©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intracellular Accumulation of -Amyloid in Cells Expressing the Swedish Mutant Amyloid Precursor Protein (*)

(Received for publication, September 20, 1995)

Bronwyn L. Martin (1) Gesine Schrader-Fischer (2) Jorge Busciglio (1) Maraid Duke (2) Paolo Paganetti (2) Bruce A. Yankner (1)(§)

From the  (1)Department of Neurology, Harvard Medical School and The Children's Hospital, Boston, Massachusetts 02115 and (2)Sandoz Research Institute, CH-3001 Berne, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

beta-Amyloid (betaA) is a normal metabolic product of the amyloid precursor protein (APP) that accumulates in senile plaques in Alzheimer's disease. Cells that express the Swedish mutant APP (Sw-APP) associated with early onset Alzheimer's disease overproduce betaA. In this report, we show that expression of Sw-APP gives rise to cell-associated betaA, which is not detected in cells that express wild-type APP. Cell-associated betaA is rapidly generated, is trypsin-resistant, and is not derived from betaA uptake, indicating that it is generated from intracellular processing of Sw-APP. Intracellular and secreted betaA are produced with different kinetics. The generation of intracellular betaA is partially resistant to monensin and a 20 °C temperature block but is completely inhibited by brefeldin A, suggesting that it occurs in the Golgi complex. Monensin, brefeldin A, and a 20 °C temperature block almost completely inhibit betaA secretion without causing increased cellular retention of betaA, suggesting that secreted betaA is generated in a post-Golgi compartment. These results suggest that the metabolism of Sw-APP gives rise to intracellular and secreted forms of betaA through distinct processing pathways. Pathological conditions may therefore alter both the level and sites of accumulation of betaA. It remains to be determined whether the intracellular form of betaA plays a role in the formation of amyloid plaques.


INTRODUCTION

The deposition of beta-amyloid (betaA) (^1)in senile plaques is a pathological hallmark of Alzheimer's disease (Glenner and Wong, 1984; Masters et al., 1985). betaA is a normal product of the metabolism of amyloid precursor protein (APP) in the secretory pathway (Haass et al., 1992; Seubert et al., 1992; Shoji et al., 1992; Busciglio et al., 1993). Mutations in APP that are linked to autosomal dominant inheritance of Alzheimer's disease have been found to alter the production of betaA. Mutation of the two amino acids proximal to the N terminus of betaA has been described in individuals from two Swedish families that develop early onset Alzheimer's disease (Mullan et al., 1992). Expression of APP containing the Swedish mutation (Sw-APP) in transfected cells increases the production of betaA by about 5-fold, suggesting a causal link between altered APP processing and the development of Alzheimer's disease (Citron et al., 1992; Cai et al., 1993). In this report, we show that the Swedish APP mutation not only increases betaA production but also results in abnormal intracellular accumulation of betaA. The processing pathways that give rise to intracellular and secreted betaA can be distinguished by their differential kinetics and sensitivities to metabolic inhibitors and temperature block.


EXPERIMENTAL PROCEDURES

Metabolic Labeling and Immunoprecipitation

COS-1 cells maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) were transiently transfected with expressor plasmids encoding APP, APP, and APP containing the Swedish mutation (KM changed to NL immediately N-terminal to the betaA sequence, designated Sw-APP) (Mullan et al., 1992). HEK 293 cells stably transfected with the APP cDNA containing the Swedish mutation were maintained in DMEM with 10% FCS and 0.25 mg/ml G418. Transfected COS cells were metabolically labeled with 125 µCi/ml [S]methionine, and immunoprecipitations were performed as described previously (Busciglio et al., 1993). For immunoprecipitation of cell-associated betaA, three semi-confluent 10-cm plates of transfected COS cells were lysed and combined, and the supernatant of the 100,000 times g centrifugation was immunoprecipitated. APP was immunoprecipitated with a polyclonal antibody to the 20 C-terminal residues of APP (RG117, 1:200) and resolved by 10% Tris-glycine SDS-polyacrylamide gel electrophoresis. betaA was immunoprecipitated with a polyclonal antibody to synthetic beta1-40 (1:200 in culture medium and 1:30 in cell lysates) and resolved by 10-20% Tris-Tricine SDS-polyacrylamide gel electrophoresis. Gel loading was normalized to the protein content of the cell lysate (Bio-Rad protein assay), and quantitation was performed by PhosphorImager scanning. The specificity of these antibodies and the protocol for peptide preabsorption have been described previously (Busciglio et al., 1993). For pulse-chase analysis, stably transfected HEK 293 cells were labeled with 100 µCi/ml [S]cysteine/methionine in cysteine/methionine-free DMEM with 2% dialyzed FCS for 20 min and then chased in DMEM with 2% FCS and 1 mM cysteine/methionine. betaA was immunoprecipitated with 5 µg of purified mouse monoclonal antibody beta1 to residues 1-40 of betaA (Paganetti and Scheller, 1994). Quantitation of pulse-chase results was performed by direct counting of the gels using an InstantImager (Canberra Packard). Values were normalized for the number of cysteine and methionine residues in the different APP metabolites.

