©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Amyloids and Are Generated Intracellularly in Cultured Human Neurons and Their Secretion Increases with Maturation (*)

(Received for publication, September 21, 1995; and in revised form, February 5, 1996)

R. Scott Turner (1)(§) Nobuhiro Suzuki (3)(¶) Abraham S. C. Chyung (2) Steven G. Younkin (3)(**) Virginia M.-Y. Lee (2)(§§)

From the  (1)Departments of Neurology and (2)Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and (3)Division of Neuropathology, Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies have demonstrated the presence of amyloid beta (Abeta) in neurons (NT2N) derived from a human embryonal carcinoma cell line (NT2) by steady state metabolic radiolabeling and immunoprecipitation. We show here that Abeta is present intracellularly since trypsin digestion of intact NT2N cells at 4 °C did not eliminate the Abeta recovered in cell lysates. To determine whether both Abeta and Abeta are produced intracellularly, quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed using COOH-terminal end-specific anti-Abeta monoclonal antibodies. Sandwich ELISA detected intracellular Abeta and Abeta in NT2N cell lysates at a ratio of 3:1, whereas secreted Abeta and Abeta were recovered in medium conditioned by NT2N cells at a ratio of approximately 20:1. Metabolic steady state and pulse-chase labeling studies demonstrated a 2-h delay in the detection of cell-associated Abeta/Abeta in the medium, suggesting that Abeta is generated at a slow rate intracellularly prior to its secretion. Finally, as NT2N cells mature over time in culture, the secretion of Abeta and Abeta increases more than 5-fold over 7 weeks. This increase in the secretion of Abeta/Abeta in NT2N cells as a function of time may recapitulate a similar phenomenon in the aging brain.


INTRODUCTION

The amyloid beta (Abeta) (^1)peptide deposited as insoluble amyloid in plaques and vascular deposits is one of the most striking neuropathological features of the Alzheimer's disease (AD) brain, and these plaques may play a critical role in the pathogenesis of AD. Abeta, purified from AD blood vessels (1) and brain parenchyma (2) and sequenced as 39- to 43-amino acid peptides of 4 kDa. Abeta was shown to be an insoluble peptide with a beta-pleated sheet secondary conformation. The amyloid precursor protein (APP), a transmembrane glycoprotein, was subsequently cloned (3) and localized to human chromosome 21(4) . Alternative mRNA splicing generates primarily 695- (APP), 751- (APP), and 770- (APP) amino acid isoforms of APP(5, 6, 7) . Some kindred of early onset familial AD (8, 9, 10, 11, 12) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (13, 14, 15) are linked with APP missense mutations which localize within or adjacent to the Abeta sequence contained within the carboxyl-terminal region of APP. Thus, it is likely that these mutations influence APP processing and Abeta generation, at least in familial AD.

Several proteolytic pathways for APP metabolism have been described. An alpha-secretase cleaves APP within the Abeta region at or near the plasma membrane and thus precludes amyloid deposition(16, 17) . A beta-secretase cleaves APP at the amino terminus of Abeta and a -secretase cleaves APP at the carboxyl terminus of Abeta, releasing Abeta from APP that is either 40 amino acids (Abeta) or 42 amino acids (Abeta) long(18) . An endosomal/lysosomal pathway generates a number of intracellular carboxyl-terminal APP fragments, the larger of which contain the entire Abeta peptide sequence and thus are also potentially amyloidogenic(19, 20, 21, 22) . Abeta is found in conditioned medium from mixed brain-cell primary cultures and in normal cerebrospinal fluid(23, 24) , suggesting that it is produced and secreted constitutively. Abeta peptides are also secreted by a number of nontransfected and APP-transfected cell lines(22, 24, 25, 26) . Transfection of cells with the APP tandem mutation linked to early onset familial AD results in a 6-fold increase in the secretion of Abeta(27, 28) , and transfection of cells with the APP mutation increases the production of Abeta(29) . These observations support the hypothesis that overproduction of Abeta or increased production of Abeta leads to familial AD. However, the exact mechanism that leads to the production and secretion of Abeta and Abeta and the identity of the alpha-, beta-, and -secretase remains elusive. This is in part due to the fact that APP metabolism is cell type-specific, and overexpression of APP may affect its metabolism. For example, Abeta was detected associated with cell lysate as well as in the conditioned medium from cultured postmitotic CNS neuronal cells (NT2N) produced by the terminal differentiation of a teratocarcinoma cell line (NT2) with retinoic acid(26, 30) . By contrast, cell associated Abeta has not been demonstrated convincingly in the less differentiated NT2 cells, and many APP-transfected or nontransfected cell lines although Abeta has been recovered from the conditioned medium of a variety of cultured cells(22, 24, 25, 26) . However, it was recently reported that Abeta is found in a human neuroblastoma cell line (SY5Y) (31) .

