COMMUNICATION
Amyloid Precursor Protein Processing in Sterol Regulatory Element-binding Protein Site 2 Protease-deficient Chinese Hamster Ovary Cells*

Sandra L. RossDagger , Francis MartinDagger , Lizette SimonetDagger , Frederick JacobsenDagger , Rohini DeshpandeDagger , Robert Vassar§, Brian Bennett§, Yi Luo§, Scott WoodenDagger , Sylvia HuDagger , Martin Citron§, and Teresa L. BurgessDagger

From the Departments of Dagger  Mammalian Cell Molecular Biology and § Neuroscience, Amgen Inc., Thousand Oaks, California 91320-1789

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Amyloid peptides of 39-43 amino acids (Abeta ) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta - and gamma -secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma -secretase activity. Identification of the beta - and/or gamma -secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma -secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma -secretase.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

A characteristic neuropathological feature of Alzheimer's disease (AD)1 are amyloid plaques, containing Abeta , a 39-43-amino acid protein proteolytically derived from a large type 1 transmembrane protein, the amyloid precursor protein (APP) (for review see Ref. 1). APP undergoes a series of proteolytic processing steps in the biosynthetic and/or endocytic pathway. alpha - and gamma -Secretase cleavages generate the non-amyloidogenic peptide, p3, whereas beta - and gamma -secretase cleavages lead to the production of the amyloidogenic Abeta peptide (Fig. 1). A number of different mutations in APP have been identified that lead to rare forms of early onset familial Alzheimer's disease (FAD). All of these mutations appear to alter the proteolytic processing of the precursor, increasing the total production of the 42-amino acid form of Abeta , Abeta 42 (for review see Ref. 2).


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Fig. 1.   Schematic drawing of APP processing. A typical N-terminal (NH2) signal peptide (open box) is removed before APP is processed by one of two mutually exclusive pathways. alpha -Secretase cleaves at residue 17 of Abeta (gray box) preventing the generation of the amyloidogenic Abeta peptides. If, however, beta -secretase cleaves APP (at position 1 of Abeta ), subsequent cleavage by one or more gamma -secretases generates Abeta 39-Abeta 43. TMD, transmembrane domain; alpha APPs and beta APPs, secreted fragments generated by alpha - and beta -secretase cleavage, respectively.

Despite intensive study of APP processing, none of the secretase activities has been definitively identified, purified, or cloned, although many enzymes have been proposed as potential secretases (for review see Ref. 3). Secretase candidates have been difficult to identify because specific inhibitors are not generally available and it is unclear to which protease classes beta - and gamma -secretases belong. In addition, it is not known whether beta - and gamma -secretases are each just one enzyme or are a collection of different enzymes. For example, it has been suggested, but not proven, that gamma -secretase cleavages at positions 40 and 42 are carried out by different proteases (4, 5). Finally, more than 10 years after the cloning of APP it is still puzzling how gamma -secretase cleaves APP within the transmembrane domain (6).

Recently, Brown and Goldstein (7) noted numerous similarities between the sterol regulatory element-binding protein (SREBP) site 2 protease (S2P) and the gamma -secretase(s). Based on these similarities, they hypothesized that either the S2P may be the gamma -secretase or that the two proteases may be closely related (7). The substrate of the S2P, SREBP, is initially synthesized as an endoplasmic reticulum membrane-bound precursor. Following a two-step cleavage, the cytosolic domain of SREBP is released and targeted to the nucleus where it transcriptionally regulates genes containing the sterol regulatory element (SRE), including numerous genes involved in cholesterol and fatty acid metabolism (8).

