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Control of APP processing and A␤ generation level by BACE1 enzymatic activity and transcription Yu Li,*,#,1 Weihui Zhou,*,1 Yigang Tong,* Guiqiong He,*,§ and Weihong Song*,2 *Department of Psychiatry, Brain Research Center, The University of British Columbia, Vancouver, BC, Canada; and #Department of Pathology, §Department of Anatomy, Chongqing University of Medical Sciences, Chongqing, China Deposition of amyloid ␤ protein (A␤) is one of the characteristic features of Alzheimer's disease (AD) neuropathology. Beta-secretase, a ␤-site APP cleaving enzyme 1 (BACE1), is essential for A␤ biosynthesis. Although inhibition of BACE1 is considered a valid therapeutic target for AD, the enzymatic dynamics of BACE1 in regulating APP processing and A␤ generation has not yet been fully defined. To examine this issue, tightly controlled inducible BACE1 gene expression was established in the neuronal cell line N2ABP1 and the non-neuronal cell line E2BP1 using an ecdysone-inducible system. The BACE1 protein level was increased in a time- and dosage-dependent manner in the inducible BACE1 stable cells by treatment with inducer ponasterone A. The generation of APP CTF␤, the ␤-secretase product, increased proportionally with the level of BACE1 protein expression. However, A␤40/42 production sharply increased to the plateau level with a relatively small increase in BACE1 expression. Although further increasing BACE1 expression increased ␤-secretase activity, it had no additional effect on A␤ production. Furthermore, we found that BACE1 mRNA levels and BACE1 promoter activity were significantly lower than APP mRNA levels and APP promoter activity. Our data demonstrate that lower BACE transcription is responsible for the minority of APP undergoing the amyloidogenic pathway and relatively lower A␤ production in the normal conditions, and that a slight increase in BACE1 can induce a dramatic elevation in A␤ production, indicating that the increase in BACE1 can potentially increase neuritic plaque formation in the pathological condition.—Li, Y., Zhou, W., Tong, Y., He, G., Song, W. Control of APP processing and A␤ generation level by BACE1 enzymatic activity and transcription. FASEB J. 20, 285–292 (2006) Key Words: BACE1 䡠 secretase 䡠 transcription 䡠 APP processing 䡠 amyloid ␤ 䡠 Alzheimer’s disease

zyme 1 (BACE1), is essential for A␤ biosynthesis. BACE1 is a type 1 membrane-associated aspartyl protease of 501 amino acids (1– 4). BACE1 cleaves APP at the major Asp⫹1 site of A␤ to generate C99 and at a minor Glu⫹11 site to release the C89 fragment (1). The major site of BACE1 cleavage is located between Met596 and Asp597 of the APP695 isoform. BACE1 is the ␤-secretase that processes APP in vivo (5–7). In addition to APP, BACE1 substrates include several other proteins: the low density lipoprotein receptor-related protein (LRP) (8), ␤ amyloid precursor-like protein-1 (APLP1) (9), -2 (APLP2) (10), a Golgi-resident sialyltransferase ST6Gal I (11), and the cell adhesion protein P-selectin glycoprotein ligand-1 (PSGL-1) (12). BACE1 undergoes a complex set of post-translational modifications during its maturation including removal of the pro-peptide (13–17), phosphorylation (15, 18, 19), and glycosylation (15, 18, 20, 21). BACE1 is also processed between Leu228 and Ala229 to generate stable N- and C-terminal fragments that remain covalently associated via a disulfide bond. Such proteolysis occurs primarily in the pancreas, liver and muscle, while holoprotein is predominantly in the brain (22). Inhibition of BACE1 shedding does not regulate APP processing at the ␤-site, and the shedding seems to have no major physiological significance in A␤ generation (23). BACE1 forms a dimer (24) prior to its full maturation and pro-peptide cleavage (25). Dimerization of BACE1 may help APP binding and cleavage. Reticulon family members were found to be binding partners of BACE1 which block access of BACE1 to APP and reduce APP cleavage (26). The degradation of BACE1 is mediated by the ubiquitin proteasome pathway and the proteasomal degradation of BACE1 regulates APP processing and A␤ generation (27). Although genetic analysis has failed to uncover any BACE1 coding sequence mutations in patients with familial AD (28, 29), increased ␤-secretase activity was reported in some FAD brains (30) and greater expression levels of BACE1 were

AD is a progressive neurodegenerative disorder characterized by senile plaques with deposition of A␤ in the brain of elderly patients with aging dementia. A␤ is the major component of the neuritic plaques and is generated from ␤-amyloid precursor protein (APP) by ␤- and ␥-secretases. Beta-secretase, a ␤-site APP cleaving en-

Y.L. and W.Z. contributed equally to this work. Correspondence: Department of Psychiatry, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada; Tel: 604-822-8019; Fax: 604-822-7756; Email: [email protected] doi: 10.1096/fj.05-4986com

