lines by immunofluorescence. Cells were fixed/permeabilized in methanol for 5 min at 20 °C and stained with monoclonal antibodies 5A3/1G7. APP shows a mainly juxtanuclear Golgi distribution, whereas APPER staining pattern reveals a strong perinuclear ring extending into fine reticular structures, a distribution characteristic of ER resident proteins. APPLYS shows the accumulation of APP in vesicular structures throughout the cytoplasm. APPDD was found at the cell surface (arrowheads, APP panel) in addition to the juxtanuclear staining also present in both APP and APPLYS.

Biochemical Characterization of APP/CD3 Chimeras-- Because accurate targeting of APP chimeras to different subcellular locations is essential in this study, additional biochemical approaches were used to further define the subcellular localization of the three APP/CD3 chimeras. Recently, it became clear that selective degradation of ER membrane proteins occurs mainly through the ubiquitin-proteasome pathway (21, 22). Accordingly, if APP_ER is an ER resident protein, it should accumulate after treatment with MG-132, a specific proteasome inhibitor that has no apparent effect on lysosomal proteases (23). As shown in Fig. 4, there was no effect on APP (Fig. 4b), APP_LYS or APP (not shown), but APP_ER increased substantially after MG-132 treatment, indicating that the latter chimera is retained in the ER.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effect of protease inhibitors on the accumulation of APP and APP chimeras. Panel a, cells were incubated in the absence or presence of leupeptin (100 µM) for 8 h. At the end of the incubation period, equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, and the levels of APP were analyzed by Western blotting. A modest accumulation of APPLYS, but not APP, is apparent after leupeptin treatment. Parallel samples were blotted for -tubulin to confirm protein loading. Panel b, proteasome inhibitor MG-132 (10 µM) was added to cells for 4 h, and levels of APP were analyzed as above. The strong accumulation of APPER after MG-132 indicates that its predominant degradation route is, as expected, in the ER. Results from a representative experiment are shown.

To further confirm that the vesicular staining seen for APP_LYS corresponds to the lysosomes, the transfected cells were treated with leupeptin to inhibit lysosomal proteases. As presented in Fig. 4a, leupeptin treatment results in accumulation of APP_LYS, but not APP (Fig. 4a) or the other APP chimeras (not shown). A control immunoblot for -tubulin (Fig. 4a) shows that the modest increase in APP_LYS after leupeptin treatment is not due to differences in the amount of protein loading.

In the absence of targeting signals, APP is predicted to be mainly cell surface distributed and deficient in endocytosis (15). Consequently, cell surface accumulation of APP was further examined by surface biotinylation, followed by lysis and immunoprecipitation of total cellular APP. Surface and total amounts of APP were estimated by dividing the amount of reactivity of the biotinylated versus total pools of APP (see "Materials and Methods"). The results showed that cell surface levels of APP were ~2.5-fold higher than those seen in APP (Fig. 5), a value comparable with that seen in a C-terminally truncated APP (5). The levels of cell surface APP_ER were essentially undetectable, whereas APP_LYS was too low for accurate quantification, estimated to be approximately one-tenth of the amount of wild-type APP. The lack of cell surface molecules in APP_ER and APP_LYS forms provided further confirmation of the intracellular localization of these chimeric proteins. Lastly, cell surface accumulation of APP is correlated with a marked decrease in endocytosis, as measured by uptake of radioiodinated 1G7 antibody (5) (not shown). Moreover, secretion of sAPP (Fig. 6) was increased ~2.5-fold, a value also similar to what has previously been described for C-terminally truncated mutant APP (10). Therefore, these results show that the APP-CD3 chimeras are appropriately targeted to their predicted subcellular locations.