The similarities between the S2P and gamma -secretase are provocative. First, the substrates (SREBP and APP) of both enzymes are cleaved uniquely within the transmembrane domain (9, 10). Second, both S2P and gamma -secretase enzymes catalyze the second cleavage of an obligatory two-step cleavage reaction. Third, both enzymes appear to be ubiquitously expressed (as are the substrates). Finally, processing of SREBP is regulated by a six to eight transmembrane domain protein, SCAP (SREBP cleavage activating protein) (11), which is located in the endoplasmic reticulum (12). Dominant missense mutations in SCAP have been shown to alter SREBP processing (11, 13). Although there is no direct sequence homology, the presenilins (PS1 and PS2 (14, 15)) also contain six to eight transmembrane domains and are localized to the endoplasmic reticulum where they are thought to regulate gamma -secretase activity. Dominant missense mutations in both PS1 and PS2 account for the most common forms of FAD, and both in vitro and in vivo evidence suggest that these mutations in PS1/PS2 somehow alter gamma -secretase activity and lead to increased production of Abeta 42 (for review see Ref. 16). Furthermore, neurons from PS1 knock-out mice appear to have dramatically reduced gamma -secretase activity, suggesting that PS1 may be an activator of gamma -secretase (17), much as SCAP activates SREBP processing (11-13, 18).

Recent data have allowed us to directly test whether the gamma -secretase and the S2P are identical enzymes. Sakai et al. (9) demonstrated that a mutant Chinese hamster ovary (CHO) cell line, CHO M19 (19, 20), is deficient in the S2P activity. Second, the S2P cDNA was cloned by complementation of the M19 defect (21, 22); furthermore, Rawson et al. (22) show that CHO M19 cells do not express the S2P mRNA due to a large deletion encompassing most, it not all, of the S2P gene (22).

In this communication we show that over-expression of human (hu) S2P does not enhance secretion of Abeta from 293 cells, suggesting that either the S2P is not the gamma -secretase or that the activity is not rate-limiting for processing APP to Abeta . Furthermore, we show that CHO M19 clones process transfected hu APPsw to Abeta ; both Abeta 40 and Abeta 42 are generated in proportions indistinguishable from those produced by the wild-type CHO clones. Finally, we confirm that our CHO M19 clones are auxotrophs and that they do not express detectable S2P mRNA. These results definitively show that the S2P is not required for gamma -secretase processing of APP to Abeta 40 and Abeta 42.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmid Constructions-- The sense primer (5'-CGG AGG AGA TCT CTG AAG TGA ATC TGG ATG CAG AAT TCC GAC ATG ACT C-3') containing the Swedish mutation and BglII restriction site and the antisense primer (5'-TAT AGC AGA AGC AGC AAT CTG TAC AG-3') were used with wild-type APP695 cDNA as template. The PCR-amplified, BglII-digested DNA fragment was subcloned into the wild-type APP695 cDNA to generate pGEM3/APPsw695 (Promega). After confirming the DNA sequence, the APPsw fragment (HindIII-XmnI) was subcloned to generate pBCB/APPsw695 (23). The APPsw695 cDNA (HindIII-NotI) was subcloned into pCMVi (Cell & Molecular Technologies) to generate pCMVi/APPsw695. pRc/Neo was derived from pRc/CMV (Invitrogen). It contains the pRc/CMV 1524-bp neomycin resistance expression cassette (KpnI-SalI) fused to the pRc/CMV 2178-bp vector backbone (SalI-XhoI fragment). PCR primers for the amplification of codons 1-519 of the human S2P (22) (GenBankTM accession number AFO 19612) were synthesized: 5'-GTC TCT AGA GCT GCT ACT ATG ATT CCG GTG TCG-3' and 5'-CAT TAC CGT GCT GTA ACC ATC CAG-3'. PCR amplification was performed with random primed human fetal brain cDNA as template. The product was digested with XbaI and cloned into pcDNA3.1(-) (Invitrogen) between the XbaI and BamHI (blunt) sites to generate S2P/pcDNA3.1.