ABSTRACT

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found in the cortex of sporadic AD patients vs. agematched controls (31–34). BACE1 protein and activity levels increase with aging and in brain regions affected by amyloid deposition and remain increased despite significant neuronal and synaptic loss in AD (33, 34). The AD-associated Swedish mutant APP (Lys595-Met596 to Asn595-Leu596) is associated with increased ␤-secretase activity (35, 36). siRNA suppression of BACE1 reduced CTF␤ and A␤ production in neurons derived from both wild-type and the Swedish APP mutant transgenic mice (37). BACE1-KO mice, without developmental deficits, have abolished A␤ generation (5–7). Disruption of the BACE1 gene rescues memory deficits and cholinergic dysfunction in the Swedish APP mutant mice (38). These results suggest that therapeutic inhibition of BACE1 is a valid therapeutic target for AD. One pharmaceutical strategy in AD therapy is to reduce Aß generation by inhibiting either ␤-secretase or ␥-secretase activity. Therapeutic inhibition of ␥-secretase might have a potentially severe side effect due to its effect on other substrates, including Notch (39, 40). However, disruption of BACE1 in a BACE1-KO mouse model causes little developmental and behavioral defects (5). Therefore, BACE1 could be a better anti-amyloid production target for AD therapeutic drug design. The enzymatic dynamics of BACE1 in regulating APP processing and A␤ generation are not fully defined. In the present study, we first established two BACE1-inducible expression cell lines using an ecdysone-inducible expression system. We found that there is a linear relation between the BACE1 protein level and the ␤-secretase activity level. However, a slight increase in BACE1 induces a dramatic elevation in A␤ production and A␤ generation quickly reached a plateau despite a further increase in ␤-secretase activity. Furthermore, BACE1 transcription was significantly lower than APP transcription. Our study indicates that lower BACE1 transcription is responsible for the minority of APP undergoing the amyloidogenic pathway and relatively lower A␤ production in the normal conditions and a slight increase of BACE1 can induce a dramatic elevation in A␤ production and can potentially facilitate neuritic plaque formation in the pathological condition.

MATERIALS AND METHODS cDNA constructs Human BACE1 cDNA was cut from pBACE1-mychis with Nhe I and Sal I and subcloned into pIND (SP1)/Hygro mammalian expression vector (Invitrogen, San Diego, CA, USA) at Nhe I and Xho I. The new plasmid construct was named pI-BACE1myc. Plasmid pVgRXR expresses the heterodimeric ecdysone receptor (VgEcR) and the retinoid X receptor (RXR) (Invitrogen). The pB1P-A plasmid contains the 2.1-kb 5⬘ UTR from –1944 to ⫹292 bp of the human BACE1 gene upstream of the luciferase reporter gene (42). A 2.94 kbp fragment containing the human APP promoter region was 286

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cut from pUCR5r and subcloned into pGL3-Basic vector at the Bgl II site to generate pAPP-Luc. The resulting promoter plasmid pAPP-luc contains the human APP gene promoter upstream of the luciferase reporter gene. Cell culture and transfection HEK293, Neuro-2a (N2a), and SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine, 50 U/mL penicillin G sodium, and 50 ␮g/mL streptomycin sulfate (Invitrogen). Stable cell lines were maintained in media containing zeocin and hygromycin B. The 20E2 cell line is a Swedish mutant APP695 stable HEK cell line (27). All cells were maintained at 37°C in an incubator containing 5% CO2. For transfection, cells were grown to ⬃70% confluence and transfected with 2 ␮g plasmid DNA/35 mm plate using 4 ␮L of Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer's instructions. Generation of stable BACE1-inducible cell lines To establish stable BACE1-inducible cell lines using the ecdysone-inducible mammalian expression system, the human BACE1 cDNA with myc tag was subcloned into an inducible vector pIND (SP1)/Hygro to generate pI-BACE1myc. This vector contains modified ecdysone response elements and SP1 enhancers, and activation of the BACE1 gene transcription is dependent on the binding of a heterodimer of VgEcR and RXR receptors in the presence of a ligand such as ecdysone analog ponasterone A. Mammalian cells lack the ecdysone receptor, and the pVgRXR plasmid was stably transfected into the cells to express the heterodimer of VgEcR and RXR receptors. N2a or 20E2 cells were cotransfected with pVgRXR (zeocin-resistant) and a pI-BACE1myc (hygromycin B-resistant) and selected in 500 ␮g/mL of zeocin (Invitrogen) and 1000 ␮g/mL hygromycin B (Invitrogen) to generate BACE1-inducible cell lines E2BP1 and N2ABP1, respectively. E2BP1 cells, selected from 20E2 cells, stably express Swedish APP695 and the ecdysone and retinoid X receptors, and contain inducible BACE1mycHis cDNA. N2ABP1 cells, originated from N2A cells stably express the ecdysone and retinoid X receptors and contain inducible BACE1mycHis cDNA. After 24 h of induction with 1 ␮M of the ecdysone analog ponasterone A inducer. BACE1 proteins were robustly expressed (Fig. 1). The basal levels of the BACE1 proteins were very low or almost absent without the ligand present. These stable cell lines allowed us to efficiently control the level of human BACE1 protein expression. Ponasterone A treatment Ponasterone A, an ecdysone analog, was obtained from Invitrogen and dissolved in ethanol. N2ABP1 and E2BP1 were treated with inducer ponasterone A for BACE1 expression. N2ABP1 cells were also transfected with Swedish APP695 cDNA. ponasterone A was added into cell cultures at 0, 0.25, 0.5, 1, or 2 ␮M for 24 h, or at 1 ␮M for 0, 12, 24, 36, or 48 h. Cells were lysed with RIPA-Doc buffer containing 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, and protease inhibitor cocktail Complete (Roche, Nutley, NJ, USA). The conditioned mediums were collected for the A␤ ELISA assay. Luciferase assay The luciferase assay was performed according to the manufacturer’s protocol (Promega, Madison, WI, USA). Cells were