Treatments

The 4 °C temperature block was performed by pulse labeling of transfected cells for 10 min at 37 °C followed by a 90-min chase in unlabeled DMEM at either 4 or 25 °C. Trypsinization of cell surface betaA was performed by labeling transfected cells for 16 h followed by addition of 0.25% trypsin, 0.02% EDTA for 10-20 min at 37 °C. The reaction was stopped by 10-fold dilution in cold phosphate-buffered saline containing 5 µg/ml phenylmethylsulfonyl fluoride. The cells were centrifuged at 500 times g and washed with phosphate-buffered saline, and the cell pellet was lysed and immunoprecipitated. Brefeldin A and monensin were preincubated with transfected COS cells in methionine-free medium for 60 min followed by a 2-h labeling period in the presence of the drugs. The 20 °C temperature block was performed by preincubation in methionine-free DMEM for 1 h at 20 °C followed by labeling with 150 µCi/ml [S]methionine for 3 h at 20 °C using a 20 °C water bath in a 4 °C cold room.


RESULTS

COS cells were transiently transfected with wild-type APP and APP and Sw-APP cDNAs and metabolically labeled followed by immunoprecipitation of betaA and APP from the culture medium and cell lysate. Cells expressing Sw-APP secreted about 10-fold higher levels of the 4-kDa betaA peptide than cells expressing wild-type APP when normalized for APP (Fig. 1A), consistent with previous reports (Citron et al., 1992; Cai et al., 1993). Immunoprecipitation of the detergent-soluble lysate of cells expressing Sw-APP revealed the presence of a labeled 4-kDa peptide, which was not detected in cells expressing wild-type APP and APP or in mock-transfected cells (Fig. 1B). The 4-kDa peptide immunoreactivity was abolished by preabsorption of the betaA antibody with synthetic beta1-40 peptide (Fig. 1D) and did not appear after immunoprecipitation with an antibody to the APP C-terminal domain. When cells were incubated at 4 °C, the generation of cell-associated betaA was completely inhibited (Fig. 1C), suggesting that cell-associated betaA is produced by active APP processing and not as an artifact of proteolysis during the immunoprecipitation protocol. Cell-associated betaA was recovered in the supernatant after high speed centrifugation of the detergent-extracted lysate suggesting that it is soluble (data not shown). These results suggest that the cell-associated 4-kDa peptide is antigenically and physically similar to the secreted 4-kDa betaA peptide. The low level of cell-associated betaA precluded sequence analysis of the N terminus.


Figure 1: Accumulation of cell-associated betaA in cells expressing Sw-APP. A, secreted betaA; B, cell-associated betaA. Transfected COS cells expressing APP, APP, or Sw-APP and mock-transfected cells were metabolically labeled with [S]methionine for 16 h. The culture medium and cell lysates were immunoprecipitated for betaA as described under ``Experimental Procedures.'' C, temperature dependence of intracellular betaA generation. Sw-APP-expressing cells were pulse-labeled at 37 °C for 10 min and then chased for 90 min at either 4 or 25 °C. Incubation at 4 °C completely prevents generation of intracellular (Cell) and secreted (Sec) betaA. D, trypsin resistance of cell-associated betaA. Sw-APP-expressing cells were untreated (Cntrl) or incubated with trypsin for 10 or 20 min at 37 °C, and the cell lysates were immunoprecipitated with the beta1-40 antibody. Preabsorption of the antibody with synthetic beta1-40 peptide (Preab) abolishes immunoreactivity. E, cell-associated betaA in Sw-APP-expressing cells is not due to betaA uptake from the medium. The first three panels show cell-associated betaA in Sw-APP transfectants (Cntrl) and in non-transfected COS cells incubated for 6 or 24 h with medium containing radiolabeled betaA. The last two panels show betaA remaining in the conditioned medium after 6- and 24-h incubations with non-transfected COS cells. Radiolabeled betaA is derived from the medium of metabolically labeled Sw-APP transfectants.



