Secretion and Intracellular Generation of Truncated Abeta in beta -Site Amyloid-beta Precursor Protein-cleaving Enzyme Expressing Human Neurons*

Edward B. Lee, Daniel M. Skovronsky, Farhad Abtahian, Robert W. DomsDagger §, and Virginia M.-Y. Lee

From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, and Dagger  Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received for publication, October 2, 2002, and in revised form, December 4, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insoluble pools of the amyloid-beta peptide (Abeta ) in brains of Alzheimer's disease patients exhibit considerable N- and C-terminal heterogeneity. Mounting evidence suggests that both C-terminal extensions and N-terminal truncations help precipitate amyloid plaque formation. Although mechanisms underlying the increased generation of C-terminally extended peptides have been extensively studied, relatively little is known about the cellular mechanisms underlying production of N-terminally truncated Abeta . Thus, we used human NT2N neurons to investigate the production of Abeta 11-40/42 from amyloid-beta precursor protein (APP) by beta -site APP-cleaving enzyme (BACE). When comparing undifferentiated human embryonal carcinoma NT2- cells and differentiated NT2N neurons, the secretion of sAPP and Abeta correlated with BACE expression. To study the effects of BACE expression on endogenous APP metabolism in human cells, we overexpressed BACE in undifferentiated NT2- cells and NT2N neurons. Whereas NT2N neurons produced both full-length and truncated Abeta as a result of normal processing of endogenous APP, BACE overexpression increased the secretion of Abeta 1-40/42 and Abeta 11-40/42 in both NT2- cells and NT2N neurons. Furthermore, BACE overexpression resulted in increased intracellular Abeta 1-40/42 and Abeta 11-40/42. Therefore, we conclude that Abeta 11-40/42 is generated prior to deposition in senile plaques and that N-terminally truncated Abeta peptides may contribute to the downstream effects of amyloid accumulation in Alzheimer's disease.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease (AD)1 is characterized by the accumulation of aggregated Abeta peptides in senile plaques and vascular deposits. Abeta has classically been described as a 4-kDa peptide, derived from proteolytic processing of APP, varying in length from 40 to 42 amino acids due to C-terminal heterogeneity. The generation of the longer Abeta 1-42 peptide is specifically increased by several mutations linked to familial Alzheimer's disease (1). Furthermore, Abeta 1-42 is more fibrillogenic than Abeta 1-40 in vitro (2), consistent with the finding that both diffuse and senile plaques are composed of primarily Abeta peptides that terminate at position 42 (3). However, several N-terminal truncations are also found in Abeta peptides derived from AD brains, demonstrated as early as the first biochemical isolation of Abeta peptides from senile plaques (4). The relative importance of N-terminally truncated Abeta peptides in the pathogenesis of AD is unknown. Interestingly, some individuals with sporadic AD (5), familial AD (5, 6), and Down's syndrome (7) preferentially accumulate N-terminally truncated Abeta species. Also, overexpression of APP harboring disease-associated mutations within the Abeta domain (i.e. A692G and E693G) results in increased secretion of Abeta 11-40/42 (8, 9).

Whereas some truncations may be due to the partial degradation of full-length Abeta after secretion of peptides into the extracellular milieu, Abeta peptides beginning with glutamine at position 11 are derived from the membrane-bound beta -site APP-cleaving enzyme (BACE) (10-12). To generate full-length Abeta , BACE cleaves APP between the methionine and aspartate at position 1 (Asp-1) of the N terminus of Abeta (beta -cleavage), resulting in the secretion of a large N-terminal ectodomain, sAPPbeta , and the retention of a 99-amino acid C-terminal fragment, C99. To generate N-terminally truncated Abeta , BACE cleaves APP between tyrosine and glutamate at position 11 (Glu-11) within the Abeta domain (beta '-cleavage), resulting in the secretion of a slightly larger N-terminal ectodomain, sAPPbeta ', and the retention of an 89-amino acid C-terminal fragment, C89. C89 can also be produced by proteolysis of C99 by BACE at Glu-11 (11). Alternatively, APP may also be cleaved at position 16 by alpha -secretase (13, 14), resulting in secretion of the N-terminal sAPPalpha along with the retention of an 83-amino acid C-terminal fragment, C83. Membrane-bound C-terminal fragments are subjected to further proteolysis within the transmembrane domain by gamma -secretase, with cleavage typically occurring at either position 40 or 42 within the Abeta region. Whereas all of the components of gamma -secretase have not been identified, the presenilin proteins are necessary for secretion of Abeta peptides and have been postulated to contain the active site of gamma -secretase (1). However, multiple gamma -secretases may exist as presenilin is not needed for Abeta 1-42 production early in the secretory pathway (15).

Untransfected non-neuronal cells and murine cells are not well suited toward the study of BACE-derived N-terminally truncated Abeta . Many non-neuronal cells preferentially use the alpha -secretase pathway at the expense of beta -cleavage of APP (13, 14), although the secretion of full-length and N-terminally truncated Abeta can be increased upon overexpression of either APP or BACE (11, 12, 16, 17). Furthermore, due to low BACE activity and low APP expression, intracellular Abeta is difficult to detect in non-neuronal cells (16, 18). In contrast, neuronal cells cleave a larger proportion of APP by BACE (19-21), although overexpression of human APP in rodent cells does not lead to the generation of Abeta 11-40/42 from human APP (22), consistent with evidence that beta '-cleavage is species-specific (23). Furthermore, rodent neuronal cells preferentially cleave endogenous APP at position 11, whereas the presence of beta '-cleavage in human neuronal cells has not been adequately addressed. Therefore, rodent cell culture models may not accurately reflect the proteolytic processing of APP in human neurons.

NT2N neurons are a post-mitotic, terminally differentiated human neuronal cell culture model derived from retinoic acid treatment of the human embryonal carcinoma cell line NTera2/c1.D1 (NT2-) (24-27). Their high APP expression and beta -secretase activity make them amenable to biochemical analysis of both neuronal specific and human-specific characteristics of APP processing (18, 21). We have shown that NT2N neurons generate Abeta 1-40/42 intracellularly prior to secretion and that endogenous secretion of Abeta 1-40/42 from NT2N neurons increases with age in culture (19, 20). Furthermore, a detergent-insoluble pool of intracellular Abeta accumulates with time in NT2N neurons (28). In this study, we found that sAPP and Abeta secretion increase upon neuronal differentiation of NT2N neurons, correlating with BACE expression. Given a recent report (29) that BACE protein expression and activity are increased in AD, we sought to refine further our understanding of APP metabolism in NT2N neurons by examining the effect of exogenous expression of BACE in undifferentiated NT2- cells and differentiated NT2N neurons. We found that the increase in Abeta production due to BACE overexpression was more pronounced in NT2N neurons than in non-neuronal NT2- cells. Furthermore, we found that Abeta 11-40/42 is produced endogenously by NT2N neurons and that BACE overexpression increases the secretion of both full-length and truncated Abeta peptides. We further demonstrate that Abeta 11-40/42 is generated intracellularly, indicating that Abeta 11-40/42 is produced prior to deposition in senile plaques. The effect of BACE expression on the generation of both full-length and N-terminally truncated Abeta underscores the role of BACE activity in the generation of Abeta peptides and indicates that N-terminally truncated Abeta peptides may contribute to the pathogenesis of AD.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Undifferentiated NTera2/c2.D1 cells were maintained as described previously (26, 27) in Opti-MEM (Invitrogen) containing 5% fetal bovine serum (FBS), 100 units/ml penicillin, and 10 µg/ml streptomycin sulfate. Cells were differentiated by twice weekly 10 µM retinoic acid treatments for 5 weeks and then replated (replate 2 neurons) in DMEM with high glucose, 5% FBS, and mitotic inhibitors (10 µM uridine, 10 µM 5-fluoro-2'deoxyuridine, 1 µM cytosine arabinoside, Sigma) to obtain nearly pure NT2N neurons (26). Greater than 99% pure neurons (replate 3 neurons) (27) were isolated by mechanical separation of neurons after brief trypsinization of mixed cultures and replated into 6-well plates coated with Matrigel and poly-D-lysine.

