Alzheimer's disease, a progressive neurodegenerative disorder of the elderly, is characterized by the presence of parenchymal deposits of A, ()a 39- to 43-amino acid peptide derived from APP(1, 2, 3, 4) . APP are integral membrane glycoproteins that mature through the secretory pathway(5) . A fraction of newly synthesized APP appears on the cell surface (6, 7, 8, 9) and some of these molecules are cleaved by -secretase (7, 8) within the A sequence (10, 11, 12, 13) , resulting in the release of the APP ectodomain (APP). In a cell-type specific manner, APP is generated in the trans-Golgi network or other late compartments of the constitutive secretory pathway (14, 15, 16, 17) . A fraction of APP are directly sorted, or reinternalized from the cell surface, to endosomal/lysosomal compartments(7) , where a complex set of A-containing membrane-bound fragments accumulate(7, 18) . Finally, it is fully established that A (4 kDa) and a truncated form of A (3 kDa) are released constitutively in vitro and in vivo(19, 20, 21, 22, 23) .

The biochemical mechanism(s) and cellular compartments involved in A production have not been fully elucidated. Despite earlier excitement created by the discovery of potential amyloidogenic fragments generated in endosomal-lysosomal pathways, several lines of evidence now suggest that lysosomal degradation of APP is unlikely to contribute to the production of A (reviewed in (24) ). However, agents that interfere with pH gradients (i.e. ammonium chloride and chloroquine) inhibit the production of A(19, 21) , suggesting that A may be generated in acidic compartments (i.e. endosomes and/or late Golgi). Indeed, biochemical studies by Koo and Squazzo (23) confirmed that A production and release involves the endocytosis of full-length APP from the cell surface and subsequent recycling. In this model, -secretase cleavage occurs within endocytic compartments while -secretase cleavage of the residual 100-amino acid membrane fragment occurs virtually simultaneously with A formation and release. However, the stoichiometry of A contributed by reinternalized APP to A generated in the biosynthetic pathway is not known.

To identify potential intracellular compartments involved in A production, we examined the metabolism of APP with a double mutation at codons 670 and 671 (of APP-770) described in two large Swedish pedigrees with familial Alzheimer's disease. Cells expressing this mutant APP secrete 6-8-fold higher levels of A relative to cells expressing wild-type APP(25, 26) . In preceding efforts, we examined the trafficking and metabolism of APPwt and APPswe in polarized epithelial cells (MDCK)(27) . We demonstrated that APPswe was cleaved at the -secretase site and that the resulting soluble derivative, termed APP, was detectable in cell lysates. These results lead to the suggestion that ``-secretase'' cleavage occurs in an intracellular compartment. In the present report, we extend the observations in MDCK cells by characterizing the maturation of APPswe in transiently and stably transfected N2a cells, a mouse peripheral neuroblastoma line. We demonstrate that N2a cells expressing APP harboring the Swedish mutations secrete high levels of A peptides and APP derivatives relative to cells expressing APPwt. Interestingly, we observed a concomitant diminution in the levels of secreted APP. One scenario to explain this intriguing result is that normal -secretase substrates are disabled by the prior action of -secretase. This model predicts that -secretase must cleave APPswe in an intracellular compartment proximal to the cellular site(s) of -secretase activity. We now confirm that -secretase cleavage occurs within the Golgi apparatus, as early as the medial compartment.

MATERIALS AND METHODS

Plasmid Construction and Expression

Mouse N2a neuroblastoma cells were transfected using a high-efficiency CaPO co-precipitation procedure(30) . To generate cell lines that express human APPwt and APPswe, N2a cells were transfected with pAPPwt or pAPPswe and stable transfectants were selected in medium containing 0.4 mg/ml G418 (Life Technologies, Inc.) (the pCB6 vector contains a gene encoding resistance to the neomycin analog, G418). Expression of c-Myc-modified human APP was assayed by immunoblotting and immunoprecipitation with a polyclonal antiserum Myc-I raised against the synthetic peptide MEQKLISEEDLN(31) .