To determine whether cell-associated betaA in cells expressing Sw-APP is intracellular or associated with the cell surface, cells were treated with trypsin prior to lysis and immunoprecipitation. The level of cell-associated betaA was only slightly decreased by trypsinization (Fig. 1D), suggesting that it is mostly intracellular. In some labeling experiments, the 3-kDa peptide was detected in a cell-associated form. In contrast to the 4-kDa peptide, the cell-associated 3-kDa peptide was trypsin-sensitive (Fig. 1D), suggesting that it is mostly associated with the plasma membrane. We then determined whether intracellular betaA could be due to uptake of betaA from the medium. Conditioned medium from Sw-APP-expressing cells containing high levels of radiolabeled betaA was incubated for 6 and 24 h with non-transfected COS cells. The level of labeled betaA in the medium was only slightly decreased after this incubation period (Fig. 1E). A low level of radiolabeled betaA was detected in association with nontransfected cells after incubation with radiolabeled betaA but did not exceed 20% of the level of cell-associated betaA in Sw-APP-transfected cells (Fig. 1E). Thus, most of the intracellular betaA produced from Sw-APP is not derived by uptake of betaA from the medium. These results suggest that cell-associated betaA is intracellular and is predominantly generated from intracellular processing of Sw-APP.

The kinetics of production of intracellular and secreted betaA were examined by pulse-chase labeling of HEK 293 cells stably transfected with Sw-APP. Intracellular and secreted betaA both appeared by 30 min of chase time (Fig. 2, A and B). Intracellular betaA continued to accumulate after 45- and 60-min chase times and then declined by 120 min. In contrast, betaA in the medium continued to accumulate up to 120-min chase time (Fig. 2, A and B). Intracellular betaA reached a maximum of about 15% of secreted betaA at 60-min chase time. We also examined the time course of appearance of the 11.5- and 9-kDa C-terminal fragments of APP, which are metabolic intermediates in the processing of APP to betaA and the 3-kDa peptide, respectively (Busciglio et al., 1993; Haass et al., 1993; Higaki et al., 1995). The 11.5-kDa fragment appeared by 15-min chase time, which slightly preceded the appearance of intracellular and secreted betaA (Fig. 2, A and B). In contrast, the alpha-secretase-generated 9-kDa C-terminal fragment accumulated more slowly, reaching a maximal level after 90-min chase time (data not shown). The differential kinetics of production of intracellular and secreted betaA suggest that they are processed differently and provide further evidence that intracellular betaA is not a contaminant of extracellular betaA or a product of betaA endocytosis.


Figure 2: Pulse-chase kinetic analysis of Sw-APP metabolism to secreted and intracellular betaA and C-terminal APP fragments. A, intracellular (IC) and secreted (SEC) 4-kDa betaA and the 11.5-kDa C-terminal APP fragment (CTD) after 20 min of pulse labeling followed by chase in unlabeled medium for the indicated times. Shown are fluorographs of 10% Tris-Tricine (IC) or 13% Tris-Tricine (SEC) SDS gels. B, quantitative pulse-chase analysis of the appearance of intracellular (bullet) and secreted betaA (circle) and the 11.5-kDa C-terminal fragment (). Values are the mean ± S.E.; n = 3. A and B are from separate experiments.



To determine the sites of generation of intracellular betaA from Sw-APP, we examined the effects of inhibitors of processing and transport in the secretory pathway. Pretreatment of cells with brefeldin A causes resorption of the proximal Golgi into the endoplasmic reticulum and inhibits anterograde transport and maturation of APP in the secretory pathway (Caporaso et al., 1992; Gabuzda et al., 1994). Brefeldin A completely inhibited the production of both intracellular and secreted betaA from Sw-APP, suggesting that these processing steps are unlikely to occur in the endoplasmic reticulum or proximal Golgi (Fig. 3A). We also examined the effects of the ionophore monensin, which inhibits the maturation of newly synthesized proteins at the trans-Golgi and their transport past the trans-Golgi network (Tartakoff, 1983). Incubation with 7.5 µM monensin almost completely inhibited betaA secretion in both Sw-APP and wild-type APP-expressing cells but only partially inhibited the generation of intracellular betaA in cells expressing Sw-APP (Fig. 3A). PhosphorImager analysis showed that monensin inhibited betaA secretion by 99 ± 0.2% but inhibited the generation of intracellular betaA by only 49 ± 5%. A monensin dose-response analysis showed that betaA secretion was almost completely inhibited by 2.5 µM monensin, whereas intracellular betaA production was only partially inhibited by 2.5 µM monensin, and no further inhibition was evident up to 10 µM monensin (Fig. 3B). Cell-associated APP increased in monensin-treated cells, as previously reported (Gabuzda et al., 1994). The differential sensitivity of intracellular betaA to monensin and brefeldin A is consistent with a site of generation in the trans-Golgi. In contrast, the inhibition of betaA secretion by monensin, without increased cellular retention of betaA, is consistent with the generation of secreted betaA in a post-Golgi compartment.