To date, the cell types responsible for the production of Abeta that forms amyloid deposits in the AD brain are not known. Since Abeta deposits are found exclusively in the brain, and APP is found predominantly in neurons, it is reasonable to hypothesize that Abeta produced by human CNS neurons from APP is the likely source of beta amyloid in senile plaques. Since NT2N cells resemble human CNS neurons(32, 33) , express primarily the APP isoform and produce Abeta, they may provide a unique system to test this hypothesis. In the present study, we demonstrated that Abeta is indeed produced intracellularly before secretion. We also showed increase secretion of Abeta and Abeta in NT2N cells as a function of time in culture.


EXPERIMENTAL PROCEDURES

Cell Culture

NT2 cells derived from a human embryonal carcinoma cell line (Ntera2/cl.D1) were grown and passaged twice weekly in Opti-Mem (Life Technologies, Inc.) supplemented with 5% fetal bovine serum and penicillin/streptomycin as described previously(32, 33) . To begin differentiation, 2.5 times 10^6 cells were seeded in a 75-cm^2 flask and fed with Dulbecco's modified Eagle's medium (DMEM) HG (Life Technologies, Inc.) containing 10 µM retinoic acid, 10% fetal bovine serum, and penicillin/streptomycin twice weekly for 5 weeks. The cells were then replated (Replate 1 cells) at a lower density. After 2 days, cells were enzymatically and mechanically dislodged, counted, and replated at a density of 7.5 times 10^6 cells per 10-cm dishes (Replate 2 cells) on polylysine and Matrigel in medium containing mitotic inhibitors (1 µM cytosine arabinoside, 10 µM fluorodeoxyuridine, and 10 µM uridine). To obtain 99% pure neurons (Replate 3 cells), Replate 2 cells were plated at a density of 25 times 10^6 cells per 75-cm^2 flasks for about 1 week after which they were removed enzymatically and mechanically to generate nearly pure NT2N neurons. Cultures of Replate 2 or Replate 3 NT2N cells were used for experiments when they were 3-4 weeks old except for those used in the maturation studies.

Metabolic Labeling and Immunoprecipitation

Cultured NT2N cells were incubated in methionine-free DMEM (Life Technologies, Inc.) for 30 min before the addition of [S]methionine (100-400 µCi/ml) (ICN Radiochemicals) to the methionine-free DMEM. For pulse-labeling studies, NT2N cells were labeled with [S]methionine continuously for 0-24 h. For pulse-chase studies, cells were labeled with [S]methionine for 3 or 4 h, washed twice with methionine-containing DMEM, and chased in the same medium for 0-24 h. Cell lysates were prepared as described elsewhere(20) . Protein was determined by the bicinchoninic acid procedure (Pierce). Conditioned medium was centrifuged at 100,000 times g for 30 min at 4 °C, and the proteins in the supernatant were either immunoprecipitated with antibodies directly or they were precipitated with an equal volume of saturated ammonium sulfate, pH 7.5, at 4 °C for 2 h. After recentrifugation at 100,000 times g for 30 min at 4 °C, the pellet was dissolved in 1 ml of 1 times radioimmune precipitation buffer (20) followed by immunoprecipitation as described previously(26) . Abeta and APPs were separated either on 7.5% Laemmli SDS-PAGE gels or on 10/16.5% step gradient Tris-Tricine gels, whereas Abeta was separated either on 10/16.5% step-gradient or 16.5% Tris-Tricine gels(20) . Gels were stained with Coomassie Blue R (Pierce), treated with ENHANCE (DuPont NEN), dried, and placed on PhosphorImager plates (Molecular Dynamics) for 72 h. Quantitation of S-labeled proteins was performed using the ImageQuant software provided with the PhosphorImager. Gels were also analyzed by standard autoradiographic methods.