Metabolic Labeling and Immunoprecipitation Analysis

Cell-associated and soluble APP derivatives were immunoprecipitated as described previously(11) . To immunoprecipitate full-length APP and APP COOH-terminal fragments, we used 369, a polyclonal antiserum raised against the entire cytoplasmic tail of APP(32) , or Myc-I, a polyclonal serum raised against a 12-amino acid epitope in the c-Myc oncoprotein (see above). To immunoprecipitate full-length and soluble APP derivatives, we used a monoclonal antibody (mAb), P2-1, which recognizes an epitope in the NH-terminal cysteine-rich region of human APP(33) . 6E10, a mAb that recognizes residues 1-17 of human A (34) was used to immunoprecipitate soluble APP generated following cleavage by -secretase, while 4G8, a mAb that recognizes residues 17-28 of A (34) was used to immunoprecipitate A and related p3 peptides. Antibody 54 was used to immunoprecipitate the soluble APP derivative generated following cleavage of APPswe(35, 36) . Polyclonal anti-VSV serum (28) was used to immunoprecipitate VSVG protein. Quantification of immunoprecipitated polypeptides was performed by phosphorimaging using Molecular Dynamics software.

A Immunoprecipitation and Radiosequencing Analysis

Cell Surface Labeling Studies

To assess the level of cell surface-bound APP, radiolabeled cell lysates were subject to immunoprecipitation with 369 antiserum. The immunoprecipitates were boiled in immunoprecipitation buffer containing 1% SDS to dissociate APP-related polypeptides from the antibody. The resulting soluble fraction was subsequently incubated with streptavidin-agarose beads (Pierce) to recover biotinylated APP, as described previously(27) . To examine the fraction of soluble derivatives which were biotinylated, i.e. that were released from the cell surface, equivalent aliquots of medium collected from cells incubated at 37 °C following biotinylation was incubated with either mAb 22C11, an antibody that recognizes a region between residues 66 and 81 of APP (6, 38) or mAb 6E10 (see above). Immunoprecipitated proteins were dissociated by boiling in immunoprecipitation buffer containing 1% SDS. The resultant soluble fraction was adjusted to 1 immunoprecipitation buffer and subjected to a second round of binding to streptavidin-agarose beads.

Endoglycosidase H Digestion

RESULTS

Expression and Metabolism of Human APP and the ``Swedish'' Variant in N2a Cells

Figure 1: Metabolism of human APP-695 in mouse N2a cells. A, analysis of APP metabolism in transiently transfected cells. N2a cells transfected with plasmid vector or plasmids encoding human APPwt (Wt) or APPswe (Swe) were labeled for 3 h with [S]methionine. Full-length APP were immunoprecipitated from cell lysates with antibody 369. Secreted APP molecules were immunoprecipitated from conditioned medium (Supt) using either mAb P2-1 or 6E10. Note that 6E10 immunoprecipitated considerably lower amounts of APP from APPswe as compared with P2-1. A and p3 were immunoprecipitated from conditioned medium using mAb 4G8. B, characterization of termini generated by -secretase cleavage of APPswe. Aliqouts of [S]methionine-labeled conditioned medium from cells transiently transfected with empty vector (lane 1), APPwt cDNA (lane 2), or APPswe cDNA (lane 3) were immunoprecipitated with antibody 54, specific for the carboxyl terminus of APP. APPwt and APPswe panels, radiosequencing of A molecules from conditioned medium of cells expressing APPwt or APPswe, respectively. C, analysis of APP metabolism in stable N2a lines. Stable N2a cell lines 695.12 (Wt) and Swe.1 (Swe), expressing human APPwt or APPswe, respectively, were labeled for 3 min (lanes 1 and 2) or 3 h (lanes 3-10) with [S]methionine. Full-length human APP were immunoprecipitated from cell lysates using Myc-I antiserum or mAb P2-1. Secreted APP were immunoprecipitated with either mAb P2-1 or 6E10. Note that the vast majority of APP derived from APPswe (lane 6) was not immunoprecipitated by 6E10 (lane 8). A and p3 were immunoprecipitated from conditioned medium using mAb 4G8. The 95-kDa APP species (marked by an arrowhead) observed in lysates of Swe.1 cells (lane 4) likely represents APP(27) .