View larger version (46K): [in this window] [in a new window]   Fig. 5.   Cell surface biotinylation. Confluent cultures of stably transfected CHO cells were surface-biotinylated on ice, lysed and immunoprecipitations for APP were performed as described under "Materials and Methods." The levels of biotinylated APP were analyzed by phosphorimaging after enhanced chemiluminescence using an antibody against biotin. The relative amounts of APP at the cell surface are shown as the ratio of biotinylated versus total APP. The results are the mean ± S.E. of three experiments.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Release of sAPP from CHO cells transfected with wild-type APP or CD3/APP chimeras. Levels of sAPP, normalized to the actual rates of APP synthesis of the different cell lines. The results are the mean ± S.E. of three experiments.

Levels of A_1-40 and A_1-42 in CD3/APP Chimeras-- We next examined the levels of A_1-40 and A_1-42 in cell lysates and conditioned media from cells stably transfected with APP or APP chimeras using sandwich ELISA (see "Materials and Methods"). In comparison with APP, all APP chimeras showed differences in the levels of A_1-40 and A_1-42, although in different ways. First, with respect to APP, there was a dramatic decrease in the secreted and intracellular pools of A_1-40 in APP_LYS- and APP-expressing cells (Fig. 7a). Because all three proteins presumably share similar processing steps through the secretory pathway up to the TGN, the differences in A_1-40 production are likely to originate late in the secretory pathway (i.e. a post-TGN compartment) and/or the endocytic pathway. Neither APP_LYS nor APP undergo efficient endocytosis, because in the former construct, the molecules were sorted away from the cell surface levels (Fig. 5) and in the latter construct, internalization was substantially impaired. Taken together, these results suggest that the endocytic pathway is a major site of production and subsequent release of A_1-40 (~60-70% of the total detected), the remaining presumably originated in the early secretory pathway (i.e. ER, Golgi, and/or TGN, the three compartments shared by both chimeras).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Levels of intra- and extracellular A1-40 and A1-42 from CHO cells expressing APP or CD3/APP chimeras. Cell lysates and conditioned media were assayed by ELISA for A1-40 (a) and A1-42 (b) as described under "Materials and Methods." Values for both secreted and intracellular A are normalized to the levels obtained from wild-type APP (which is assigned a value of 1). Results represent the mean ± S.E. from four independent experiments (secreted A) or from three independent experiments (intracellular A). A1-40: APP versus APPER, p < 0.05 (secreted), p < 0.01 (intracellular); APP versus APPLYS, p < 0.01 (secreted and intracellular); APP versus APP, p < 0.01 (secreted and intracellular). A1-42: APP versus APPER, p < 0.001 (secreted), p < 0.001 (intracellular); APP versus APPLYS, p = not significant; APP versus APP, p = not significant. Statistical analysis was carried out by analysis of variance (F = 19.54, p < 0.0001) coupled with post-test Tukey-Kramer.

In contrast, however, levels of A_1-40 from APP_ER-expressing cells showed different changes with respect to wild-type APP. Specifically, although intracellular levels of A_1-40 were almost 75% higher, secreted levels were approximately one-half those of APP cells (Fig. 7a). This suggested that only a portion of intracellularly generated A_1-40 molecules are released. Moreover, because APP_ER is retained in the ER and endocytic processing is largely absent, the high levels of intracellular A_1-40 are unexpected. We hypothesized that the accumulation of A_1-40 in APP_ER transfected cells derives from its retention in the ER, where there is an enhancement of substrate available for -secretase activity. To test this hypothesis, we measured the half-life of APP and the three APP chimeras by pulse-chase labeling. Indeed, both APP and APP_ER showed markedly longer half-lives than wild-type APP (135.1 ± 3.38 min and 86.6 ± 4.3 min, respectively, versus 53.3 ± 6.9 min, n = 3, p < 0.001, analysis of variance) (Table I).

View this table: [in this window] [in a new window]   Table I Half-life of APP/CD3 chimeras Data from three independent experiments. Values for half-life are expressed in minutes as mean ± S.D.