Trypsin Treatment of NT2N cells

NT2N cells were metabolically labeled with 0.5 mCi/ml [S]methionine for 16 h as described above. After rinsing twice in PBS, the NT2N cells were incubated on ice for 20 min either with PBS alone, or with 10 µg/ml of trypsin (Life Technologies, Inc.) in PBS in the presence or absence of 0.1% Triton X-100. The trypsin in the cultures was inactivated by the addition of 100 µg/ml soybean trypsin inhibitor. The treated NT2N cells were then washed with ice-cold PBS, scraped into cell lysis buffer, and processed for immunoprecipitation with 6E10, an anti-Abeta antibody, as described above.

Antibodies and Antisera for Immunoprecipitation

Antibodies used for immunoprecipitation included a goat polyclonal antisera (Karen) raised to APPs (a kind gift of Dr. Barry Greenberg), a monoclonal antibody (6E10) specific for Abeta1-17(34) , and polyclonal antisera SGY2134 and 2332 which bind most avidly to amino acids 1-17 of the Abeta peptide(24, 28) .

Sandwich ELISA

For immunodetection of Abeta in conditioned medium or cell lysates, a sandwich ELISA was performed as described previously(29) . In brief, Abeta in samples were captured with BAN-50, a mAb which is specific for the first 10 amino acid of Abeta. The presence of Abeta and Abeta in samples was detected specifically by horseradish peroxidase-conjugated BA-27, a mAb specific for Abeta and horseradish peroxidase-conjugated BC-05, a mAb specific for Abeta. This sandwich ELISA has a detection limit of <1 fmol/well. The monoclonal antibodies BAN50, BA-27, and BC-05 were prepared as described previously(29) .


RESULTS

Abeta Is Produced Intracellularly in NT2N Cells

To demonstrate that the Abeta recovered from NT2N cells is indeed produced intracellularly and not in association with the cell surface, we treated cultures of NT2N cells with trypsin at 4 °C. Under such conditions, cell surface-associated but not intracellular Abeta is expected to be destroyed. Fig. 1shows that a similar amount of Abeta was recovered from NT2N cells treated or untreated with trypsin (Fig. 1, compare lanes 1 and 2). By contrast, if the NT2N cells were treated with trypsin and Triton X-100, intracellular Abeta was completely eliminated (Fig. 1, lane 3). This experiment provides evidence that the Abeta found in NT2N cell lysates is produced intracellularly.


Figure 1: Abeta is produced intracellularly in NT2N cells. Culture dishes containing >99% NT2N cells (Replate 3) were metabolically labeled with 500 µCi/ml [S]methionine for 16 h. Cells were rinsed twice with PBS and incubated on ice for 20 min with PBS alone (lane 1), with 10 µg/ml trypsin (lane 2), or with 10 µg/ml trypsin and 0.1% Triton X-100 (lane 3). The cells were processed for immunoprecipitation and gel electrophoresis as described under ``Experimental Procedures.'' All lanes were immunoprecipitated with the anti-Abeta antibody 6E10.



Abeta and Abeta Are Both Present Intracellularly in NT2N

To assess whether or not Abeta and Abeta are present in NT2N cell lysates, we utilized a previously developed sandwich ELISA to quantify the amount of Abeta and Abeta. Using BAN-50/BA-27 and BAN-50/BC-05, we detected on average 18.3 fmol/mg Abeta and 7.4 fmol/mg Abeta, respectively, in 1-week-old cultures of NT2N cells (n = 6; Table 1). About a 30% increase in intracellular Abeta (33.5 fmol/mg) and Abeta (10.3 fmol/mg) was observed in NT2N cells that were about 6 weeks old. By contrast, neither Abeta nor Abeta was found in the less differentiated NT2 cells. These results confirm our previous finding that intracellular Abeta can be detected in NT2N but not NT2 cells.