Stable Cell Lines-- A derivative of 293 cells (Cell & Molecular Technologies Inc.) was co-transfected with pCMVi-APPsw695 and pRSV-Puro by the calcium phosphate method (24). Individual puromycin-resistant clones were expanded and analyzed for high APP production by Western dot-blot with monoclonal antibody 22C11 (Boehringer). APPsw 293 clone 101 was used in all experiments reported here; similar results were obtained with clone 117. Wild-type CHO K1 (CHO WT) and CHO M19 cells (19, 20) were co-transfected with pBCB/APPsw695 and pRc/Neo or with pRc/Neo alone using the polyamine LT-1 (Mirus). Individual G418 resistant colonies were expanded and assayed for high APP production by Western blot with antibody 22C11.

Transient Transfections and Cell Culture-- 293 cells over-expressing APPsw (clone 101) were transiently transfected with S2P/pcDNA3.1, pcDNA3.1, or a beta -galactosidase-containing vector using DMRIE-C (Life Technologies, Inc.). 293 clone 101 cells were grown in Dulbecco's modified Eagle's medium with 10% FBS and 1% pen/strep/gln containing 5 µg/ml puromycin. CHO clones were grown in F12 with 10% fetal bovine serum, 10 µg/ml gentamycin, and 0.5 mg/ml G418.

Electrophoretic Separation of Abeta 40 and Abeta 42, Immunoprecipitations, and Westerns-- Tris-Bicine urea gels were used to resolve Abeta 40 and Abeta 42 as described (25) using synthetic peptides from Quality Controlled Biochemicals, Inc. as standards. Proteins were transferred to polyvinylidene difluoride membranes as described (26).

For 293 cells over-expressing APPsw and transiently transfected with human S2P or vector control, 20 µl of 24-h conditioned medium was analyzed on Tris-Bicine urea gels. Following transfer, the blot was boiled in phosphate-buffered saline for 5 min and blocked for 2 h in 5% milk. The blot was probed overnight with 10 µg/ml 6E10 (Senetek), washed, and then treated with biotinylated anti-mouse IgG (Amersham Pharmacia Biotech) for 30 min. The blot was washed, incubated with streptavidin linked to horseradish peroxidase (Amersham Pharmacia Biotech), washed, and exposed to ECL film. For CHO clones, 48-h conditioned medium samples were collected from nearly confluent plates. One-ml samples were immunoprecipitated overnight with 10 µg of 4G8 (Senetek) and analyzed on gels/Westerns as described above.

Quantitative Analysis of S2P mRNA-- S2P mRNA expressed during transient transfection experiments was quantified using solution hybridization to [35S]UTP riboprobes (27). Riboprobes corresponding to hu S2P amino acids 437-519 and 250-321 (22) in both sense and antisense orientations were used to quantify the S2P mRNA.

RT-PCR-- cDNA template generated with random primers and total cellular RNA from CHO M19 and WT clones was used to amplify S2P or alpha -tubulin by PCR. Codons 254-519 of S2P (22) were amplified with sense, 5'-GTT GGG GTG CTC ATC ACT GAA-3', and antisense, 5'-CAT TAC CGT GCT GTA ACC ATC CAG-3', primers yielding a product of 790 bases. alpha -Tubulin was amplified with sense, 5'-AAG AAG TCC AAG CTG GAG TTC TC-3', and antisense, 5'-GTG GTG TTG CTC AGC ATG CAC AC-3', primers yielding a product of 700 base pairs.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Over-expression of the SREBP S2P Does Not Enhance Production of Abeta 40 or Abeta 42 from APPsw-- To test the hypothesis that the S2P may be identical to the APP gamma -secretase, we generated 293 cells stably expressing human APP695 with the Swedish mutation (APPsw) (28). If S2P is the same enzyme as the gamma -secretase, transient over-expression in cells containing substrate (e.g. APP-over-expressing cell lines) could lead to an increase in the production of either Abeta 40, Abeta 42, or both.