In contrast to A_1-40 levels, A_1-42 levels (Fig. 7b) were surprisingly unchanged in both APP_LYS- and APP-expressing cells as compared with wild-type APP. This was seen in both intracellular and secreted pools of A_1-42, a finding that suggests that even the secreted pool of A_1-42 was generated predominantly in the ER/IC.

APP_ER behaved very differently from the other three constructs. As with A_1-40, the intracellular levels of A_1-42 were substantially higher in APP_ER cells as compared with wild-type APP and the other two APP/CD3 chimeras (Fig. 7b). In contrast to A_1-40, even the secreted levels of A_1-42 were also higher. In summary, with respect to wild-type APP, levels of A_1-42 from APP_ER-expressing cells were increased ~10- and ~4-fold in the intracellular and secreted pools, respectively.

Are Lysosomes Involved in the Generation of A?-- Lysosomes have been hypothesized as a possible site of A production. Several studies have indirectly addressed this question, but, to date, the role of the lysosomes in A production is still unclear (24-26). Efficient targeting of APP to lysosomes enabled us to directly address this question. Accordingly, production of A_1-40 did not increase when APP was directly sorted to the lysosomes from the TGN, indicating that lysosomes are not involved in A generation (Fig. 7a). The same can be concluded of A_1-42. Because no differences are found between APP, APP_LYS, and APP (Fig. 7b), the lysosomal compartment is unlikely to be a major source of either A_1-40 and A_1-42 generation.

A_1-42/A_1-40 Ratios Increase in All APP Chimeras-- Combining all the A results of our study (Fig. 7), we have analyzed the relative ratios of A_1-42/A_1-40 expressed in CHO cells. Extracellularly, A_1-42 is consistently a minor fraction of total A, and wild-type APP showed the expected ~1:10 ratio of A_1-42 to A_1-40. The intracellular ratio is ~6:10, a value similar to that reported in neurons (13). However, there was an increase in the 42:40 ratios in both intracellular (between 2-3:1) and secreted (~4:10) pools for all APP chimeras. And despite the differences in absolute levels of each A species, the relative levels are surprisingly similar to each other. Intracellularly, this was achieved by either a large increase in A_1-42 (APP_ER) (although attenuated by a slight increase in A_1-40) or by a decrease in A_1-40 (APP_LYS and APP). A similar, although more modest trend, is seen extracellularly. The secreted 42:40 ratio increase was achieved by either a decrease of A_1-40 and an increase in A_1-42 (APP_ER) or by a decrease in A_1-40 secretion (APP_LYS and APP).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A is the major component of senile plaques in the Alzheimer's brain, and it is thought to play an important role in the pathogenesis of the disease (1). Consequently, much effort has been dedicated to the study of A generation in a variety of models. Here, we have chosen to generate several chimeric APP proteins predicted to localize to particular organelles, to study the role of such subcellular locations in the generation and subsequent secretion of A_1-40 and A_1-42. Although several recent reports have successfully used chimeric APP proteins to study the proteolytic processing of APP (27-29), our study is the first to use a common approach to target APP to multiple organelles and directly analyze the intracellular formation and secretion of A_1-40 and A_1-42. The strategy that we followed for APP subcellular targeting is based on a well characterized model, that of the  chain of the CD3 receptor (Fig. 1) (15). Because the targeting of the APP chimeras to specific organelles is central to our approach, we ascertained by morphological and biochemical studies the predicted localization of the chimeric proteins. Accordingly, several lines of evidence demonstrated that the targeting motifs from CD3 are fully functional when fused to the APP transmembrane and extracellular domains, thereby providing a valuable tool for the analysis of A formation in different subcellular organelles. Thus, we showed that APP_ER is localized to the ER with impaired post-translational processing and is turned over more slowly. In the case of APP_LYS, we showed that this chimeric protein is predominantly sorted to lysosomes, bypassing the cell surface, after maturation. Finally, we demonstrated that in the absence of the two targeting motifs, APP accumulates at the cell surface and, not surprisingly, showed impaired internalization. As expected, a consequence of the latter abnormality is an increase in APPs in the medium (16).