Abeta Is Produced Intracellularly Prior to Its Secretion

To demonstrate that Abeta is synthesized intracellularly before secretion, we isolated intracellular Abeta and Abeta recovered in the cultured medium after continuous [S]methionine metabolic radiolabeling or pulse-labeling with [S]methionine followed by a cold chase in NT2N cells. For the continuous radiolabeling studies, the level of intracellular Abeta and APP as well as the secreted forms of APP fragments (APPs) and Abeta were determined after different lengths of time (up to 24 h) of labeling with [S]methionine. Intracellular full-length APP and secreted APPs were recovered by immunoprecipitation using a goat antisera to recombinant APP (Karen). Abeta was recovered from cell lysates and conditioned medium using either rabbit antisera raised to Abeta (rabbit SGY2134 or 2332) or a mAb that binds to an epitope within the first 17 amino acid of Abeta (6E10). Consistent with our previous study demonstrating that NT2N cells produce predominantly APP695 and a small amount of APP751 and APP770(26) , the immunoprecipitable APP protein bands in this study appeared as a single sharp band at 95 kDa and a more diffuse broader band at 100 kDa (Fig. 2A). The latter protein band probably represents glycosylated APP695 and the small amount of unmodified APP751 and APP770. The synthesis of APP is constant but the amount of radiolabeled APP recovered from cell lysates reached a steady state level by about 8-16 h (Fig. 2, A and C). Similarly, cell-associated Abeta recovered from NT2N cells also reached a steady state level of production by about 8-16 h (Fig. 2, A and C). By contrast, Abeta was detected in the medium only after a delay of almost 2 h (Fig. 2, B and D). Furthermore, the amount of Abeta recovered from the medium continued to increase beyond 8 h (Fig. 2B and 3B). These results support a temporal relationship between Abeta that is produced intracellularly and then secreted into the medium. A similar delay also was observed in the release of APPs into the medium (Fig. 2, B and D). However, the majority of the APPs secreted into the medium by NT2N cells appeared to be the result of alpha-secretase cleavage since antibody 92 (18) (specific to the beta-secretase-cleaved APP fragment) detected only a small amount of a slightly lower molecular mass (93 kDa) protein by immunoblots (data not shown). Thus, APP appears to be cleaved by both the alpha- and beta-secretases during metabolism in NT2N cells.


Figure 2: NT2N cell-associated Abeta precedes its recovery from conditioned medium. Replate 3 NT2N cells were metabolically labeled with 200 µCi/ml [S]methionine for 1, 3, 8, or 24 h. Proteins from cell lysates (A) or conditioned medium (B) were immunoprecipitated sequentially with Karen (for APP) or 6E10 (for Abeta). Radiolabeled immunoprecipitates were subsequently separated on 7.5% Laemmli SDS-PAGE gels (APP) or 16.5% Tris-Tricine gels (Abeta), stained, dried, and exposed to PhosphorImager plates (72 h) for quantitation and to autoradiographic film (14-21 days). Molecular mass markers are in kilodaltons. Panels C and D are quantitation of intracellular APP and Abeta, and secreted APPs and Abeta, respectively. Counts from five experiments for cell lysates (A) and 10 experiments for medium (B) were converted to percent of maximal counts at 24 h. Because results from experiments using a number of polyclonal anti-Abeta (e.g. 2332, SGY2134) antibodies in Replate 2 and Replate 3 NT2N cells were similar, the data were pooled for statistical analysis (mean ± standard error).



To demonstrate unequivocally that Abeta is produced intracellularly before secretion, the NT2N cells were pulsed with [S]methionine for 3 or 4 h and then chased for different lengths of time. Notably, it was necessary to pulse label the NT2N cells for a longer period of time, since at shorter labeling times, intracellular Abeta was at too low a level to be detected. Nevertheless, it became evident that the levels of intracellular APP and Abeta decreased with increasing chase times and that only a small amount of intracellular APP and Abeta was detectable after a 24 h chase time (Fig. 3, A and C). By contrast, the levels of Abeta and APPs secreted into the medium increased with increasing chase time (Fig. 3, B and D). These studies demonstrate definitively that Abeta is produced intracellularly before secretion.


Figure 3: Newly synthesized Abeta is rapidly secreted into the medium in NT2N cells. NT2N cells (Replate 3) were pulsed with 400 µCi/ml [S]methionine for 16 h and chased for 0, 1, 3, 8, and 24 h. Radiolabeled proteins from cell lysates (A) or conditioned medium (B) were immunoprecipitated sequentially with Karen (for APP) followed by 6E10 (for Abeta). Immunoprecipitated APP was separated on 7.5% Laemmli gels, and Abeta was separated on 16.5% Tris-Tricine gels which were exposed to PhosphorImager plates (72 h) or exposed to x-ray film for 2 weeks for quantitation. Molecular mass markers are in kilodaltons. Panels C and D are quantitation of experiments shown in Panels A and B. Counts from six experiments were converted to percent of maximal counts at 24 (A) or 8 (B) hours.