Finally, and to confirm that endoproteolytic cleavage by -secretase occurs between Leu and Asp of APPswe, we characterized the carboxyl terminus of the secreted soluble derivative and the amino terminus of A. To confirm the authenticity of the -secretase-generated soluble derivative, we subjected parallel aliquots of conditioned medium shown on Fig. 1A, lanes 4-6, to immunoprecipitation analysis with antibody 54, specific for APP(35, 36) . This antibody fails to detect -secretase-cleaved APPswe(35, 36) , and hence is specific for an epitope of the APPswe soluble derivative which is ``exposed'' upon truncation of the full-length precursor by -secretase. As anticipated, antibody 54 recovered high levels of 95-kDa species from medium of cells expressing APPswe (Fig. 1B, lane 3); this result is in sharp contrast to the isolation of nonspecific polypeptides of 85 and 95 kDa from medium of cells transfected with empty vector or wtAPP cDNA (Fig. 1B, lanes 1 and 2, respectively). To analyze the amino terminus of A derived from cells expressing APPwt and APPswe, we radiolabeled cells with [H]phenylalanine and immunoprecipitated A and p3 from the conditioned medium; the isolated 4-kDa band was subjected to radiosequencing. Predominant peaks of radioactivity in cycles 4, 19, and 20 clearly show that >95% of the 4-kDa peptides secreted by cells expressing either APPwt and APPswe initiate at Asp (Fig. 1B, panels APPwt and APPswe). Taken together with studies demonstrating that the membrane-bound fragment generated by -secretase initiates at Asp(39) , our demonstration that the carboxyl terminus of the soluble derivative extends to Leu fully confirms our view that -secretase cleaves APPswe between Leu and Asp.

In order to further characterize intracellular trafficking and processing of APPwt and APPswe APP in N2a cells, we generated 20 and 18 independent stably transfected cell lines which expressed variable levels of APPwt or APPswe, respectively. We did not observe any toxicity-related phenotypes in the N2a lines expressing either APPwt or APPswe. Biosynthetic rates of c-Myc epitope-tagged transgene-derived polypeptides in individual cell lines were assessed by short (3 min) pulse-labeling with [S]methionine and immunoprecipitation analysis with Myc-1 antibody. Two cell lines, 695.12 and Swe.1, that express essentially indistinguishable levels of newly synthesized APPwt and APPswe, respectively (Fig. 1C, compare lanes 1 and 2, respectively), were selected for further analysis. Immunoprecipitation of APP-related species using APP NH-terminal antibody, P2-1, from lysates of 695.12 and Swe.1 cells labeled for 3 h, revealed the presence of accumulated 105- and 120-kDa forms (Fig. 1C, lanes 3 and 4). Interestingly, we also detected a species with an apparent molecular mass of 95 kDa in lysates of cell line Swe.1 (Fig. 1C, lane 4), similar to a species observed in lysates of MDCK that constitutively express APPswe(27) . Analysis of soluble APP derivatives in radiolabeled conditioned medium of lines 695.12 and Swe.1 (Fig. 1C, lanes 5-8) fully supported our findings in transient assays. For example, relative to the level of soluble derivatives in 695.12 cell medium (Fig. 1C, lane 5) the total level of soluble derivatives in Swe.1 cell medium (Fig. 1C, lane 6) was clearly elevated; by phosphorimaging analysis, the total level of secreted derivatives generated by line Swe.1 was increased 3-fold relative to line 695.12, and the vast majority of these species exhibited accelerated electrophoretic mobility. Moreover, the levels of APP in Swe.1 cell medium was clearly diminished relative to the level of APP in 695.12 cell medium (Fig. 1C, compare lanes 8 and 7, respectively); phosphorimaging analysis showed 3-fold higher levels of APP in 695.12 cell medium compared to Swe.1 cell medium. Hence, we argue that the principal contributor to the elevated level of accumulated derivatives secreted by Swe.1 cells are species which are generated following cleavage at the -secretase site. Finally, a 4-fold elevation of accumulated 4-kDa A was evident in Swe.1 cell medium relative to 695.12 cell medium. However, and in contrast to our findings in transiently transfected N2a cells, a corresponding diminution in the levels of the p3 fragment was not observed. At present, there is not a satisfactory explanation for this phenomenon. Earlier studies have suggested that the penultimate precursor for p3 is the membrane-retained fragment generated by -secretase(24) . It is also conceivable that a fraction of -secretase-generated, carboxyl-terminal derivatives are substrates for cleavage by -secretase and that this truncated fragment is ultimately cleaved by -secretase to generate p3. Thus, we would propose that the trafficking and proteolysis of the -secretase-generated fragment is intrinsically different in stable and transiently transfected N2a cells, and may reflect differences in expression levels of transgene-encoded APPswe or the corresponding -secretase-generated carboxyl-terminal fragment. Despite this inconsistency in p3 levels between stable and transiently transfected N2a cells, these data do not detract from our principal findings that the total levels of secreted derivatives generated by the stable APPswe cell line are increased relative to cells expressing APPwt and that over 80% of these species exhibited accelerated electrophoretic mobility. Moreover, concomitant dimunition in APP levels in medium of the stable cell line expressing APPswe strongly supports our view that potential -secretase substrates are disabled by the prior action of -secretase.