Western Blot Analysis-- Cell lysates were collected in RIPA buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 5 mM EDTA in TBS, pH 8.0) in the presence of protease inhibitors (1 µg/ml each of pepstatin A, leupeptin, L-1-tosylamido-2-phenylethyl chloromethyl ketone, 1-chloro-3-tosylamido-7-amino-2-heptanone, soybean trypsin inhibitor, and 0.5 mM phenylmethylsulfonyl fluoride) and briefly sonicated. Protease inhibitors were also added to conditioned media samples. Samples were centrifuged at 100,000 × g for 20 min at 4 °C, electrophoresed on 7.5% Tris-glycine acrylamide gels, and transferred to nitrocellulose. When indicated, samples were immunoprecipitated with Karen, a goat polyclonal antibody raised against sAPP, prior to electrophoresis. APP and total sAPP were probed with Karen. sAPPalpha and sAPPbeta ' were probed with Ban50, a mouse monoclonal antibody recognizing Abeta residues 1-10, or with NAB228, a mouse monoclonal antibody recognizing Abeta residues 1-11.2 sAPPbeta was specifically probed with C5A4/2, a rabbit polyclonal antibody raised against a synthetic peptide (CSEVKM) corresponding to the C terminus of sAPPbeta (11). sAPPbeta ' was specifically probed with C10A4, a rabbit polyclonal antibody raised against a synthetic peptide (CHDSGY) corresponding to the C terminus of sAPPbeta '. The specificity of C5A4 and C10A4 was determined by their lack of immunoreactivity with full-length APP or C-terminal APP fragments, and by blocking experiments in which only peptides with the corresponding free C terminus are able to block immunoreactivity (data not shown). Immunoblots were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences) after application of species-specific horseradish peroxidase-conjugated anti-IgG antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). However, for quantification of APP and sAPP, Karen immunoblots were labeled with 125I-protein A (PerkinElmer Life Sciences) after application of a rabbit anti-goat IgG linker. Radiolabeled APP and sAPP were quantified using PhosphorImager analysis (Amersham Biosciences). For detection of BACE, crude membrane fractions were prepared by first collecting cells in hypotonic buffer (10 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 7, protease inhibitors) followed by centrifugation at 100,000 × g for 20 min at 4 °C. After an additional wash with hypotonic buffer, membrane proteins were extracted by sonication of the pellet in 1 M NaCl, 40 mM Tris, 4 mM EDTA, protease inhibitors, 0.5% Triton X-100, pH 7. The fraction was cleared by another round of centrifugation, and the BACE-containing supernatants were electrophoresed as above and immunoblotted with a rabbit polyclonal anti-BACE (CT) antibody (ProSci, Poway, CA).

Metabolic Labeling and Immunoprecipitation-- Cells were incubated in methionine-free DMEM (Invitrogen) for 30 min, labeled with [35S]methionine (250 µCi/ml in methionine-free DMEM supplemented with 5% dialyzed FBS; PerkinElmer Life Sciences) for 90 min, and chased for 1 h. Cells were treated with 10 µM PMA in Me2SO (Sigma) during the chase period and/or with 10 µM TAPI in Me2SO (Peptides International, Louisville, KY) 30 min prior to and throughout the chase period. Protease inhibitors were added to conditioned media, and sAPP was immunoprecipitated with Ban50 prior to electrophoresis on 7.5% Tris-glycine acrylamide gels. For C-terminal fragment analysis, cells were labeled for 2 h with [35S]methionine in the presence of 200 µM MG132 (Peptides International), rinsed, and lysed in 1000 µl of RIPA buffer containing protease inhibitors for immunoprecipitation. Lysates were briefly sonicated, and both lysates and media were cleared by centrifugation at 100,000 × g for 20 min at 4 °C. C-terminal fragments were immunoprecipitated with 2493, a rabbit polyclonal antibody recognizing the C-terminal region of APP, and resolved on 10/16.5% step gradient Tris-Tricine gels. Gels were fixed in 50% methanol, 5% glycerol, dried, and exposed to PhosphorImager plates for visualization. Finally, to detect both secreted and intracellular Abeta , two 10-cm dishes of replate 2 NT2N neurons were infected with recombinant Semliki Forest virus encoding wild type APP695, prepared as described previously (18, 30). Cells were infected in serum-free medium for 1 h, cultured in complete growth medium for 14 h, and then labeled with [35S]methionine (500 µCi/ml) for 8 h. Conditioned media and RIPA cell lysates were collected, cleared by centrifugation, and immunoprecipitated with 4G8 (Senetek, Maryland Heights, MO) prior to electrophoresis on 10/16.5% step gradient Tris-Tricine gels.

Northern Analysis-- Total RNA was extracted from NT2- cells and NT2N neurons with the Trizol Reagent (Invitrogen) as per manufacturer's protocol. RNA concentrations were determined by optical density readings, and equal amounts of RNA were separated on 1% agarose-formaldehyde gels. RNA was transferred to a nitrocellulose membrane (Amersham Biosciences) and hybridized with a radiolabeled probe generated by either PstI digestion or AccI-HincII double digestion of a BACE cDNA. The blot was washed and exposed to a PhosphorImager plate for visualization. Glyceraldehyde-3-phosphate dehydrogenase levels were obtained by using a glyceraldehyde-3-phosphate dehydrogenase probe purchased from Ambion (Austin, TX). 28 S and 18 S ribosomes were visualized by staining a duplicate gel with ethidium bromide.

Stable Transduction of NT2- Cells-- NT2- cells were stably transduced with a vesicular stomatitis virus surface glycoprotein (VSV-G) pseudotyped self-inactivating lentiviral vector.3 To generate the virus, QBI 293A cells were plated on poly-D-lysine-coated 10-cm dishes. Cells were transfected with pMD.G (containing the VSV-G envelope glycoprotein), pCMVDelta R8.2 (containing viral structural, enzymatic, and accessory genes), and SIN-EFp-GFP/SIN-EFp-BACE (containing minimal human immunodeficiency virus-based viral sequences, the elongation factor 1-alpha promoter, and either a GFP or BACE cDNA) using standard CaPO4 techniques. Media conditioned with viral particles were harvested over 3 days, centrifuged at 1000 rpm for 5 min, and passaged through a 0.45-µm filter to remove any cellular debris. Viral supernatants were added to NT2- cells, and transduced NT2- cultures were subcloned by limited dilution into 96-well plates. Uniform expression was verified either by direct fluorescence for GFP-expressing cells or indirect immunofluorescence with BaceN1, a rabbit polyclonal antibody raised against the N terminus of BACE (32), for BACE-expressing cells.

Immunoprecipitation/Mass Spectrometry-- Media conditioned for 10 days were collected in the presence of protease inhibitors and cleared by centrifugation at 100,000 × g for 20 min at 4 °C. Media were immunoprecipitated with 4G8, and immunoprecipitated material was eluted with a saturated solution of alpha -cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid, 50% acetonitrile. Data were collected on an ABI/Perspective (Framingham, MA) Voyager DE-PRO MALDI-TOF instrument in the positive-ion mode at the Protein Microchemistry/Mass Spectrometry Facility of the Wistar Institute (Philadelphia). Samples were spotted to a 100-well plate using alpha -cyano-4-cinnamic acid matrix (Sigma) at 10 mg/ml. Reflector mode with the accelerating potential at 20 kV was used. External calibration was performed on all samples.