Analysis of Abeta 40 and/or Abeta 42 secretion from 293 cells transiently transfected with S2P by quantitative urea-gel/Western blot was carried out on conditioned media samples (Fig. 2). Synthetic Abeta 40 and Abeta 42 peptides are well separated by this gel system with Abeta 42 migrating faster than Abeta 40 (Fig. 2, lanes 4 and 5), and our linear range of detection is from about 20 pg to 10 ng. Because this gel system does not separate peptides strictly on the basis of size, it is possible to identify the bands only by co-migration with known peptide standards (and by antibody specificity). (Some of the slower migrating bands in the conditioned media samples are dimers of the 40 and 42 peptides, but others, both slower and faster migrating in Figs. 2 and 3B, have not been identified by us, but see Ref. 26.)


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Fig. 2.   Over-expression of human S2P does not alter Abeta production. 293 cells over-expressing APPsw695 were transiently transfected with the human S2P cDNA in pcDNA3.1 or vector controls. Conditioned media collected between 48 and 72 h post-transfection were loaded on Tris-Bicine urea gels and transferred to polyvinylidene difluoride membranes. Synthetic Abeta 40 and Abeta 42 peptides (500 pg of each; lanes 4 and 5, respectively) co-migrate with Abeta peptides generated by vector control cells, human S2P transfected cells, and beta -galactosidase control vector. No qualitative or quantitative difference in Abeta processing is detected when S2P is expressed.

APPsw-expressing 293 cells, transiently transfected with either vector control (Fig. 2, lane 1) or a beta -galactosidase-containing vector (lane 3), secrete Abeta 40 and Abeta 42 in a ratio of about 10:1 as expected (29). Transient expression of human S2P in these cells (lane 2) did not lead to any detectable increase in Abeta 40 or Abeta 42 production nor to a change in the ratio of the two Abeta species (Fig. 2, compare lane 2 to control lanes 1 and 3). These results suggest that over-expression of S2P does not lead to increased gamma -secretase activity in these cells.

To show that the S2P cDNA was being expressed during this transient assay, we first determined that the transient transfection efficiency in this experiment was about 80% (based on beta -galactosidase expression; data not shown). Next we performed a quantitative solution hybridization assay to detect S2P mRNA (see "Experimental Procedures" and Ref. 27) in the same cells from which the conditioned media were collected. This assay revealed that S2P transfected cells contained on average about 800 copies of S2P mRNA, whereas untransfected cells contained less than 20 copies per cell; nonetheless, no change in Abeta 40 or Abeta 42 production could be detected (Fig. 2).

CHO Cells Lacking S2P Expression Process Hu APPsw to Abeta 40 and Abeta 42-- Recent data indicate that the cholesterol auxotroph CHO M19 contains a deletion spanning most, if not all, of the S2P gene and thus represents a cellular S2P gene knock-out (22). If the activity of S2P is responsible for the gamma -secretase generation of Abeta 40 and/or Abeta 42 from APP, then the CHO M19 cells should be unable to generate Abeta 40 and/or Abeta 42.

Because endogenous production of Abeta is very low in CHO cells, and it is not recognized by the 6E10 antibody used in our gel assay, both CHO WT and CHO M19 cells (19, 20) were stably transfected with pBCB/APPsw695 which encodes hu APPsw. Individual geneticin-resistant clones, which secreted roughly equivalent amounts of hu APPs and vector control clones for CHO WT and CHO M19, were selected for further analysis (Fig. 3A).


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Fig. 3.   CHO M19 cells that lack S2P expression generate Abeta . A, cell lysates from CHO M19 and CHO WT clones stably expressing APPsw from pBCB/APPsw695 contain both endogenous (APP751/770) and APPsw695. Non-ionic detergent extracts containing 25 µg of total protein from the indicated clones were loaded in each lane. B, 48-h conditioned media samples immunoprecipitated with monoclonal antibody 4G8 were separated on a Tris-Bicine/urea gel to resolve Abeta 40 and Abeta 42. Abeta 40 and Abeta 42 synthetic peptides (500 pg of each) were run separately in lanes 8 and 9, respectively, or were mixed and immunoprecipitated together (lane 7) to serve as migration standards for the experiment. Medium from CHO WT or CHO M19 clones stably transfected with the control vector (pBCB) or expressing APPsw are shown.