In the aggregate, we have demonstrated that in transient and stably transfected N2a cells, the levels of soluble derivatives and A in medium of cells expressing APPswe are elevated relative to respective species secreted by cells expressing APPwt. Moreover, the vast majority of soluble derivatives in medium of cells expressing APPswe are APP, molecules generated following cleavage at the -secretase site. The production of elevated levels of APP from cells expressing APPswe is essentially identical to results obtained by expression of chimeric human placental alkaline phosphatase-APP molecules harboring the Swedish substitutions in H4 glioblastoma cells. These studies demonstrated that human placental alkaline phosphatase-APPswe were sensitive to endoproteolytic cleavage at the -secretase site and that high levels of chimeric human placental alkaline phosphatase-APP species were secreted into the conditioned medium(40) . More importantly, we document that the total level of APP derivatives is diminished in medium of cells expressing APPswe. This result can be accommodated by a model in which a significant population of substrates which might normally be subject to cleavage by -secretase are disabled by the prior action of -secretase. Implicit in this model is that -secretase must cleave APPswe substrates in an intracellular compartment proximal to the cellular site(s) of -secretase activity. Thus, we argue that APPswe is obligatorily cleaved by -secretase in an intracellular compartment wherein -secretase is either inactive or absent.

Kinetics of - and -Secretase Processing of APPwt and APPswe APP in N2a Cells

()

Figure 2: Kinetics of APP processing in N2a cells. A, analysis of APP carboxyl-terminal fragments generated by proteolytic processing. Detergent lysates of transiently transfected N2a cells (Fig. 1A) were analyzed by immunoprecipitation with either Myc-I or 369 antiserum. B and C, kinetics of cleavage and secretion of APP molecules. Parallel dishes of N2a cells transfected with plasmids encoding APPwt (Wt) or APPswe (Swe) were pulse-labeled for 10 min with [S]methionine and chased for the times indicated. At each time point, medium was collected and lysates were prepared from cell monolayer. Carboxyl-terminal APP fragments were immunoprecipitated from lysates with 369 antiserum. Secreted APP molecules were immunoprecipitated from conditioned medium using mAb P2-1. The 13.5-kDa fragment whose levels were elevated in cells expressing APPswe is marked by an arrow. The 12-kDa band (marked by an arrowhead) likely represents the carboxyl-terminal fragment generated after -secretase cleavage of APP.