Abeta Levels in NT2N Cell-conditioned Medium Increase with Neuronal Maturation

Since Abeta and Abeta are produced intracellularly in NT2N cells before secretion, we sought to determine the levels of Abeta and Abeta secretion into the cultured medium in a 24-h period using a sandwich ELISA with Abeta carboxyl-terminal-specific antibodies. Table 1shows that, after 24 h, both Abeta and Abeta are secreted into the medium. However, we noted that much higher levels of Abeta were secreted into the medium. In fact, out of the total pool of Abeta/Abeta secreted by the NT2N cells, only about 5% is Abeta, which is in sharp contrast to the ratio of these Abeta peptides produced intracellularly in NT2N cells. Table 1shows that about 20-30% of the intracellular Abeta is Abeta, versus only 4-6% of the secreted Abeta is Abeta. This suggests that some of the intracellular Abeta is either degraded inside the cell or converted to Abeta or differentially modified. We next compared the levels of both Abeta and Abeta in samples of 24-h conditioned medium from NT2N cells of different ages. We also observed a steady increase in the secretion of both Abeta and Abeta from NT2N cells over time in culture (Fig. 4). A 5-7-fold increase in both Abeta and Abeta can be observed in cultures that are about 6-7 weeks old. A plateau also was reached by this time and a longer time in culture resulted in a slight decrease in the levels of Abeta and Abeta which probably was a consequence of cell death (data not shown). The ratio of secreted Abeta to Abeta remained constant with cellular maturation and Abeta representing 5.1 ± 1.2% (mean ± standard deviation, n = 92) of the total (Fig. 5). It is presently unclear whether this increase in Abeta peptides represents greater secretion or decreased clearance of Abeta with maturation. Increased Abeta secretion is unlikely to be due to cellular proliferation, since NT2N cells are postmitotic and the total protein per culture dish as well as the total amount of APP were relatively constant over time.


Figure 4: Soluble Abeta levels increase with NT2N cell maturation. Six ml of serum-free DMEM were conditioned for 24 h by 10-cm dishes of 7.5 times 10^6 NT2N cells (Replate 2). Medium was harvested weekly for 13 weeks, stored at -70 °C, and analyzed by a sandwich ELISA using BAN-50/BA-27 and BAN-50/BC-05 for specific quantitation of Abeta (A) or Abeta (B), respectively. The means ± standard error (n = 4-8 dishes) are indicated, and the data are representative of three separate experiments.




Figure 5: The proportion of secreted Abeta and Abeta remains constant with NT2N cell maturation. NT2N cell-conditioned medium was analyzed by ELISA as described in the legend to Fig. 4, and the proportions of Abeta and Abeta are expressed as a percent of total Abeta.




DISCUSSION

In this study, we show for the first time that both Abeta and Abeta are produced intracellularly in NT2N cells. Although cell-associated Abeta has been detected in NT2N cells and human neuroblastoma SY5Y cells(31) , no other cell line has been shown to produce detectable levels of intracellular Abeta and Abeta. The presence of Abeta in the neuron-like NT2N cells and its subsequent secretion suggests that human CNS neurons may contribute to the formation of amyloid plaques, since recent studies have shown that Abeta is the major Abeta species in amyloid plaque cores (35, 36) .

Several lines of evidence were presented here to demonstrate that both Abeta and Abeta are produced intracellularly prior to secretion. First, Abeta was recovered from NT2N cell lysates even after intact NT2N cells were treated with trypsin. Such treatment would eliminate cell surface associated Abeta but not intracellular Abeta. Indeed, the loss of Abeta following trypsin treatment of detergent permeabilized NT2N cells further confirmed the intracellular location of Abeta in NT2N cells. Second, the steady state metabolic labeling experiments showed that Abeta was detected intracellularly before it was recovered in the medium. In fact, there was a lag time of about 2 h before the detection of secreted radiolabeled Abeta and APPs from the medium. Third, the pulse-chase experiments showed that newly synthesized APP and freshly produced Abeta from cell lysates could be chased into the medium after a lag time of about one hour. Finally, our ELISA data showed that both Abeta and Abeta peptides were present at much higher levels in NT2N cell-conditioned medium than in NT2N cell lysate suggesting that Abeta and Abeta that were produced intracellularly eventually accumulated in the medium.

