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Phorbol Esters Affect Multiple Steps in β-Amyloid Precursor Protein Trafficking and Amyloid β-Protein Production
Molecular Medicine volume 3, pages 204–211 (1997)
Amyloid β-protein (Aβ), the major constituent of amyloid deposits found in Alzheimer’s disease, is derived from the β-amyloid precursor protein (βPP). Constitutive proteolysis by α-secretase and secretion of soluble βPP (βPPs) are stimulated by protein kinase C (PKC) activation, whereas Aβ production and release are inhibited. The cellular mechanism that underlies the PKC-mediated down-regulation of Aβ generation is unclear. Because endocytic processing of βPP from the cell surface is a major pathway of Aβ production, the effect of PKC activation by phorbol 12,13-dibutyrate (PDBu) on endocytic trafficking of βPP was examined.
Materials and Methods
In this study, trafficking of βPP was assayed in Chinese hamster ovary cells (CHO) cells stably transfected with full-length βPP751.
Treatment with PDBu resulted in a rapid and striking reduction of up to 80% in the amount of βPP at the cell surface. This loss of cell-surface molecules could not be accounted for by changes in the trafficking of cell-surface βPP molecules, as determined by a radiolabeled antibody assay. Rather, the decrease in βPP was due primarily to a reduction in the sorting of βPP to the cell surface. This alteration was correlated with accelerated intracellular α-secretase-mediated βPP cleavage and accelerated βPP trafficking in the exocytic pathway.
The data suggest that the displacement of βPP away from the cell surface after phorbol ester treatment reduces the substrate available for endocytic processing and, in turn, results in the inhibition of Aβ production.
Alzheimer’s disease is characterized by the deposition of amyloid β-protein (Aβ) in parenchyma and blood vessel walls and the intracellular accumulation of neurofibrillary tangles. Aβ is derived by proteolysis of the larger β-amyloid precursor protein (βPP), a 100–140 kDa integral membrane protein. Processing of βPP in the constitutive pathway results in the secretion of a large N-terminal soluble product (βPPs) and a membrane-retained C-terminal fragment of approximately 10 kDa (1). Release of the N-terminal ectodomain of βPP results from cleavage by “α-secretase,” an as yet unidentified protease that cleaves βPP within the Aβ sequence (2). Thus, formation of a full-length, 40–43 residue Aβ peptide is precluded by α-secretase proteolysis. Both intracellular and cell-surface βPP molecules are substrates for α-secretase; indeed, accumulating evidence suggests proteolysis of βPP occurs in both cellular compartments (3).
Generation and release of Aβ from βPP occurs constitutively after two proteolytic events, designated “β- and γ-secretase” cleavages at the Aβ N- and C-termini, respectively. In addition to βPPs, Aβ and a 3-kDa fragment (p3) are normally produced and released from a variety of cells both in vivo and in vitro (4). The precise cellular compartments in which Aβ is generated are unclear, although processing in an acidic compartment appears to be required. Both secretory (5) and endocytic (6) pathways have been shown to contribute to Aβ production; however, the major source of Aβ derived from wild-type βPP in cultured cells appears to be the endocytic pathway (6,7).
Activation of protein kinase C (PKC) by a variety of agents stimulates α-secretase-mediated βPPs secretion (8). This alteration, however, does not require direct phosphorylation of βPP, which suggests an indirect mode of action of PKC on regulating βPP cleavage (9,10). Acceleration of βPP metabolism through the α-secretory pathway by PKC activation results in a corresponding decrease in β-secretory processing and Aβ production (11,12). The cellular and molecular basis of the PKC-mediated reduction of Aβ production remains to be clearly defined. In view of our results indicating a major role of the endocytic pathway in Aβ production, this study was designed to determine how processing of βPP in this pathway is affected by PKC activation. To date, cell-surface receptors have shown heterogeneous and unpredictable responses to phorbol esters with respect to trafficking in the receptor-mediated pathway (13). The results showed that PKC activation by phorbol esters leads to a rapid and major reduction in βPP sorting to the cell surface. In turn, this loss in cell-surface βPP results in a corresponding reduction in available substrate for endocytic processing and Aβ production.