Recently, it has become clear that the sites of generation and the rates of secretion of different A forms are complex and may be cell type dependent. Specifically, in neuronal cells, A_1-42 is produced in the ER/IC compartment (12, 13, 30), whereas A_1-40 is apparently derived from the TGN or beyond (12). In contrast, APP₆₉₅ transfected COS-7 cells generate both A forms at the plasma membrane (12) and are undetectable intracellularly. A nonneuronal cell type, kidney 293 cells, when stably transfected with APP, shows detectable levels of intracellular A_1-42 but not A_1-40 (31). Therefore, our analysis is important from the standpoint that the different intracellular processing pathways can be simultaneously analyzed with respect to both A_1-40 and A_1-42. Our results indicate that in CHO cells (1) both secretory and endocytic processing contribute to the production of A_1-40, whereas A_1-42 derives mainly from the secretory pathway (2) both endocytic and, to a lesser extent, ER/IC-derived A forms can enter the secretable pool, and (3) lysosomes are not a major site of A generation.

Our studies showed clearly that both A_1-40 and A_1-42 species are readily detectable in APP-transfected CHO cells and that the secretory pathway plays a role in the generation of both forms (Fig. 7, a and b). This is apparent from the fact that both A_1-40 and A_1-42 are increased when APP is artificially retained in the ER (APP_ER) via the presence of a retention signal engineered into the cytoplasmic domain. However, we reasoned that the majority of A_1-40 (~70-75%) originates from endocytic processing because of the loss of A_1-40 in the APP_LYS and APP cells as compared with wild-type APP cells (Fig. 7a). Furthermore, the fact that retention of APP in the ER results in accumulation of intracellular A_1-40 (Fig. 7a, compare APP_ER with APP) indicates that the ER itself contains an A_1-40-specific -secretase activity. This is a surprising finding, because A_1-40-specific -secretase activity has only been reported previously at the TGN or at the plasma membrane. One explanation of our finding is that this population of ER-derived A_1-40 is generated from secondary cleavage of A_1-42, a postulate that is consistent with evidence arguing distinct A_1-40 and A_1-42 -secretase activities (32).

In contrast, consistent with published reports, A_1-42 may be derived primarily from processing in the early secretory pathway (12, 13). As shown in Fig. 7b, APP and both APP_LYS and APP produce and secrete comparable amounts of A_1-42, indicating that a main site of production is in a compartment common to all three APP forms, i.e. ER, Golgi, or the TGN. Again, the fact that retention of APP in the ER causes intracellular accumulation of A_1-42 points to that organelle as a major site for A_1-42 production. However, our results cannot rule out the late secretory and/or the endocytic pathways as additional sites of production for A_1-42. Specifically, because the APP chimeras have very different cytosolic tails, direct comparison to APP may be misleading. This is evident from the fact that all three APP chimeras show substantially higher A_1-42/A_1-40 ratios. Therefore, the possibility remains that the decrease seen in A_1-42 production from APP_LYS and APP with respect to APP_ER, rather than to wild-type APP, may be due to the deficient endocytosis from the former chimeras and not only to the accumulation of APP_ER in the ER. This concept would be consistent with the observation that secretion of both A_1-40 and A_1-42 is diminished when the endocytic signal is removed from the cytoplasmic domain of APP (33). We should emphasize that retaining APP in the ER is equally artificial, and we cannot ascertain the degree to which A_1-42 generation has been abnormally increased. Furthermore, the normal interactions with proteins that associate with the APP cytoplasmic domain are absent in the chimeras. Thus, the Fe65/X11 family of proteins that bind to APP at or near the YENPTY domain and alter APP trafficking, translocation to the plasma membrane, sAPP secretion, and A production (34, 35) are ineffectual in the APP chimeric molecules. Interestingly, overexpression of X11 as well as the Y743A mutation (in the NPTY motif) both decreased the turnover of APP but with different effects on A generation (33, 35). Although APP_ER also increased the APP half-life, direct comparison between these three conditions is not possible. First, as mentioned above, APP_ER contains a completely different cytoplasmic domain. Second, the other studies did not examine the levels of intracellular A. Third, the mechanisms underlying the reduced turnover of APP are different in all three cases: in APP_ER, the molecule is postulated to remain in the ER; in APP Y743A mutant, a lysosomal targeting signal may be lost (33); and in X11, the effect is presumably due to delayed protein maturation (35).