To evaluate the kinetics of cleavage of APPwt and APPswe at the - or - secretase sites specifically, we performed pulse-chase analyses. Parallel dishes of N2a cells were transiently transfected with cDNAs expressing APPwt or APPswe. Cell monolayers were pulse-labeled with [S]methionine for 10 min, then chased at 37 °C for varying periods of time up to 45 min. At the end of each chase period, medium was saved and detergent soluble lysates were prepared from cell monolayers. Lysates or conditioned medium were subjected to immunoprecipitation with Ab369 or P2-1 antibodies, respectively. Fig. 2, B and C, depicts the production of APP carboxyl-terminal fragments in cell lysates and the appearance of APP in the medium, respectively. Analysis of the carboxyl-terminal fragments generated in cells expressing APPwt revealed the nearly concomitant generation of both 12- and 13.5-kDa fragments at 20 min into the chase period, species which are continually generated (or accumulate) over the next 25 min (Fig. 2B, left panel). We suggest that the 13.5-kDa fragment is the penultimate precursor of A and that the kinetics of appearance is consistent with previous studies which demonstrated A generation from plasma membrane-bound APP after endosomal recycling(23) . In parallel, APP first appear in the conditioned medium of cells expressing APPwt APP at 30 min into the chase period (Fig. 2C, left panel). Taken together with the kinetics of appearance of the 12-kDa carboxyl-terminal fragment, we argue that the APP species are produced by endoproteolytic cleavage by -secretase, an activity which occurs late in the secretory pathway, and predominantly on the plasma membrane (see below, Fig. 3). In striking contrast to the results obtained in cells expressing APPwt, cells expressing APPswe contain a prominent 13.5-kDa fragment which first appears within 10 min into the chase period (Fig. 2B, right panel). Notably, a 12-kDa carboxyl-terminal fragment is also present, albeit at somewhat lower levels, in cells expressing APPswe, and the kinetics of appearance of this species fragment is indistinguishable to that observed in cells expressing APPwt. Interestingly, APP first appear in the conditioned medium within 20 min of chase (Fig. 2B, right panel), a rate considerably accelerated relative to the secretion of APP species from cells expressing APPwt. Moreover, the soluble derivatives in medium of cells expressing APPswe clearly exhibit accelerated migration relative to APP. Our interpretation of this result is that these APP species which are produced early in the secretory pathway are liberated from specific intracellular sorting/retention signals contained within the core protein and hence rapidly transit through the secretory pathway prior to secretion. Thus, the early production of the 13.5-kDa carboxyl-terminal fragment and accelerated appearance of secreted APP supports our view that -secretase cleavage of APPswe is initiated early in the secretory pathway. Interestingly, we have failed to recover APP carboxyl-terminal fragment(s) that encompasses sequences between Ala (in APP-695) and the myc-epitope tag, a species which would be generated by the action of -secretase (data not shown). The reason for this discrepancy is unclear but suggests that -secretase-generated 56-59 amino acid fragment is subject to rapid degradation.

Figure 3: Cell surface biotinylation and release assay. Transiently transfected N2a cells were labeled with [S]methionine and cell surface biotinylated. One set of cells were incubated at 37 °C for 10 min, while the other set was kept at 4 °C. Conditioned medium and cell lysates were analyzed as described under ``Materials and Methods.'' Lanes 1 and 2 represent secreted APP molecules immunoprecipitated with mAb P2-1 from conditioned medium collected during the 3-h [S]methionine labeling period. Lanes 3-6 represent cell-surface biotinylated APP molecules immunoprecipitated with 369 antiserum from lysates of cells incubated at 37 °C for 0 or 10 min. Lanes 7-10 represent mAb 22C11 or 6E10 immunoprecipitates of biotinylated APP molecules secreted during 10 min incubation at 37 °C.

-Secretase Cleavage Does Not Occur on the Plasma Membrane

-Secretase Cleavage of APPswe Is Initiated in the Medial Golgi Compartment

Figure 4: Kinetics of -secretase cleavage of APPswe molecules. A, kinetic analysis of carboxyl-terminal APP fragments in Swe.24, an N2a line expressing APPswe. Parallel dishes of Swe.24 cells transiently transfected with plasmid encoding VSVG protein were pulse labeled for 5 min with [S]methionine and chased for the times indicated. Carboxyl-terminal fragments of APP were immunoprecipitated with 369 antiserum from cell lysates prepared at each time point. B, kinetics of acquisition of endoglycosidase H resistance by APP molecules. Full-length APP molecules were immunoprecipitated from cell lysates of selected time points with Myc-I antiserum. One-half of the sample was incubated with endoglycosidase H (Endo H). Arrowhead indicates the endoglycosidase H-resistant APP species. C, kinetics of maturation of VSVG. VSVG protein was immunoprecipitated from cell lysates with rabbit anti-VSV serum. Arrowhead indicates the mature 67-kDa species which is resistant to endoglycosidase H digestion (data not shown; 41).