Sandwich ELISA Analysis-- Secreted Abeta was detected using ELISA protocols described previously (20, 33). Briefly, Ban50 (anti-Abeta 1-10) or BNT77 (anti-Abeta 11-28) were used as capturing antibodies. After application of culture media samples, horseradish peroxidase-conjugated BA-27 and BC-05 were used to report Abeta species ending at position 40 and 42, respectively. For quantification of Abeta levels, synthetic Abeta 1-40 and Abeta 1-42 purchased from Bachem Bioscience Inc. (King of Prussia, PA) were serially diluted in unconditioned cell culture media to generate standard curves. Monoclonal antibodies, Ban50, BNT77, BA-27, and BC-05 were prepared as described previously (33-35). To detect intracellular Abeta , cells were washed thoroughly with phosphate-buffered saline and scraped into RIPA buffer containing protease inhibitors. Lysates were sonicated and spun at 100,000 × g for 20 min at 4 °C. Abeta from RIPA lysates was captured with either JRF/cAbeta 40/10 or JRF/cAbeta 42/26, monoclonal antibodies specific for Abeta 40 and Abeta 42, respectively (36), supplied by Dr. M. Mercken (Janssen Research Foundation, Beerse, Belgium). To detect full-length Abeta , captured peptides were reported with horseradish peroxidase-conjugated JRF/Abeta N/25, a monoclonal antibody directed against Abeta 1-7. To detect full-length and N-terminally truncated Abeta , captured peptides were reported with horseradish peroxidase-conjugated m266, a monoclonal antibody recognizing residues 13-28 of Abeta (37).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

sAPP and Abeta Production Correlates with Neuronal Differentiation-- APP processing differs between non-neuronal and neuronal cells, underscoring the importance of using neuronal systems to study APP metabolism and Abeta generation (18-20, 28). Prior to studying BACE activity and beta '-cleavage in NT2N neurons, we quantified the differences in the expression and proteolytic processing of APP between NT2- cells and NT2N neurons. Whereas both NT2- cells and NT2N neurons express high levels of APP, the two cell types express different isoforms of APP (Fig. 1A). We have shown previously (19) that NT2- cells predominantly express the 751- and 770-amino acid isoforms of APP (APP751/770) that appear as a doublet corresponding to immature (~110 kDa) and mature N- and O-glycosylated APP (~125 kDa). NT2N neurons predominantly express the shorter 695-amino acid isoform of APP (APP695), the majority of which is immature APP695 (~95 kDa) with relatively less mature APP695 (~110 kDa). Despite the difference in isoform expression, densitometric quantification indicated that when normalized for total protein content, NT2- cells expressed APP at 98% ± 5 (S.E.) compared with NT2N neurons. However, despite equivalent total APP expression, we found that proteolytic processing of APP was more efficient in NT2N neurons compared with NT2- cells. To measure total sAPP, derived from both alpha - and beta -secretase cleavage of APP, media conditioned by NT2- cells or NT2N neurons for 24 h were immunoprecipitated with a polyclonal antibody raised against the N-terminal domain of APP. We found that NT2N neurons secreted more total sAPP relative to NT2- cells (Fig. 1B). Densitometric quantification of total sAPP indicated that NT2N neurons secreted over 5-fold more sAPP than NT2- cells (Fig. 1C). Differentiation had an even larger effect on Abeta secretion, as NT2N neurons secreted over 20-fold more Abeta than NT2- cells (Fig. 1D), determined by sandwich ELISA for Abeta 1-40. Because differences in APP expression cannot explain the increase in Abeta secretion, differences in either beta - or gamma -secretase activity are likely to be responsible for the more efficient proteolysis of APP in NT2N neurons.


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Fig. 1.   Dissociation between APP expression and sAPP/Abeta secretion. A, protein-corrected RIPA lysates from NT2- cells and replate 3 NT2N neurons were separated on a 7.5% Tris-glycine gel and immunoblotted with Karen for full-length intracellular APP. The bands represent immature APP751/770 (*, left lane), mature APP751/770 (arrowhead, left lane), immature APP695 (*, right lane). and mature APP695 (arrowhead, right lane). B, after normalization to intracellular full-length APP expression levels, total sAPP was purified from media conditioned from either NT2- cells or replate 3 NT2N neurons for 24 h by immunoprecipitation with Karen, separated on a 7.5% Tris-glycine gel, and immunoblotted with Karen. C, total sAPP was quantified by Karen immunoblots of conditioned media, analyzed by PhosphorImager, corrected for intracellular APP expression, and shown as mean values relative to NT2- cells ± S.E. One-way analysis of variance revealed p < 0.0001; post hoc analysis showed p < 0.01 (*) compared with NT2- cells. D, Abeta 1-40 levels were quantified by Ban50/BA-27 ELISA, normalized to intracellular APP expression levels, and shown as mean values relative to NT2- cells ± S.E. One-way analysis of variance revealed p value of 0.0015. Post hoc analysis showed p < 0.01 (*) compared with NT2- cells.

TAPI-insensitive sAPP Production in NT2N Neurons-- The increased production of sAPP secretion by NT2N neurons indicated that BACE and/or alpha -secretase activity might be elevated in NT2N neurons upon neuronal differentiation of the NT2- cells. To distinguish between these possibilities, we first addressed the presence of alpha -secretase by testing the pharmacologic responses of NT2- cells and NT2N neurons upon alpha -secretase inhibition. alpha -Secretase has been attributed to members of a family of proteases that contain a disintegrin and a metalloprotease domain (ADAM), including ADAM10 and tumor necrosis factor-alpha converting enzyme. alpha -Cleavage is enhanced by phorbol ester-induced stimulation of tumor necrosis factor-alpha converting enzyme via protein kinase C and can be inhibited by metalloprotease inhibitors. Therefore, we treated metabolically labeled NT2- cells and NT2N neurons with either phorbol 12-myristate 13-acetate (PMA) or the specific metalloprotease inhibitor, TAPI. sAPP from media samples was immunoprecipitated using Ban50, a monoclonal antibody that recognizes the first 10 amino acids of Abeta , thereby recognizing both sAPPalpha and sAPPbeta '. In NT2- cells, PMA treatment increased sAPP secretion by 2.15 ± 0.29-fold, whereas TAPI treatment inhibited sAPP production to 0.54 ± 0.04-fold of untreated NT2- cells (Fig. 2, A and B). The magnitude of sAPP inhibition in NT2- cells was comparable with that reported for other non-neuronal cells (38). Thus we concluded that alpha -secretase activity is present in NT2- cells, and a large proportion of the base-line sAPP secreted by NT2- cells is derived from alpha -secretase. Whereas NT2N neurons also exhibited PMA-induced up-regulation of alpha -secretase activity (1.80 ± 0.37), TAPI had no effect on sAPP production (0.95 ± 0.14; Fig. 2, A and B). Furthermore, in both NT2- cells and NT2N neurons, TAPI was able to prevent PMA-induced sAPPalpha production, demonstrating that TAPI is able to inhibit PMA-induced alpha -secretase in both non-neuronal and neuronal cells. However, the inability of TAPI to decrease sAPP generation by NT2N neurons below base-line levels indicated that a relatively small proportion of APP is normally proteolyzed by alpha -secretase in NT2N neurons.


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Fig. 2.   TAPI-insensitive secretion of sAPP in NT2N neurons. A, NT2- cells and replate 3 NT2N neurons were metabolically labeled for 90 min and chased for 60 min. Cells were treated with 10 µM PMA during the chase period and/or 10 µM TAPI 30 min prior to and throughout the chase period. Conditioned media were immunoprecipitated with Ban50 and separated on 7.5% Tris-glycine gels. A representative gel out of three separate experiments is shown. B, sAPPalpha /sAPPbeta ' levels were quantified by PhosphorImager and shown as mean values ± S.E. normalized to control, untreated cells. C, total sAPP was immunoprecipitated with Karen from media conditioned for 24 h by NT2- cells and replate 3 NT2N neurons, corrected for cell lysate concentration or intracellular APP expression. Samples were then separated on a 7.5% Tris-glycine gel and immunoblotted with Ban50 (top panel), NAB228 (2nd panel), C5A4/2 (3rd panel), or C10A4 (bottom panel). The specific sAPP fragments recognized by these antibodies are labeled.

Because Ban50 recognizes both sAPPalpha and sAPPbeta ', the ability of Ban50 to immunoprecipitate TAPI-insensitive sAPP from NT2N neurons suggested that NT2N neurons may produce sAPPbeta ' endogenously. Therefore, to demonstrate more directly the presence of BACE-derived sAPP fragments, we used a panel of antibodies that recognize different sAPP species to analyze media conditioned for 24 h by NT2- cells and NT2N neurons. To better compare the relative abundance of sAPP species, media samples were corrected for either lysate protein concentration or intracellular full-length APP levels prior to analysis. Despite preferential utilization of alpha -cleavage over beta -cleavage in non-neuronal cells (13, 14), we detected less sAPP with Ban50 from NT2- cells compared with NT2N neurons (Fig. 2C, top panel), consistent with the possibility that NT2N neurons produce sAPPbeta ' endogenously. This result was confirmed with a second monoclonal antibody, NAB228, that recognizes the first 11 amino acids of Abeta (Fig. 2C, 2nd panel). C5A4/2, a polyclonal antibody that specifically recognizes the C terminus of sAPPbeta , showed the presence of sAPPbeta in media conditioned by NT2N neurons (Fig. 2C, 3rd panel). In contrast, the amount of sAPPbeta in media conditioned by NT2- cells was undetectable. Finally, a polyclonal antibody that specifically recognizes the C terminus of sAPPbeta ' demonstrated the presence of endogenous sAPPbeta ' produced by NT2N neurons (Fig. 2C, bottom panel). Therefore, not only is alpha -secretase activity relatively low in NT2N neurons, as determined pharmacologically, but BACE cleavage at both Asp-1 and Glu-11 is readily detected as a product of normal APP metabolism from NT2N neurons.