Materials and Methods
Cell Culture and Metabolic Labeling
Chinese hamster ovary (CHO) cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. Stably transfected CHO cell line expressing wild-type βPP751 has been established previously (6). In pulse-chase experiments, confluent βPP-transfected CHO cells were labeled with 35S-methionine for 10 min and chased for 20 min or 2 hr with or without 1 µM phorbol-12,13-dibutyrate (PDBu). This labeling paradigm examines only the effect of PDBu on post-translational events. Immunoprecipitations of Aβ (media) and full-length βPP (cell lysates) were carried out with 1282, an antibody recognizing Aβ, and a C-terminal antibody, CT15, respectively (14,15) (Fig. 1). βPPs from media and saponin buffer (see below) were immunoprecipitated by antibodies B5 (16) and 1736 (12), which recognize the extracellular domain of βPP or α-secretase cleaved βPPs, respectively (Fig. 1).
To detect intracellularly cleaved βPPs, CHO cells pulse-labeled for 10 min were quickly chilled with ice-cold Dulbecco’s phosphate buffered saline (DPBS) after a 10- or 20-min chase period. The cells were then incubated at 4°C for 40 min with 0.1% saponin in DPBS supplemented with protease inhibitors as described (17). Saponin was used because this mild detergent permeabilizes cells without solubilizing cell membranes, thereby allowing soluble intracellular molecules to diffuse out of the cells. Immunoprecipitations using antibodies B5 and 1736 were then carried out from the saponin buffer supplemented with 10% calf serum, to recover βPPs released from the permeabilized cells. In surface iodination experiments, transfected CHO cells were radioiodinated with Na125I as described previously (18). After labeling, the cells were chased with normal CHO medium, with or without PDBU, for 2 hr. Aβ was then immunoprecipitated from the chase media with antibody 1282. The immunoprecipitated material was separated by SDS-PAGE (6% tris-glycine or 16.5% tris-tricine gels for βPPs or Aβ, respectively). Dried gels were either quantitated by densitometry after fluorographic enhancement or exposed directly to phosphorimager screen. All labeling experiments were repeated two to four times.
Kinetics of Surface βPP Trafficking
To determine the kinetics of βPP secretion and internalization from the cell surface, a method that quantitatively assesses both the release of βPPs into media and the internalization of βPP from the cell surface using radiolabeled 1G7 monoclonal antibody was performed as described (19). 1G7 is a monoclonal antibody that recognizes the extracellular domain of βPP within the midregion and was added at a concentration of ≈7 nM to confluent CHO cells in 12-well tissue culture plates. In brief, after incubation at 4°C, the cells were either lysed immediately (time 0) or placed in prewarmed (37°C) medium with or without PDBu. After 5, 10, 30, and 60 min, the media were collected and cells rapidly chilled with ice-cold DPBS at pH 2.8. After an additional 5-min wash with acidic buffer to detach residual surface-bound antibody, the cells were lysed in 0.2M NaOH. Radioactivity was then determined from the resultant three fractions: medium, acid wash, and acid resistant lysate, which represent secreted, cell-surface, and intracellular βPP pools, respectively. TCA precipitable counts were used from media and lysate samples. Specific binding from all the antibody-binding experiments was calculated by subtracting the radioactivity from untransfected CHO cells performed in parallel. The results were then expressed in the three fractions at each time point as a percentage of the total radioactivity obtained at time 0. All experiments were performed in triplicate and the results are expressed as average (± SEM) of three repetitions.
Three methods were used to determine the amount of βPP on the cell surface of transfected CHO cells. In the first approach, radioiodinated 1G7 antibody was added to confluent CHO cells precooled to 4°C as described (19). In all experiments, parallel sets of transfected and untransfected CHO cells were pretreated with PDBu for 0, 5, 10, or 30 min prior to antibody binding. In the second approach, full-length βPP was immunoprecipitated with CT15 antibody immediately after surface radioiodination from control cells or from cells treated with 1 µM PDBu for 15 min before labeling. In the third approach, the arrival of βPP to the cell surface from newly synthesized molecules was assayed by pulse labeling transfected CHO cells for 10 min, followed by chase periods of 10 and 20 min with or without PDBu. After rapid cooling in ice-cold DPBS, surface βPP molecules were recoved by incubating the cells with 5A3/1G7 monoclonal antibodies for 1 hr at 4°C. 5A3/1G7, which recognize nonoverlapping epitopes in the extracellular domain of βPP (6), were used together to obtain higher signal. The cells were then lysed in the presence of 2-fold excess cold unlabeled transfected CHO cells. Antibody-bound cell-surface βPP was then precipitated by incubating the lysates with precleared anti-mouse agarose beads (1). Immunoprecipitated material was fractionated by SDS-PAGE and visualized by autoradiography or phosphorimaging.