Figure 3: Effects of monensin, brefeldin A, and a 20 °C temperature block on the generation of intracellular and secreted betaA. A, PhosphorImager scans of intracellular betaA (IC) and secreted betaA (SEC) in COS cells expressing wild-type or Swedish mutant APP under control conditions (CNTL) and after treatment with 3 µg/ml brefeldin A (BFA) or 7.5 µM monensin (MON). B, monensin dose response of intracellular and secreted betaA. Note that monensin almost completely inhibits secretion of betaA but only partially inhibits generation of intracellular betaA. Values are expressed as percent of IC betaA in the absence of monensin (control) and represent the mean ± S.E. of three independent experiments. C, incubation at 20 °C almost completely inhibits generation of secreted betaA but only partially inhibits generation of intracellular betaA. Values are molar ratios of betaA:APP normalized to the 37 °C value and represent the mean ± S.E., n = 3. *, p < 0.01 relative to control by ANOVA.



To further assess the site of intracellular betaA generation, we examined the effects of a 20 °C temperature block, which results in protein retention in the trans-Golgi (Matlin and Simons, 1983). APP synthesis occurred at 20 °C but was significantly reduced (data not shown). Hence, determinations of betaA were normalized for the level of APP. The 20 °C temperature block almost completely inhibited betaA secretion but only partially inhibited the generation of intracellular betaA (Fig. 3C). The level of intracellular betaA generated at 20 °C was similar to that observed in the presence of monensin (Fig. 3, B and C). These results provide additional evidence that intracellular and secreted betaA are generated at distinct sites in Golgi and post-Golgi compartments, respectively.


DISCUSSION

These experiments suggest that APP harboring the Swedish mutation is processed to betaA at an early step in the secretory pathway giving rise to a stable intracellular pool of betaA. Hence, the Swedish mutation results in both increased secretion and intracellular accumulation of betaA. Although intracellular betaA is a small fraction of the total betaA produced, it may nevertheless play a potentially important pathogenic role in plaque formation. The appearance of betaA in a cell-associated form has also been observed in a neuronal cell line (Wertkin et al., 1993). Although we have not observed intracellular betaA in transfected cells that overexpress wild-type APP, we cannot exclude the possibility that there is a small pool that is below the limits of resolution. Nevertheless, our results demonstrate that the Swedish mutant APP gives rise to significantly increased accumulation of intracellular betaA.

Several lines of evidence suggest that the intracellular and secreted forms of betaA arise through distinct processing pathways. First, the kinetics of generation of intracellular and secreted betaA are different. Second, the generation of intracellular betaA is inhibited by brefeldin A but is partially resistant to monensin and a 20 °C temperature block, suggesting that it occurs in the Golgi complex, most likely the trans-Golgi. In contrast, both monensin and a 20 °C temperature almost completely inhibit the secretion of betaA without increasing cellular retention of betaA. Thus, secreted betaA is generated in a post-Golgi compartment, which is distinct from the Golgi site of generation of intracellular betaA. These findings are consistent with previous reports, which suggest that secreted betaA is produced in a post-Golgi compartment (Busciglio et al., 1993; Haass et al., 1993; Higaki et al., 1995; Yamazaki et al., 1995), and are also consistent with the recent observation that the cellular site of beta-secretase cleavage is altered in MDCK cells expressing Sw-APP (Lo et al., 1994).

The effects of the Swedish mutation on APP metabolism suggest that inherited mutations alter not only the level but also the sites of accumulation of betaA. Increased betaA production has also been demonstrated to result from aberrant APPs produced by deletion mutations, incorrect APP isoform expression, and APP overexpression (Zhong et al., 1994). Furthermore, we have previously demonstrated that inhibition of energy metabolism markedly increases the generation and intracellular accumulation of potentially amyloidogenic C-terminal fragments of APP (Gabuzda et al., 1994). Thus, a variety of genetic and non-genetic pathological situations can alter the processing of APP and affect both the level and cellular sites of accumulation of betaA. It remains to be determined whether intracellular accumulation of betaA predisposes to betaA aggregation and plays a role in the formation of amyloid plaques.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AG09229 and NS30352, the Alzheimer's Association, and Sandoz Pharma LTD. 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.

§
To whom correspondence should be addressed: Dept. of Neurology, Enders 260, The Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-7220; Fax: 617-738-1542.

(^1)
The abbreviations used are: betaA, beta-amyloid; APP, amyloid precursor protein; Sw-APP, Swedish mutant amyloid precursor protein; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.