BACE Expression in NT2- Cells and NT2N Neurons-- To better understand the secretion and intracellular generation of BACE-derived Abeta peptides in human neurons, we characterized BACE expression in NT2- cells and NT2N neurons. Expression of BACE mRNA was determined by Northern analysis of NT2- cells and NT2N neurons. BACE mRNA expression was clearly present in NT2N neurons, as seen by the presence of 7.0-, 4.4-, and 2.6-kb bands (Fig. 3A). NT2- cells, however, showed markedly less BACE expression, most notably demonstrated by the absence of a 7.0-kb band. The 4.4-kb band, possibly representing one of the several identified BACE splice variants (39, 40), was somewhat reduced in NT2- cells relative to NT2N neurons. Two different radiolabeled BACE cDNA fragment probes yielded the same results (Fig. 3A and data not shown).


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Fig. 3.   BACE expression in NT2- cells and NT2N neurons. A, mRNA from NT2- cells and replate 3 NT2N neurons were electrophoresed on formaldehyde-agarose gels and hybridized with a radiolabeled PstI BACE cDNA fragment and visualized by PhosphorImager. Equal mRNA loading was determined by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and by 28 S and 18 S ribosome levels. B, crude membrane fractions from stably transduced NT2- cells and replate 2 NT2N neurons expressing BACE (NT2-/NT2B30-/NT2B17-, undifferentiated cells; NT2N/NT2B30N/NT2B17N, neurons) were separated on a 7.5% Tris-glycine gel and immunoblotted for BACE. C, total sAPP from media conditioned for 24 h from NT2- cells or replate 3 NT2N neurons was immunoprecipitated with Karen, electrophoresed on a 7.5% Tris-glycine gel, and immunoblotted for sAPPbeta with C5A4/2, showing sAPPbeta derived from either APP751/770 or APP695. D, C-terminal APP fragments were immunoprecipitated with 2493 from NT2- cells and replate 2 NT2N neurons that were metabolically labeled for 2 h, separated on a 10/16.5% discontinuous gradient Tris-Tricine gel, and exposed to a PhosphorImager screen. The bands corresponding to C99, C89, and C83 are labeled.

In agreement with the Northern analysis, BACE protein was undetectable in NT2- cell lysates, whereas NT2N neuron lysates exhibited faint immunoreactivity (Fig. 3B). This suggested that low BACE expression limits Abeta production in NT2- cells. To determine the effect of BACE expression on Abeta production from endogenous APP, NT2- cells were stably transduced using a pseudotyped self-inactivating lentiviral vector encoding BACE or GFP. This viral vector is non-toxic to NT2- cells and NT2N neurons and results in stable transgene expression in differentiated neurons for over 5 months in vivo.3 Several subclones uniformly expressing GFP or BACE were isolated (data not shown). As a control, GFP-expressing NT2G7- cells were selected to ensure that the process of transduction and subcloning did not alter APP processing. Two subclones, NT2B30- and NT2B17-, were chosen to represent low and high BACE-expressing clones, respectively (Fig. 3B), and to control for transgene insertion effects. Subclones retained their ability to differentiate into neurons upon retinoic acid exposure, and transgene overexpression was maintained in differentiated neurons (Fig. 3B).

To assay BACE cleavage at Asp-1, NT2- and NT2N conditioned media were immunoprecipitated with polyclonal antisera to the APP ectodomain to collect total sAPP. The immunoprecipitate was then subjected to SDS-PAGE and immunoblotted with C5A4/2, a rabbit polyclonal antibody specific for the C terminus of sAPPbeta (Fig. 3C). NT2- cells did not secrete appreciable levels of sAPPbeta , consistent with the fact that BACE expression is nearly absent in NT2- cells. Overexpression of BACE in NT2- cells, however, resulted in the detection of sAPPbeta in a dose-dependent manner. Unlike untransduced NT2- cells, sAPPbeta was detected from untransduced NT2N neuron-conditioned media. The shift in electrophoretic mobility between sAPPbeta recovered from BACE-expressing NT2- cells and NT2N neurons reflects the shorter isoform of APP expressed in NT2N neurons. Furthermore, sAPPbeta secretion by NT2B30N and NT2B17N neurons, which express exogenous BACE as a consequence of retroviral transduction, was increased relative to control NT2N neurons. In addition to increased cleavage at Asp-1, we determined that beta '-cleavage at Glu-11 was also present in BACE-expressing cells by immunoprecipitating C-terminal APP fragments from metabolically labeled NT2- cells, NT2N neurons, and BACE subclones with 2493, a polyclonal antibody raised against the C terminus of APP. BACE overexpression resulted in increased C99 and C89 relative to control cultures in both undifferentiated cells and neurons, corresponding to increased proteolysis of endogenous APP at residues Asp-1 and Glu-11, respectively. Significantly, C89 levels increased in a dose-dependent manner in that higher BACE-expressing clones had higher C89 levels. GFP expression was found to have no effect on sAPPbeta secretion or C-terminal fragment production in both undifferentiated cells and neurons (data not shown).

Secretion of Full-length and N-terminally Truncated Abeta -- Given the differences in endogenous BACE expression between NT2- cells and NT2N neurons, we sought to determine the effect of overexpressing BACE on Abeta production in these cells and to demonstrate the presence of N-terminally truncated Abeta peptides derived from BACE cleavage of APP at Glu-11. Media conditioned for 24 h were subjected to sandwich ELISA analysis and normalized for APP expression. Two sandwich ELISA systems were used in which either Ban50 (anti-Abeta 1-10) or BNT77 (anti-Abeta 11-28) monoclonal antibodies were used to capture Abeta . The Ban50 ELISA was used to detect full-length Abeta , whereas the BNT77 ELISA was used to detect both full-length and N-terminally truncated Abeta species. Both ELISA systems demonstrated the production of Abeta in NT2- cells upon the overexpression of BACE (Fig. 4A) at levels similar to that secreted by untransduced NT2N neurons. However, the effect of BACE overexpression was more pronounced in NT2N neurons, increasing Abeta secretion 5-8-fold over untransduced NT2N neurons. Furthermore, the differences in Abeta concentration as detected by Ban50 or BNT77 indicated that a large proportion of secreted Abeta peptides are N-terminally truncated. To identify N-terminally truncated Abeta peptides secreted by NT2N neurons, conditioned media were immunoprecipitated by 4G8 and subjected to MALDI-TOF mass spectrometry. A control mixture of synthetic Abeta 1-40 and Abeta 11-40 yielded peaks of expected mass (4329.27 and 3152.29 Da, respectively; Fig. 4B). Furthermore, both Abeta 1-40 and Abeta 11-40 were readily detected from untransduced NT2N neurons (4328.56 and 3151.57 Da, respectively; Fig. 4C) and BACE-overexpressing NT2N neurons (4329.91 and 3150.94 Da, respectively; Fig. 4D), consistent with the ELISA data. Other peaks corresponding to C-terminally truncated Abeta Abeta 1-34, Abeta 1-37, Abeta 1-38, and Abeta 1-39 with masses of 3784.71, 4076.02, 4132.14, and 4230.10 Da, respectively) were also identified in the mass spectra of neuronal medium (Fig. 4, C and D). However, since these C-terminally truncated Abeta peptides contain intact N termini, the increased concentration detected by BNT77 is primarily due to the presence of Abeta 11-40/42 in culture media.