βPP Trafficking after PDBu Treatment
To examine whether PKC activation by PDBu affects the release or internalization of βPP from the cell surface, the trafficking of βPP molecules was assayed by radioiodinated 1G7 antibody binding. In CHO cells expressing wild-type βPP, cell-surface βPP molecules are normally rapidly secreted or internalized (19). Approximately 30% of cell surface molecules are eventually secreted into the medium (Fig. 2A). When PDBu is added at the beginning of the chase period, βPP secretion approximately doubled at each time point of the assay (Fig. 2A). This increase in βPP release correlated with a decrease in βPP internalization of ∼30% (Fig. 2B). The characteristically short residence time of βPP on the cell surface was not altered by PDBu treatment (Fig. 2C).
Release of Aβ after PDBu Treatment
Because Aβ can be derived from cell-surface βPP processed in the endocytic pathway, one would predict that the changes in βPP internalization demonstrated above should result in a reduction in Aβ release from surface precursors. Indeed, by using selective cell-surface iodination, addition of PDBu at the beginning of the chase period decreased recovery of labeled Aβ from medium by ∼30% (Fig. 3). The magnitude of this decrease is consistent with the reduction of βPP internalization after PKC activation (Fig. 2b). However, by metabolic labeling with 35S-methionine, Aβ release was inhibited by ∼80% (Fig. 3), a result comparable to that reported by others (3). Therefore, when the production of Aβ is measured from total cellular βPP labeled by 35S-methionine, PDBu inhibition of Aβ release is substantially greater than when only the cell-surface βPP pool is examined by selective surface radioiodination.
Cell Surface βPP after PDBu Treatment
The above results suggest that the effect of PDBu on trafficking of cell-surface βPP molecules alone is insufficient to account for the overall decrease in Aβ production. Therefore, the amount of βPP on the cell surface after PDBu treatment was examined. As detected by surface radioiodination and immunoprecipitation, the level of cell-surface βPP was markedly decreased 15 min after the addition of PDBu (Fig. 4A). To more accurately determine the time-dependent changes in surface βPP expression, transfected CHO cells were pretreated with PDBu from 5 to 30 min and then assayed using radioiodinated monoclonal antibody 1G7 binding. Surprisingly, an 80% decline in cell-surface βPP was seen within 5 min of adding PDBu and remained at approximately the same level up to 30 min following treatment (Fig. 4B). Since changes in endocytic trafficking are minor (Fig. 2), in the range of 30%, this reduction in cell-surface βPP must be due to other factors, in particular, to diminished sorting of molecules to the cell surface.
Further evidence for this mechanism was provided by assaying for the arrival of βPP to the cell surface from newly synthesized molecules. Using this approach, the levels of cell-surface βPP derived from newly synthesized molecules were dramatically decreased after PDBu treatment (Fig. 4C). Specifically, treatment with PDBu for 10 to 20 min after the initial pulse labeling period resulted in ∼80–90% reduction in the amount of labeled cell-surface βPP. This experimental paradigm was such that the labeled molecules must have been newly synthesized and recently transported to the cell surface, in contrast to earlier experiments (Fig. 2, 4A and B) that examined the fate of the steady-state pool of βPP already present at the cell surface when PDBu treatment was instituted. Thus, the latter pool is the summation of the decrease in nascent βPP arriving to the cell surface, as well as to the increase in βPPs being released from the cell surface.
Intracellular Cleavage of βPP
To confirm the above postulate that targeting of βPP to the cell surface in the secretory/exocytic pathway is altered, intracellular βPPs was immunoprecipitated from saponin-treated CHO cells after treatment with PDBu using a pulse-chase paradigm. Treatment with low concentrations of saponin (0.1%) permeabilized but did not solubilize cell membranes because in control experiments, full-length βPP was virtually undetectable from the saponin buffer (Fig. 5A). Therefore, predominantly soluble nonmembrane-bound βPP molecules, i.e., βPPs, were analyzed in this assay. As expected, secretion of βPPs into the medium was markedly increased by PDBu (Fig. 5B). The levels of βPPs immunoprecipitated by both B5 and 1736 antibodies were increased by 2- to 3-fold, which is consistent with the generation of increased amounts of α-secretase cleaved species. Concomitantly, as determined by saponin permeabilization, the amount of intracellular βPPs detected by both B5 and 1736 immunoprecipitations was markedly decreased, by ∼70%. This finding suggests that there was a redistribution of the intracellular pool of βPPs molecules into the medium, in other words, an acceleration of both α-secretory cleavage and release of βPPs, bypassing the cell surface.