Previously we showed that, after a 15-min pulse, a 95-kDa band corresponding to APP695 was detected in the NT2N cells(26) . Here, we showed that prolonged labeling resulted in the appearance of diffused bands from about 95-110 kDa. This higher molecular mass material most likely represents glycosylated APP695, although the presence of small amounts of APP751 and APP770 cannot be ruled out. Further, prolonged pulse labeling was required for the detection of intracellular Abeta before chase since the level of intracellular Abeta was quite low even after a 3-4-h pulse. Thus, it was not possible to determine the exact time course of the reduction of the intracellular pool of Abeta even though we were able to monitor and quantitate the accumulation of secreted Abeta over time after pulse labeling.

Our data show that as much as 30% of the total Abeta produced intracellularly in the NT2N cells is Abeta. Currently, it is unclear whether or not Abeta and Abeta are produced in the same or different intracellular compartments, or whether intracellular Abeta is the precursor of intracellular Abeta. The detection of both Abeta and Abeta in the intracellular compartment of the NT2N cells will provide a useful system to dissect the pathways leading to the generation of these two forms of Abeta. Our observation that 30% of the total intracellular Abeta is Abeta while only about 5-6% of the total Abeta released into the medium is Abeta suggests a hitherto unanticipated complexity in Abeta metabolism. For example, some of the Abeta could be degraded intracellularly before secretion, thereby reducing the amount of Abeta recovered in the medium. Alternatively, multiple pathways could be involved in the production of secreted Abeta especially since the production of Abeta has been shown to involve both secretory and lysosomal pathways(16, 17, 18, 19, 20, 21, 22) . One such pathway involves the rapid reinternalization of membrane inserted APP into early endosomes followed by excision and release of the Abeta peptide(21) . Since intracellular Abeta has not been detected in cultured cells that undergo APP reinternalization, and since Abeta is the major Abeta species recovered from the medium(29) , we speculate that this secretory pathway may lead to the production of Abeta but not Abeta. Thus, by selectively increasing the production of secreted Abeta, the amount of Abeta recovered in the medium would increase. This increase would alter the ratio of Abeta and Abeta such that Abeta to Abeta would be higher in the medium than in the cell lysate. We also noted a 5-7-fold increase in Abeta and Abeta secretion as the NT2N cells matured in culture. This increase in Abeta and Abeta secretion was not due to a concomitant increase in APP synthesis or intracellular Abeta and Abeta since the amount of intracellular Abeta and Abeta is increased by only about 30% even after 6 weeks in culture. Thus, it appears that APP processing is altered as NT2N cells age in culture such that the production of secreted Abeta and Abeta is dramatically increased. It is unclear at the present time whether this is due to increased beta-secretase or reduced alpha-secretase activities. Future studies will directly address this important issue since the aging of NT2N cells in culture may recapitulate key aspects of the aging brain by increasing the production of Abeta peptides leading to amyloid deposition. Finally, our ability to detect intracellular Abeta and Abeta suggests that the NT2N cells may be a unique system to identify the beta- and -secretases that produce Abeta and Abeta in neurons.


FOOTNOTES

*
Supported by National Institutes of Health NIA Grants AG-09215 (to V. M.-Y. L.) and AG06656 (to S. G. Y.). 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.

§
A Howard Hughes Medical Institute Physician Postdoctoral Fellow. Current address: VAMC GRECC and Dept. of Neurology, University of Michigan Medical Center, Ann Arbor, MI 48109.

Current address: Discovery Research Division, Takeda Chemical Industries, Ltd., Tsukuba, Japan.

**
Current address: Mayo Clinic, Jacksonville, FL 32224.

§§
To whom correspondence and reprint requests should be addressed: Dept. of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Maloney Building, Rm. A009, 36th and Spruce Sts., Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909.

(^1)
The abbreviations used are: Abeta, amyloid beta; AD, Alzheimer's disease; APP, amyloid precursor protein; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We thank Drs. D. Cooke, R. Doms, T. Golde, and J. Q. Trojanowski for critical review of the manuscript. We also thank C. D. Page for some of the cultured cells used in this study, and Dr. L. Otvos for synthetic Abeta peptides used for generation of antisera.


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