DISCUSSION

A, the principal component of parenchymal amyloid deposits in Alzheimer's disease, is derived from integral membrane glycoproteins, APP. Although A is normally secreted by a variety of cultured cells, the molecular mechanisms involved in A production have not been fully clarified. The present report provides several insights into the cellular compartments involved in -secretase cleavage and the production of A. We have assessed the metabolism of a APP (APPswe) harboring a double mutation at codons 670 and 671 (of APP-770) in N2a cells. Using surface biotinylation and release approaches, we demonstrate that relative to the steady-state levels of surface-bound wild-type APP, the levels of APPswe are diminished. Moreover, surface-bound APPswe and APPwt molecules are released from the plasma membrane after cleavage by -secretase, but not by -secretase. Finally, using kinetic approaches, we provide compelling evidence that -secretase cleavage of APPswe occurs early in the secretory pathway and unequivocally demonstrate that a population of APPswe molecules are cleaved at the -secretase site within the Golgi apparatus, as early as the medial compartment. Moreover, and consistent with the cell surface labeling and release studies, cleavage of both the APPwt and APPswe substrates occurs several minutes later, concomitant with the appearance of soluble APP derivatives in the conditioned medium. The principal conclusions of this work are summarized in Fig. 5.

Figure 5: Model depicting sites of - and -secretase cleavage of APPwt and APPswe in N2a cells. The middle panel depicts selected cellular compartments of the central vacuolar pathway involved in APP trafficking/processing. For APPwt, the majority of secreted APP are generated by cleavage of full-length molecules by -secretase on the plasma membrane. Low levels of secreted A presumably occurs via endocytic recycling of plasma membrane-bound APP(23) . For APPswe, -secretase cleaves full-length membrane bound molecules in the medial Golgi and compartments proximal to the plasma membrane. Resulting soluble APP traffic through the secretory pathway and are released into the medium. As a consequence of early cleavage by -secretase, the levels of full-length APPswe that arrive on the plasma membrane is considerably diminished. Finally, the membrane-bound, -secretase-generated 100-amino acid carboxyl-terminal fragment is cleaved by -secretase at, or near the plasma membrane to release high levels of soluble A. Asterisks indicate the position of the Swedish mutation in APPswe; solid box, A domain.

Our finding that -secretase cleavage of APP harboring the Swedish mutations is initiated in the Golgi apparatus provides a conceptual framework for developing a model to explain the increase in A secretion by APPswe cells. We propose that the production of the membrane-bound 13.5-kDa A-containing carboxyl-terminal fragment early in the secretory pathway liberates the fragment from potential sorting/retention signals in the APP lumenal domain. Support for a role for APP lumenal sequences in intracellular trafficking has recently emerged from studies of soluble APP molecules expressed in MDCK cells (45, 46) . Thus, in sharp contrast to APP or APP with a deletion of the cytoplasmic sequence, YENPTY, in which only 20 or 60%, respectively, of newly synthesized molecules reach the cell surface and are secreted(9) , we suggest that the 13.5-kDa fragment is efficiently shunted through the secretory pathway. These molecules would subsequently encounter -secretase near, or at, the plasma membrane immediately prior to A release.

FOOTNOTES

*

This work was supported by National Institutes of Health Grants AG 05146 and NS 20471 from the United States Public Health Service, and by grants from the Adler Foundation and the Alzheimer's Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Recipient of an Alzheimer's Association Zenith Award. To whom correspondence should be addressed: The Johns Hopkins University School of Medicine, Neuropathology Laboratory, 558 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205-2196. Tel.: 410-955-5632; Fax: 410-955-9777.

()

The abbreviations used are: A, -amyloid; APP, amyloid precursor protein; APPwt, wild-type human APP-695 protein; APPswe, human APP-695 protein bearing the Swedish double mutation; APP, soluble APP derivatives; APP, -secretase cleaved soluble APP; APP, -secretase cleaved soluble APP; mAb, monoclonal antibody; MDCK, Madin-Darby canine kidney; N2a, mouse Neuro2a neuroblastoma cells; VSVG, vesicular stomatitis virus G protein; Tricine, N-[2-hydroxyl-1,1-bis(hydroxymethyl)ethyl]glycine.

()

S. Sisodia, T. Golde, and S. Younkin, unpublished observations.

ACKNOWLEDGEMENTS

Addendum-While our manuscript was under review, we learned of studies reported by C. Haass and colleagues (47) which concluded that in human embryonic kidney 293 cells, APPswe is cleaved in Golgi-derived vesicles, most likely within secretory vesicles.

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