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Fig. 4.   BACE expression increases full-length and N-terminally truncated Abeta secretion. A, media conditioned for 24 h by NT2- cells, NT2N neurons (5-week-old replate 2 neurons) and stably transduced subclones (NT2G7-/NT2B30-/NT2B17-, undifferentiated cells; NT2G7N/NT2B30N/NT2B17N, 5-week-old replate 2 neurons) were assayed for Abeta production by sandwich ELISA. Full-length Abeta was measured by Ban50 ELISA, whereas full-length and N-terminally truncated Abeta was measured by BNT77 ELISA, shown as mean values corrected for APP expression ± S.E. from six cultures over three independent collections. A control mixture of Abeta 1-40 and Abeta 11-40 (B), NT2N conditioned media (C), and NT2B30N conditioned media (D) were immunoprecipitated with 4G8 and subjected to MALDI-TOF analysis to identify different secreted Abeta species. Several peaks corresponding to C-terminally truncated Abeta peptides, in addition to Abeta 1-40 and Abeta 11-40, are labeled.

Intracellular Generation of Truncated Abeta -- Previous analysis of several cell lines indicated that both high expression of APP and high beta -secretase activity were co-requisites for intracellular Abeta detection (18, 28). We therefore tested whether BACE overexpression increases intracellular Abeta in NT2N neurons. Because the BNT77 ELISA did not have the sensitivity required for accurate intracellular Abeta quantification, a more sensitive ELISA was utilized in which Abeta peptides from cell lysates were captured with either JRF/cAbeta 40 or JRF/cAbeta 42, specific for Abeta 40 and Abeta 42, respectively. Captured peptides were detected with either JRF/Abeta N, recognizing Abeta 1-7 (to measure full-length Abeta ) or m266, recognizing Abeta 13-28 (to measure full-length and N-terminally truncated Abeta ). BACE overexpression resulted in the presence of intracellular Abeta from undifferentiated NT2B30- and NT2B17- cells, in contrast with NT2- and NT2G7- cells (Fig. 5). The amount of intracellular Abeta from BACE-expressing non-neuronal cells was comparable with the amount of intracellular Abeta from untransduced NT2N neurons. Similar to secreted Abeta , BACE overexpression resulted in a marked increase in intracellular Abeta in NT2N neurons. Furthermore, N-terminally truncated Abeta peptides comprised a large proportion of intracellular Abeta . The difference between Abeta concentration as determined by JRF/Abeta N and m266 indicated that N-terminally truncated Abeta is present intracellularly not only in BACE-overexpressing cells but in untransduced NT2N neurons as a result of normal metabolism of endogenous APP.


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Fig. 5.   Intracellular accumulation of truncated Abeta peptides. Abeta from NT2- cells, NT2N neurons (5-week-old replate 2 neurons), and stably transduced subclones (NT2G7-/NT2B30-/NT2B17-, undifferentiated cells; NT2G7N/NT2B30N/NT2B17N, 5-week-old replate 2 neurons) was extracted with RIPA and assayed by sandwich ELISA for intracellular Abeta . Full-length Abeta was measured by JRF/Abeta N ELISA, whereas full-length and N-terminally truncated Abeta was measured by m266 ELISA, shown as mean values ± S.E. from four to six cultures over three independent collections. Undifferentiated cultures were corrected for expression of APP, whereas neuronal cultures were corrected for expression of neuronal specific enolase.

To demonstrate further the presence of intracellular N-terminally truncated Abeta species, NT2N, NT2B30N, and NT2B17N neurons were metabolically labeled, and both media and cell lysates were immunoprecipitated with 4G8. Due to the lower sensitivity of 4G8 immunoprecipitation, we overexpressed APP in NT2N neurons to increase the production of Abeta . As shown in Fig. 6A, overexpression of APP in NT2N neurons resulted in a large increase in secreted Abeta . However, by using both Ban50 and BNT77 ELISAs, we found that the relative amount of truncated Abeta secreted by NT2N neurons upon APP overexpression was reduced. That is, although 24.5% of Abeta produced from endogenous APP is truncated, only 8.7% of Abeta is truncated upon APP overexpression. Despite this relative decrease in truncated Abeta , immunoprecipitates from NT2N media revealed three bands (Fig. 6B) corresponding to Abeta 1-40/42 (upper 4-kDa band), Abeta 11-40/42 (middle 3.2-kDa band), and p3 (Abeta 17-40/42, lower 3-kDa band). As expected, Abeta 1-40/42 and Abeta 11-40/42 were increased in a dose-dependent manner upon BACE expression, consistent with both the ELISA and mass spectral analysis. Immunoprecipitates from neuronal lysates demonstrated that full-length Abeta increased as a result of BACE expression. Furthermore, Abeta 11-40/42 was also recovered from BACE-expressing NT2N cell lysates in a dose-dependent manner, as shown by the presence of a 3.2-kDa band that co-migrated with the Abeta 11-40/42 recovered from media. Importantly, p3 was not detected from NT2N neuron lysates, indicating that p3 does not accumulate intraneuronally and that lysates were not contaminated with media. Thus, although the relative amount of truncated Abeta recovered by 4G8 immunoprecipitation was altered by APP overexpression, these results nonetheless confirm that N-terminal heterogeneity of Abeta is part of the intracellular processing pathway of APP and not solely due to partial degradation of secreted Abeta peptides.


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Fig. 6.   Secreted and intracellular Abeta 11-40/42 in NT2N neurons. A, media conditioned overnight by NT2N neurons (left) or NT2N neurons transduced with Semliki Forest virus to overexpress APP695 (right) were assayed by Ban50 or BNT77 ELISA to determine the concentration of either full-length Abeta (Ban50) or full-length and truncated Abeta (BNT77), shown as mean values ± S.E. from six cultures over two collections. B, NT2N, NT2B30N, and NT2B17N neurons were transduced with Semliki Forest virus to overexpress APP695 and metabolically labeled for 8 h. Media and RIPA lysates were immunoprecipitated with 4G8, electrophoresed on a 10/16.5% discontinuous gradient Tris-Tricine gel, and exposed to a PhosphorImager screen. Full-length APP, C-terminal APP fragments, full-length Abeta 1-40/42, truncated Abeta 11-40/42, and p3 are labeled. The contrast of the bottom of the gel has been enhanced to demonstrate the increase in Abeta production due to BACE overexpression. The contrast was further enhanced (bottom) to demonstrate the presence of N-terminally truncated Abeta .


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insoluble amyloid deposits from AD brains are heterogeneous in morphology and composition. Although the seeding and maturation of senile plaques in vivo is not well understood, increased beta -cleavage has been implicated in the pathogenesis of AD, either due to a pathogenic mutation (K595N/M596L, APPsw) at the beta -cleavage site of APP in familial AD (41-43) or by increased BACE expression in sporadic AD (29). Alternatively, other familial AD-associated mutations in either APP or presenilin increase the production of Abeta 1-42 relative to Abeta 1-40 (1). Despite the predominance of Abeta 1-40 in cerebral spinal fluid (CSF) (44), Abeta 1-42 is more abundant in senile plaques (3), consistent with its ability to aggregate more readily than Abeta 1-40 in vitro (2). A third class of familial AD mutations, located in the middle of the Abeta domain, has been postulated to increase the amyloidogenicity of Abeta peptides (9). Interestingly, these mutations also appear to increase the production of Abeta 11-40/42 (8, 9). Similar to C-terminal extensions to Abeta , N-terminal truncations have been shown to reduce solubility although increasing sedimentation and beta -pleated sheet structure of Abeta peptides relative to full-length Abeta (45-47). Furthermore, cyclization of the N-terminal glutamate in Abeta 11-40/42 protects the peptide from degradation by most aminopeptidases (48). Therefore, N-terminally truncated Abeta peptides may accelerate the seeding and maturation of senile plaques and thus exacerbate the progression of Alzheimer's disease, particularly in some genetic settings.