The data presented above provide compelling evidence that PKC activation by phorbol esters results in major and rapid alterations in βPP trafficking in the endocytic pathway. In particular, an 80–90% decrease in the targeting of βPP molecules to the cell surface was seen within minutes of PDBu treatment. Surprisingly, the reduction in the amount of cell-surface βPP after PDBu treatment was caused by a major decrement in sorting of βPP to the cell surface and only secondarily from an increase in secretion of cell-surface molecules. This loss of cell-surface βPP molecules derived from the exocytic pathway would lead to a corresponding decrease in substrate available for subsequent endocytic processing. Thus, the effect of phorbol esters on trafficking of βPP molecules at the cell surface appears to be relatively minor. Finally, the data showed that the perturbations of βPP sorting to the cell surface are caused principally by an increase in intracellular α-secretase cleavage and an increase in the trafficking of βPPs out of the cell, and to a much lesser degree, from increased α-secretase activity at the cell surface.
The results also showed that PKC activation by PDBu alters a number of steps in Aβ generation. For wild-type βPP, where the endocytic pathway is hypothesized to be the major contributor to Aβ production, the net decrease in trafficking of βPP to cell surface is likely to underlie the inhibition of Aβ production by markedly reducing the substrate available for endocytic processing. Therefore, reduced internalization of cell-surface βPP only plays a minor role in this decrement in Aβ release. Moreover, the results are consistent with the hypothesis that processing of βPP in the endocytic pathway is a major route for Aβ production and release. In the “Swedish” APP double missense mutation, where Aβ production appears to be shifted to the secretory pathway, similar mechanisms occurring within the secretory pathway may underlie the reduction in Aβ production after treatment by phorbol esters (20).
The data presented here are consistent with the report that upon PKC activation, βPP is redistributed from the trans Golgi network (TGN) to other cellular locations through increased formation of secretory vesicles from the TGN (21). The results reported here complement this recent finding in demonstrating a large displacement of βPP away from the cell surface and an increase in the trafficking of βPP out of the cell. However, the data also demonstrated that the acceleration in α-secretase cleavage that accompanies this perturbation in βPP sorting is only minimally increased at the plasma membrane. Therefore, α-secretase activity enhanced by PKC activation must occur in an intracellular compartment that remains to be defined (22). Two potential mechanisms mediating this PKC effect come to mind: a direct PKC-mediated activation of intracellular α-secretase activity, or a displacement of βPP to the compartment where α-secretase is located.
Finally, in addition to phorbol esters, a number of other agents have been shown to both increase βPPs secretion and decrease Aβ production (8). These include treatment by cytokines, muscarinic activation, and inhibition of protein phosphatases. In view of the multiple effects of PKC activation by PDBu on βPP trafficking and processing, one would hypothesize that the other agents will show a range of cellular alterations affecting βPP and Aβ production and release. Understanding the multitude of cellular perturbations that alter βPP trafficking should generate additional potential targets for inhibiting Aβ generation.
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I thank Sharon Squazzo and Leslie Caromile for invaluable technical assistance, Drs. Christian Haass, Albert Hung, Sam Sisodia, and David Teplow for helpful discussions, and Drs. Dale Schenk and Dennis Selkoe for use of various antisera. This work was supported by grants from the Alzheimer’s Association (ZEN 94-011) and the National Institutes of Health (AG12376 and NS01812).
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Koo, E.H., Greengard, P. Phorbol Esters Affect Multiple Steps in β-Amyloid Precursor Protein Trafficking and Amyloid β-Protein Production. Mol Med 3, 204–211 (1997). https://doi.org/10.1007/BF03401673
- Phorbol 12,13-dibutyrate (PDBu)
- Endocytic Process
- Constitutive Proteolysis
- Endocytic Trafficking
- Exocytic Pathway