Most non-neuronal cell lines, including these CHO cells, express the 751 or 770 splice variants of APP. Endogenous intracellular APP expressed by these cells is detected as the upper band in extracts from all clones (Fig. 3A). It is well separated from the smaller APPsw which is detected in all of the pBCB/APPsw695 transfected clones (lower band in lanes 2, 3, 5, and 6) but not in the vector control clones (lanes 1 and 4).

Conditioned media from these six clones were collected and concentrated by immunoprecipitation and the hu Abeta 40 and hu Abeta 42 production was analyzed on the urea-gel/Western described above (Fig. 3B). As expected, CHO WT clones expressing the APPsw processed and secreted hu Abeta 40 and hu Abeta 42 at a ratio of about 10:1 (Fig. 3B, lanes 2 and 3). The CHO M19 clones also processed and secreted hu Abeta 40 and hu Abeta 42 in a ratio of about 10:1 and at a level reflecting total hu APPsw expression (Fig. 3B, lanes 5 and 6). These data demonstrate directly that the S2P-deficient CHO M19 cells are not deficient in gamma -secretase activity.

Following the collection of the conditioned media used above, we incubated these cultures in serum-free media to confirm that the CHO M19 clones lack S2P expression. We found that all of the CHO M19 clones died in serum-free media (within 24-48 h) as would be expected for cholesterol auxotrophs, whereas the CHO WT clones remained attached and appeared healthy for many days (data not shown). Furthermore, we used RT-PCR to demonstrate that only the CHO WT clones express mRNA for the S2P (Fig. 4). alpha -Tubulin-specific primers were used as a positive control, and RT-PCR generated a product of the expected 700 base pairs in CHO WT (lanes 1-3) and CHO M19 clones (lanes 4-6). In contrast, the S2P specific primers led to the generation of a 790-base pair fragment in the CHO WT clones (lanes 7-9), but no product was generated from cDNA derived from any of the CHO M19 clones (lanes 10-12). Similarly, Northern blot analysis for S2P expression showed that CHO WT cells expressed the ~4-kilobase S2P transcript, CHO M19 cells were negative (data not shown).


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Fig. 4.   Expression of S2P mRNA in CHO and M19 cells by RT-PCR. Gene-specific primers for either alpha -tubulin, lanes 1-6, or S2P, lanes 7-12, were used to amplify first-strand cDNA from the indicated clones. A band migrating at the expected 700 base pairs is detected for alpha -tubulin in all of the clones (CHO WT, lanes 1-3; CHO M19, lanes 4-6) whereas only the CHO WT clones generate a 790-base pair fragment expected for S2P (lanes 7-9). None of the CHO M19 clones contain detectable S2P mRNA (lanes 10-12).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

S2P stands out among the many proteases suggested to be gamma -secretase because it has several properties that make it an ideal candidate. Only S2P and gamma -secretase have been postulated to cleave substrates within the membrane in vivo (6, 9, 10). S2P is widely expressed as is gamma -secretase. S2P activity is (indirectly) enhanced by a transmembrane protein, SCAP, which is located in the endoplasmic reticulum (12), and gamma -secretase activity appears to require another endoplasmic reticulum transmembrane protein, PS1 (17). We therefore directly tested the hypothesis that the gamma -secretase is the same enzyme as the SREBP S2P (7). First we show that over-expression of the recently cloned S2P in cells expressing APPsw does not lead to any enhancement of the production of Abeta 40 or Abeta 42 (Fig. 2) suggesting that either the S2P is not the gamma -secretase or that gamma -secretase activity is not limiting. The immediate substrates of gamma -secretase are the ~10- and ~12-kDa C-terminal fragments (CTFs) generated by alpha - and beta -secretase cleavage of APP, respectively (see Fig. 1 and Ref. 30). Using antibodies that recognize these CTFs, we can detect both of these substrates in the 293 APPsw-expressing cells (data not shown), thus it would appear that the generation of additional Abeta (and p3) by over-expression of the authentic gamma -secretase could occur in these cells. Although we did not directly show that the S2P protein was enzymatically active in our transfected 293 cells, using a similar protocol, Rawson et al. (22) documented that S2P cleavage was restored and cholesterol auxotrophy of M19 cells was corrected in CHO cells expressing S2P. Because over-expression of the S2P mRNA in 293 cells did not lead to generation of additional Abeta 40 or Abeta 42 we conclude that it is unlikely to be the same enzyme as the gamma -secretase.