Until recently, the mechanism whereby Abeta peptides are N-terminally truncated has been unclear. Abeta peptides beginning at Glu-11 were first identified from purification of Abeta peptides from human CSF (37) and are found in insoluble fractions from AD brain (5, 12). The discovery of BACE led to the realization that two alternative cleavage sites are present in the N-terminal region of Abeta and that beta '-cleavage is species-specific (10, 23). Therefore, although rodent neuronal cells preferentially cleave endogenous APP at position 11 (22, 49), overexpression of human APP in rodent cells does not result in the formation of human Abeta 11-40/42 (22). Many modified Abeta peptides accumulate in brains of tg2576 mice, a transgenic mouse model overexpressing APPsw, including isomerized Asp-1 (L-iso-Asp), stereoisomerized Asp-1 (rectus Asp), and pyroglutaminated Glu-3. However, pyroglutaminated Glu-11 is conspicuously absent from tg2576 brains (50). Therefore, although many of the mechanisms for N-terminal modification of human Abeta are present, the generation of Abeta 11-40/42 is currently missing from both rodent cell culture and transgenic models. Given the limitations of rodent models, we investigated the generation of Abeta 11-40/42 in human NT2N neurons. We found that the secretion of sAPP and Abeta correlates with the expression of BACE that occurs upon neuronal differentiation. The increased secretion of sAPP was predominantly due to the secretion of sAPPbeta and sAPPbeta '. Additionally, NT2N neurons produce the N-terminally truncated Abeta 11-40/42 from normal metabolism of endogenous APP. Furthermore, exogenous BACE expression increased the secretion and intracellular generation of both Abeta 1-40/42 and Abeta 11-40/42. Interestingly, increasing APP expression decreased the relative amount of truncated Abeta produced by NT2N neurons. In contrast, non-neuronal cells with higher levels of BACE overexpression than reported here resulted in the preferential generation of N-terminally truncated Abeta over full-length Abeta (11, 12). Taken together, the ratio of APP to BACE expression may dictate the extent of beta '-cleavage. Regardless, the intracellular generation of Abeta 11-40/42 from normal APP processing in NT2N neurons indicates that this N-terminally truncated peptide is generated prior to deposition into insoluble aggregates in AD.

Additional Abeta peptides were recovered from NT2N neuron medium with truncated C termini. These C-terminally truncated Abeta species are also found in human CSF (51) and AD brain homogenates (5, 12), indicating that they may also contribute to amyloid formation. The close correlation between Abeta peptides found in NT2N neuronal medium and human CSF further validates the NT2N neuronal culture system as a useful model to study the generation of N- and C-terminally truncated Abeta peptides. Immunoprecipitation of intracellular Abeta from BACE-expressing NT2N neurons yielded a faint band slightly smaller than full-length Abeta (see Fig. 6). Although obscured somewhat by the intense signal derived from full-length Abeta , this truncated Abeta peptide appeared to be present in NT2N media samples, indicating that it corresponds to one of the C-terminally truncated Abeta peptides identified by mass spectrometry. APP and presenilin mutations that are known to affect C-terminal gamma -secretase cleavage also result in the increased accumulation of N-terminally truncated Abeta peptides (5, 6), indicating that beta - and gamma -secretase cleavage may influence each other. Importantly, the magnitude of the increase in Abeta production upon BACE expression indicates that the level of endogenous BACE expression in NT2- cells and NT2N neurons is rate-limiting in terms of Abeta generation. Although gamma -secretase activity was not addressed directly in these experiments, the modest effect of BACE expression in non-neuronal NT2- cells compared with the effect of BACE expression in NT2N neurons indicates that gamma -secretase cleavage is enhanced in neurons.

Although secretion of Abeta from neuronal cells is high relative to non-neuronal cells, the concentration of Abeta in human CSF is below the threshold for Abeta aggregation in vitro (2). The stability and insolubility of intraneuronal Abeta lead to the hypothesis that intracellular Abeta may be the source of Abeta aggregates that seed senile plaques. Indeed, insoluble Abeta accumulates in NT2N neurons with age in culture (28), and SDS-stable oligomeric Abeta is found intracellularly prior to secretion (52). Furthermore, prior to the presence of amyloid pathology, Abeta can be detected biochemically from tg2576 mice (50) and patients with early cognitive dysfunction (53). Intracellular Abeta has been found in affected brain regions in AD brains (54-56) and in animal models of AD amyloid pathology (57-60). Finally, mRNA isolated from senile plaques is predominantly neuronal (61). These reports suggest that the nidus for senile plaque formation may be intraneuronal Abeta . Interestingly, although various N-terminally truncated Abeta species, including Abeta 11-40/42, are readily detected from detergent-insoluble preparations from AD brain, p3 is not detected (5). p3 is a major component of diffuse plaques in AD (62, 63) and in diffuse plaques in the cerebellum of Down's syndrome patients (64). However, cerebellar diffuse plaques of Down's syndrome patients do not progress to form neuritic senile plaques even though in vitro studies of the p3 peptide indicate that it is highly hydrophobic, capable of forming fibrils, and has the tinctoral properties of amyloid as determined by thioflavin T and Congo Red staining (64). We could not detect intracellular p3 from NT2N neurons, consistent with the generation of p3 at or near the plasma membrane (65, 66). These observations are also consistent with the hypothesis that the intracellular environment is necessary to convert fibrillogenic Abeta peptides into a nidus for senile plaque formation.

Multiple subcellular sites are responsible for the production of different Abeta peptides. The trans-Golgi network produces predominantly Abeta 1-40 (67, 68), although the endoplasmic reticulum/intermediate compartment produces Abeta 1-42 (28, 31, 68, 69). Endoplasmic reticulum/intermediate compartment-derived Abeta 1-42 is not secreted but rather is retained intracellularly and contributes to the accumulation of a pool of insoluble Abeta that can be recovered with formic acid. Unfortunately, the sandwich ELISAs used in this study either do not have the sensitivity (BNT77) or are incompatible (JRF/Abeta N and m266) with formic acid lysates. However, the increased production of intracellular Abeta upon BACE expression is expected to increase the accumulation of insoluble full-length and truncated Abeta peptides. Production of endoplasmic reticulum/intermediate compartment-derived Abeta is independent of presenilin, indicating that multiple gamma -secretases may responsible for gamma -secretase cleavage in different subcellular organelles (15). In contrast, BACE-deficient untransduced NT2- cells do not have appreciable levels of intracellular Abeta , although BACE overexpression increases intracellular Abeta . Therefore, BACE appears to be responsible for both secreted and intracellular Abeta . The extent of beta '-cleavage is also dependent on the subcellular localization of BACE and APP in 293 cells (12). The engineering of BACE-overexpressing NT2N neurons allows for future investigations into the subcellular site of Abeta 11-40/42 generation in neuronal cells. However, the downstream effect of Abeta 11-40/42 generation on plaque formation awaits the engineering of transgenic mice co-expressing human BACE and human APP.

Increased BACE expression has been implicated in the pathogenesis of AD (29). Interestingly, BACE expression had a more profound effect on Abeta generation in NT2N neurons than in non-neuronal NT2- cells. Therefore, even modest increases in BACE expression may precipitate amyloid formation due to overproduction of Abeta . Conversely, mild inhibition of BACE activity may have a large effect on Abeta generation, underscoring the possibility of using BACE inhibitors as a therapy for AD. However, given the endogenous production of Abeta 11-40/42 by human NT2N neurons, the effect of BACE inhibitors on both full-length and N-terminally truncated Abeta peptides needs to be determined.

    ACKNOWLEDGEMENTS

We gratefully thank Takeda Pharmaceutical, Janssen Pharmacia, and Lilly for providing monoclonal antibodies for the Abeta sandwich ELISA. We thank K. N. Liu and A. Crystal for critical reading and suggestions in the preparation of this manuscript and Dr. J. Huse and Dr. C. Wilson for valuable discussions. We thank C. D. Page, J. Bruce, and C. Li for assistance with cultured cells and lentivirus production. We are grateful to Dr. L. J. Chang, Dr. G. Kobinger, Dr. D. Watson, and Dr. J. H. Wolfe for providing transfer plasmids for lentivirus production.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Training Grants T32 AG00255 (to E. B. L.) and NIA AG11542 (to R. W. D. and V. M.-Y. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a Paul Beeson Faculty Scholar award.

John H. Ware III professor of Alzheimer's research. To whom correspondence should be addressed: Center for Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, Maloney 3, HUP, Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909; E-mail: vmylee@mail.med.upenn.edu.

Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M210105200

2 E. B. Lee and V. M.-Y. Lee, unpublished data.

3 Watson, D. J., Longhi, L., Lee, E. B., Fulp, C. T., Fujimoto, S., Royo, N. C., Passini, M. A., Trojanowski, J. Q., Lee, V. M.-Y., McIntosh, T. K., and Wolfe, J. H. (2003) J. Neuropath. Exp. Neurol., in press.

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid-beta peptide; Abeta 1-40, Abeta 1-42, 40- and 42-amino acid forms of Abeta respectively; Abeta 11-40, Abeta 11-42, N-terminally truncated Abeta peptides starting at position Glu-11; ADAM, a disintegrin and metalloprotease; APP, amyloid-beta precursor protein; APP695, 695-amino acid isoform of APP; APP751/770, 751- and 770-amino acid isoforms of APP; BACE, beta -site APP-cleaving enzyme; C83, alpha -secretase derived C-terminal fragment of APP; C89, beta -secretase derived C-terminal fragment beginning at Glu-11; C99, beta -secretase derived C-terminal fragment beginning at Asp-1; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GFP, green fluorescent protein; NT2-, undifferentiated embryonal carcinoma NTera2/c1.D1; NT2N, differentiated neuron derived from NTera2/c1.D1; PMA, phorbol 12-myristate 13-acetate; RIPA buffer, radioimmune precipitation assay buffer; sAPP, total secretase-derived N-terminal ectodomain of APP; sAPPalpha , alpha -cleavage-derived N-terminal ectodomain of APP; sAPPbeta , beta -cleavage-derived N-terminal ectodomain of APP; sAPPbeta ', beta '-cleavage-derived N-terminal ectodomain of APP; TAPI, (N-R-(2-hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-L-naphthylalanyl-L-alanine 2-aminoethyl amide; Tricine, N-[2-hydroxyl-1,1-bis(hydroxymethyl)ethyl]glycine; VSV-G, vesicular stomatitis virus surface glycoprotein; DMEM, Dulbecco's modified Eagle's medium; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; CSF, cerebral spinal fluid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Selkoe, D. J. (2001) Physiol. Rev. 81, 741-766[Abstract/Free Full Text]
2. Jarrett, J. T., Berger, E. P., and Lansbury, P. T., Jr. (1993) Biochemistry 32, 4693-4697[Medline] [Order article via Infotrieve]
3. Iwatsubo, T., Odaka, A., Suzuki, N., Mizusawa, H., Nukina, N., and Ihara, Y. (1994) Neuron 13, 45-53[Medline] [Order article via Infotrieve]
4. Masters, C. L., Simms, G., Weinman, N. A., Multhaup, G., McDonald, B. L., and Beyreuther, K. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4245-4249[Abstract]
5. Naslund, J., Schierhorn, A., Hellman, U., Lannfelt, L., Roses, A. D., Tjernberg, L. O., Silberring, J., Gandy, S. E., Winblad, B., Greengard, P., Nordstedt, C., and Terenius, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8378-8382[Abstract]
6. Russo, C., Schettini, G., Saido, T. C., Hulette, C., Lippa, C., Lannfelt, L., Ghetti, B., Gambetti, P., Tabaton, M., and Teller, J. K. (2000) Nature 405, 531-532[CrossRef][Medline] [Order article via Infotrieve]
7. Saido, T. C., Iwatsubo, T., Mann, D. M., Shimada, H., Ihara, Y., and Kawashima, S. (1995) Neuron 14, 457-466[Medline] [Order article via Infotrieve]
8. Haass, C., Hung, A. Y., Selkoe, D. J., and Teplow, D. B. (1994) J. Biol. Chem. 269, 17741-17748[Abstract/Free Full Text]
9. Nilsberth, C., Westlind-Danielsson, A., Eckman, C. B., Condron, M. M., Axelman, K., Forsell, C., Stenh, C., Luthman, J., Teplow, D. B., Younkin, S. G., Naslund, J., and Lannfelt, L. (2001) Nat. Neurosci. 4, 887-893[CrossRef][Medline] [Order article via Infotrieve]
10. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) Science 286, 735-741[Abstract/Free Full Text]
11. Liu, K., Doms, R. W., and Lee, V. M.-Y. (2002) Biochemistry 41, 3128-3136[CrossRef][Medline] [Order article via Infotrieve]
12. Huse, J. T., Liu, K., Pijak, D. S., Carlin, D., Lee, V. M.-Y., and Doms, R. W. (2002) J. Biol. Chem. 277, 16278-16284[Abstract/Free Full Text]
13. Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell, A. R., Oltersdorf, T., McClure, D., and Ward, P. J. (1990) Science 248, 1122-1124[Medline] [Order article via Infotrieve]
14. Sisodia, S. S., Koo, E. H., Beyreuther, K., Unterbeck, A., and Price, D. L. (1990) Science 248, 492-495[Medline] [Order article via Infotrieve]
15. Wilson, C. A., Doms, R. W., Zheng, H., and Lee, V. M.-Y. (2002) Nat. Neurosci. 5, 849-855[CrossRef][Medline] [Order article via Infotrieve]
16. Haass, C., Schlossmacher, M. G., Hung, A. Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E. H., Schenk, D., Teplow, D. B., and Selkoe, D. (1992) Nature 359, 322-325[CrossRef][Medline] [Order article via Infotrieve]
17. Shoji, M., Golde, T. E., Ghiso, J., Cheung, T. T., Estus, S., Shaffer, L. M., Cai, X. D., McKay, D. M., Tintner, R., Frangione, B., and Younkin, S. G. (1992) Science 258, 126-129[Medline] [Order article via Infotrieve]
18. Forman, M. S., Cook, D. G., Leight, S., Doms, R. W., and Lee, V. M.-Y. (1997) J. Biol. Chem. 272, 32247-32253[Abstract/Free Full Text]
19. Wertkin, A. M., Turner, R. S., Pleasure, S. J., Golde, T. E., Younkin, S. G., Trojanowski, J. Q., and Lee, V. M.-Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9513-9517[Abstract]
20. Turner, R. S., Suzuki, N., Chyung, A. S., Younkin, S. G., and Lee, V. M.-Y. (1996) J. Biol. Chem. 271, 8966-8970[Abstract/Free Full Text]
21. Chyung, A. S., Greenberg, B. D., Cook, D. G., Doms, R. W., and Lee, V. M.-Y. (1997) J. Cell Biol. 138, 671-680[Free Full Text]
22. Wang, R., Sweeney, D., Gandy, S. E., and Sisodia, S. S. (1996) J. Biol. Chem. 271, 31894-31902[Abstract/Free Full Text]
23. Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D. R., Price, D. L., and Wong, P. C. (2001) Nat. Neurosci. 4, 233-234[CrossRef][Medline] [Order article via Infotrieve]
24. Andrews, P. W., Damjanov, I., Simon, D., Banting, G. S., Carlin, C., Dracopoli, N. C., and Fogh, J. (1984) Lab. Invest. 50, 147-162[Medline] [Order article via Infotrieve]
25. Andrews, P. W. (1984) Dev. Biol. 103, 285-293[Medline] [Order article via Infotrieve]
26. Pleasure, S. J., Page, C., and Lee, V. M.-Y. (1992) J. Neurosci. 12, 1802-1815[Abstract]
27. Pleasure, S. J., and Lee, V. M.-Y. (1993) J. Neurosci. Res. 35, 585-602[Medline] [Order article via Infotrieve]
28. Skovronsky, D. M., Doms, R. W., and Lee, V. M.-Y. (1998) J. Cell Biol. 141, 1031-1039[Abstract/Free Full Text]
29. Holsinger, R. M., McLean, C. A., Beyreuther, K., Masters, C. L., and Evin, G. (2002) Ann. Neurol. 51, 783-786[CrossRef][Medline] [Order article via Infotrieve]
30. Liljestrom, P., and Garoff, H. (1991) Bio/Technology 9, 1356-1361[Medline] [Order article via Infotrieve]