Our results also showed that not all intracellular A_1-40 and A_1-42 is released. It has been argued that in neurons, the secreted and the intracellular pools of A are produced independently and that the secreted pool is endocytosis-dependent (12, 36). Subsequent studies (13) showed that the intracellular pool of A_1-42 derives from the ER and does not enter the secreted pool. This was shown by insertion of a KK ER-retention motif to the cytosolic tail of APP (13), an approach similar to the APP_ER construct, and by BFA treatment of wild-type APP-expressing cells, although protein traffic distal to the ER is inhibited, and therefore is more appropriate to study A production than A secretion. In our study, we showed that secretion was impaired but not abrogated for both A_1-40 and A_1-42 from APP_ER. Note that, although there is an increase in secretion of A_1-40 from APP_ER when compared with APP_LYS and APP, there is a decrease with respect to wild-type APP, indicating that accumulation of A_1-40 in the ER results in its impaired secretion. Similarly, A_1-42 from APP_ER accumulates in the ER in much higher proportions than it can be released (10-fold accumulation versus 3-fold secretion increase when compared with wild-type APP). These findings are of interest, because it has been proposed that generation of A_1-42 in the ER, at least in neurons, is a saturable process (13). In other words, it was suggested that retention of APP in the ER in neurons does not result in an increase in A_1-42 levels and, importantly, secretion of A_1-42 is abrogated. In contrast, we show that in our model both A_1-40 and A_1-42 can accumulate in the ER, indicating that saturation seems to be reached at the secretion rather than at the level of production.

Finally, we presented evidence that the lysosomes are not the site of production of either A_1-40 or A_1-42. Lysosomes contain APP degradation products, especially the C-terminal fragments that may be direct precursors to A. Although there is indirect evidence suggesting that an acidic compartment, such as the lysosomes, is necessary for A production, attempts to isolate A from endosomes/lysosomes fractions have not been successful (24). Therefore, the role of the lysosomes in A production remains poorly defined. This issue is both important and perplexing in view of the disruptions of the endosomal/lysosomal system that accompany AD (37, 38). We addressed this question by targeting APP to the lysosomes and directly measuring A_1-40 and A_1-42 production. Our results showed that in CHO cells the lysosomes are not likely to represent a major site of production for either A_1-40 or A_1-42.

In summary, we have used three different APP chimeric proteins to study the intracellular amyloidogenic processing of APP and the subsequent release of both A_1-40 and A_1-42 into the medium in cultured CHO cells. We found that secretory and endocytic processing contribute to different degrees to the production and release of both A_1-40 and A_1-42. Moreover, our results show that the production and secretion pathways may be substantially more complex than previously thought.

	ACKNOWLEDGEMENTS

We thank Drs. David Kang and Sreeganga Chandra for stimulating discussions, Dr. Claus Pietrzik and Dr. Nathalie Chevalier for critical reading of this manuscript, and W. Cox Terhorst for CD3r cDNA.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants AG12376 and NS01812 (to E. H. K.) and by the Boehringer Ingelheim Fonds (to G. B. S.).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.

^¶ To whom correspondence should be addressed: Dept. of Neurosciences 0691, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0691. E-mail: edkoo@ucsd.edu.

	ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; APP, -amyloid precursor protein; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; IC, intermediate compartment; sAPP, secreted N-terminal ectodomain of APP; A, amyloid -protein; TGN, trans-Golgi network; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin.

	REFERENCES

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