Second, using S2P-deficient CHO M19 (9, 19, 20, 22) we show here that these cells process transfected hu APPsw to both Abeta 40 and Abeta 42. Neither the amount nor the ratio of Abeta 40 or Abeta 42 produced by the mutant CHO M19 clones is different from that produced by the CHO WT clones. Based on these experimental results we conclude that the SREBP S2P is not identical to the gamma -secretase enzyme(s) responsible for the generation of the C termini of Abeta 40 and Abeta 42.

Although the similarities between gamma -secretase and the S2P are intriguing, there are a few differences. First, the substrates have different membrane insertion topology; APP is a type 1, single transmembrane protein (C terminus is cytosolic, N terminus is lumenal) whereas SREBP has two transmembrane domains (both N and C termini are cytosolic). Second, although it is not known how PS1 and/or PS2 regulate gamma -secretase activity (direct versus indirect), it is clear that SCAP directly interacts with and regulates the SREBP site 1 protease (18); thus the S2P cleavage is only indirectly regulated by SCAP.

Perhaps, as originally suggested by Brown and Goldstein (7), the S2P and gamma -secretase(s) are members of the same protease family. Rawson et al. (22) recently cloned the S2P, and although they found numerous related genes from different organisms (human, hamster, Sulfolobus sp., Drosophila sp., Caenorhabditis elegans, and Schistosoma mansoni), the S2P appears to be a novel member of a new family of metalloproteases. The cDNA encoding the S2P contains the signature sequence (HEXXH) of zinc metalloproteases; however it does not belong to any of the well recognized protease families (31, 32). Analysis of the sequences suggests that the S2P is a polytopic membrane protein with four or five transmembrane domains; even the hydrophilic active site, HEXXH, appears to be buried in a very hydrophobic region of the protein. These features, along with a serine repeat of variable length (in different species) and a cysteine-rich domain, may be hallmarks of this new family. Finding S2P related proteases based on these motifs or by direct homology screening may yet provide a fruitful end to the search for the elusive gamma -secretase(s).

    ACKNOWLEDGEMENTS

We thank our colleagues at Amgen for their contributions, especially Jean-Claude Louis and Frank Collins for their support of this research, Roland Lüthy for computational sequence analysis, Paul Denis for CTF assays, Cybil Sundgren and Michael Thomas of Amgen's DNA sequencing group, Joan Bennett for assistance in preparing the manuscript, and Vicki Gottmer for preparing the figures. Finally, we thank T. Y. Chang (Dartmouth) for generously providing the CHO M19 and WT cell lines and Rachael Neve (Harvard Medical School, McLean Hospital) for providing the APP695 cDNA.

    FOOTNOTES

* 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.

To whom correspondence should be addressed: Dept. of Mammalian Cell Molecular Biology, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320-1789. Tel.: 805-447-2493; Fax: 805-499-7464; E-mail: tburgess{at}amgen.com.

1 The abbreviations used are: AD, Alzheimer's disease; FAD, familial AD; APP, amyloid precursor protein; APPsw, Swedish mutation of APP695; CTF, C-terminal fragment; SRE, sterol regulatory element; SREBP, SRE-binding protein; S2P, SREBP site 2 protease; SCAP, SREBP cleavage activating protein; CHO, Chinese hamster ovary cells; PS1/PS2, presenilins 1 and 2, respectively; hu, human; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); Bicine, N,N-bis(2-hydroxyethyl)glycine.

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Abstract
Introduction
Procedures
Results
Discussion
References

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