31. Greenfield, J. P., Tsai, J., Gouras, G. K., Hai, B., Thinakaran, G., Checler, F., Sisodia, S. S., Greengard, P., and Xu, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 742-747[Abstract/Free Full Text]
32. Huse, J. T., Pijak, D. S., Leslie, G. J., Lee, V. M.-Y., and Doms, R. W. (2000) J. Biol. Chem. 275, 33729-33737[Abstract/Free Full Text]
33. Suzuki, N., Cheung, T. T., Cai, X. D., Odaka, A., Otvos, L., Jr., Eckman, C., Golde, T. E., and Younkin, S. G. (1994) Science 264, 1336-1340[Medline] [Order article via Infotrieve]
34. Suzuki, N., Iwatsubo, T., Odaka, A., Ishibashi, Y., Kitada, C., and Ihara, Y. (1994) Am. J. Pathol. 145, 452-460[Abstract]
35. Asami-Odaka, A., Ishibashi, Y., Kikuchi, T., Kitada, C., and Suzuki, N. (1995) Biochemistry 34, 10272-10278[Medline] [Order article via Infotrieve]
36. Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982[CrossRef][Medline] [Order article via Infotrieve]
37. Seubert, P., Vigo-Pelfrey, C., Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M., Whaley, J., Swindlehurst, C., McCormack, R., Wolfert, R., Selkoe, D., Lieberburg, I., and Schenk, D. (1992) Nature 359, 325-327[CrossRef][Medline] [Order article via Infotrieve]
38. Skovronsky, D. M., Moore, D. B., Milla, M. E., Doms, R. W., and Lee, V. M.-Y. (2000) J. Biol. Chem. 275, 2568-2575[Abstract/Free Full Text]
39. Bodendorf, U., Fischer, F., Bodian, D., Multhaup, G., and Paganetti, P. (2001) J. Biol. Chem. 276, 12019-12023[Abstract/Free Full Text]
40. Tanahashi, H., and Tabira, T. (2001) Neurosci. Lett. 307, 9-12[CrossRef][Medline] [Order article via Infotrieve]
41. Mullan, M., Crawford, F., Axelman, K., Houlden, H., Lilius, L., Winblad, B., and Lannfelt, L. (1992) Nat. Genet. 1, 345-347[Medline] [Order article via Infotrieve]
42. Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A. Y., Seubert, P., Vigo-Pelfrey, C., Lieberburg, I., and Selkoe, D. J. (1992) Nature 360, 672-674[CrossRef][Medline] [Order article via Infotrieve]
43. Cai, X. D., Golde, T. E., and Younkin, S. G. (1993) Science 259, 514-516[Medline] [Order article via Infotrieve]
44. Vigo-Pelfrey, C., Lee, D., Keim, P., Lieberburg, I., and Schenk, D. B. (1993) J. Neurochem. 61, 1965-1968[Medline] [Order article via Infotrieve]
45. Pike, C. J., Overman, M. J., and Cotman, C. W. (1995) J. Biol. Chem. 270, 23895-23898[Abstract/Free Full Text]
46. He, W., and Barrow, C. J. (1999) Biochemistry 38, 10871-10877[CrossRef][Medline] [Order article via Infotrieve]
47. Walsh, D. M., Hartley, D. M., Condron, M. M., Selkoe, D. J., and Teplow, D. B. (2001) Biochem. J. 355, 869-877[Medline] [Order article via Infotrieve]
48. McDonald, J. K., and Barret, A. J. (eds) (1986) Mammalian Proteases, pp. 3 and 305-311, Academic Press, London
49. Gouras, G. K., Xu, H., Jovanovic, J. N., Buxbaum, J. D., Wang, R., Greengard, P., Relkin, N. R., and Gandy, S. (1998) J. Neurochem. 71, 1920-1925[Medline] [Order article via Infotrieve]
50. Kawarabayashi, T., Younkin, L. H., Saido, T. C., Shoji, M., Ashe, K. H., and Younkin, S. G. (2001) J. Neurosci. 21, 372-381[Abstract/Free Full Text]
51. Wiltfang, J., Esselmann, H., Bibl, M., Smirnov, A., Otto, M., Paul, S., Schmidt, B., Klafki, H. W., Maler, M., Dyrks, T., Bienert, M., Beyermann, M., Ruther, E., and Kornhuber, J. (2002) J. Neurochem. 81, 481-496[CrossRef][Medline] [Order article via Infotrieve]
52. Walsh, D. M., Tseng, B. P., Rydel, R. E., Podlisny, M. B., and Selkoe, D. J. (2000) Biochemistry 39, 10831-10839[CrossRef][Medline] [Order article via Infotrieve]
53. Naslund, J., Haroutunian, V., Mohs, R., Davis, K. L., Davies, P., Greengard, P., and Buxbaum, J. D. (2000) J. Am. Med. Assoc. 283, 1571-1577[Abstract/Free Full Text]
54. Mochizuki, A., Tamaoka, A., Shimohata, A., Komatsuzaki, Y., and Shoji, S. (2000) Lancet 355, 42-43[CrossRef][Medline] [Order article via Infotrieve]
55. Gouras, G. K., Tsai, J., Naslund, J., Vincent, B., Edgar, M., Checler, F., Greenfield, J. P., Haroutunian, V., Buxbaum, J. D., Xu, H., Greengard, P., and Relkin, N. R. (2000) Am. J. Pathol. 156, 15-20[Abstract/Free Full Text]
56. Tabira, T., Chui, D. H., and Kuroda, S. (2002) Front. Biosci. 7, A44-A49[Medline] [Order article via Infotrieve]
57. Martin, L. J., Pardo, C. A., Cork, L. C., and Price, D. L. (1994) Am. J. Pathol. 145, 1358-1381[Abstract]
58. Masliah, E., Sisk, A., Mallory, M., Mucke, L., Schenk, D., and Games, D. (1996) J. Neurosci. 16, 5795-5811[Abstract/Free Full Text]
59. Chui, D. H., Tanahashi, H., Ozawa, K., Ikeda, S., Checler, F., Ueda, O., Suzuki, H., Araki, W., Inoue, H., Shirotani, K., Takahashi, K., Gallyas, F., and Tabira, T. (1999) Nat. Med. 5, 560-564[CrossRef][Medline] [Order article via Infotrieve]
60. Wirths, O., Multhaup, G., Czech, C., Blanchard, V., Moussaoui, S., Tremp, G., Pradier, L., Beyreuther, K., and Bayer, T. A. (2001) Neurosci. Lett. 306, 116-120[CrossRef][Medline] [Order article via Infotrieve]
61. Ginsberg, S. D., Crino, P. B., Hemby, S. E., Weingarten, J. A., Lee, V. M.-Y., Eberwine, J. H., and Trojanowski, J. Q. (1999) Ann. Neurol. 45, 174-181[CrossRef][Medline] [Order article via Infotrieve]
62. Gowing, E., Roher, A. E., Woods, A. S., Cotter, R. J., Chaney, M., Little, S. P., and Ball, M. J. (1994) J. Biol. Chem. 269, 10987-10990[Abstract/Free Full Text]
63. Higgins, L. S., Murphy, G. M., Jr., Forno, L. S., Catalano, R., and Cordell, B. (1996) Am. J. Pathol. 149, 585-596[Abstract]
64. Lalowski, M., Golabek, A., Lemere, C. A., Selkoe, D. J., Wisniewski, H. M., Beavis, R. C., Frangione, B., and Wisniewski, T. (1996) J. Biol. Chem. 271, 33623-33631[Abstract/Free Full Text]
65. Sisodia, S. S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6075-6079[Abstract]
66. Haass, C., Koo, E. H., Mellon, A., Hung, A. Y., and Selkoe, D. J. (1992) Nature 357, 500-503[CrossRef][Medline] [Order article via Infotrieve]
67. Xu, H., Sweeney, D., Wang, R., Thinakaran, G., Lo, A. C., Sisodia, S. S., Greengard, P., and Gandy, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3748-3752[Abstract/Free Full Text]
68. Hartmann, T., Bieger, S. C., Bruhl, B., Tienari, P. J., Ida, N., Allsop, D., Roberts, G. W., Masters, C. L., Dotti, C. G., Unsicker, K., and Beyreuther, K. (1997) Nat. Med. 3, 1016-1020[Medline] [Order article via Infotrieve]
69. Cook, D. G., Forman, M. S., Sung, J. C., Leight, S., Kolson, D. L., Iwatsubo, T., Lee, V. M.-Y., and Doms, R. W. (1997) Nat. Med. 3, 1021-1023[Medline] [Order article via Infotrieve]


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