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Figure 1. Inducible BACE1 stably transfected cells have robust BACE1 expression after ponasterone A treatment. Stable inducible cell lines were generated by transfecting pIBACE1myc and pVgRXR plasmids into 20E2 or N2a cells and selecting positive stably transfected cells with zeocin and hygromycin B. E2BP1 cells stably express Swedish APP695 and the ecdysone and retinoid X receptors, and contain inducible BACE1mycHis cDNA. N2ABP1 cells stably express the ecdysone and retinoid X receptor proteins and contain inducible BACE1mycHis cDNA. Cell lysates were analyzed by Western blot. The myc-tagged BACE proteins were detected by mouse monoclonal anti-myc antibody 9E10. Monoclonal anti ␤-actin antibody (AC-15) was used to detect ␤-actin. The myc-tagged BACE1 expression is robustly induced by ecdysone analog ponasterone A. harvested 48 h after transfection and lysed in 200 ␮L of 1⫻ Reporter Lysis Buffer for Dual Luciferase Assay (Promega). 2 ␮L of lysates was mixed with the firefly luciferase assay reagent II and the luminescent signal was measured using a TD 20/20 luminometer (Turner designs). Stop and Glo威 Reagent (10 ␮L) was added to the same tube. The Renilla (sea pansy) luciferase vector pCMV-Rluc (Promega) was cotransfected to normalize the transfection efficiency. The firefly luciferase activity was normalized according to Renilla luciferase activity and expressed as relative luciferase units (RLU) to reflect the promoter activity. The empty vector pGL3-basic was used as the negative control and pGL3 promoter as the positive control. Quantitative RT-PCR analysis Total RNA was isolated from cells using TRI reagent (Sigma, St. Louis, MO, USA). PowerScript reverse transcriptase (Invitrogen) was used to synthesize the first-strand cDNA from an equal amount of the RNA sample. The newly synthesized cDNA templates were further amplified by Platinum Taq DNA polymerase (Invitrogen). Twenty-five to 35 cycles of PCR were used to cover the linear range of the PCR amplification. The BACE1 gene-specific primers 5⬘-ACCGACGAAGAGCCCGAG and 5⬘-CACAATGCTCTTGTCATAG were used to amplify a 725-bp fragment of the BACE1 gene coding region. The APP gene-specific primers 5⬘-ACCGACGAAGAGTCGGAGGAG and 5⬘-CACAATGCTCTTGTCATAG were used to amplify a 250-bp fragment of the APP gene coding region. The primers 5⬘-GGACTTCGAGCAAGAGATGG-3⬘ and 5⬘-GAAGCATTTGCGGTGGAG-3⬘ were used to amplify a 462-bp fragment of the ␤-actin gene for internal controls. The samples were further analyzed on a 1.6% agarose gel. Kodak Image Station 1000 software (Perkin-Elmer, Oak Brook, IL, USA) was used to analyze the data.

fuged at 14,000 g for 10 min at 4°C and their protein concentration was determined by a microplate spectrophotometer (Milliken Ascent, Thermo lab) at a wavelength of 690 nm using DC Protein Assay kit (Bio-Red, Richmond, CA, USA). Cell lysates were subject to SDS-PAGE and transferred to PVDF membranes (Schleicher and Schell, Keene, NH, USA). The membranes were incubated with primary antibodies. All antibodies were diluted in 5% (v/v) nonfat dry milk and incubated with the PVDF membranes overnight at 4°C. Incubation with secondary peroxidase-conjugated anti-mouse or anti-rabbit antibodies was performed at room temperature for 1 h. The membranes were developed using ECL system (Habersham Pharmacia Biotech, Little Chalfont, and UK) and quantitated by Kodak Image Analysis. A␤40/42 Sandwich ELISA assay Conditioned media was collected from cells. Protein inhibitors and AEBSF were added to the media to prevent degradation of A␤ protein. The concentration of A␤40/42 was detected by ␤-amyloid 1-40 or 1-42 Colorimetric ELISA kit (Biosource International, Inc., Camarillo, CA, USA) according to the manufacturer’s instructions.

RESULTS Inducible expression of BACE1 in E2BP1 and N2ABP1 cells To assess inducible BACE1 expression, the stable cell lines were treated with vehicle solution control or ponasterone A for 24 h at 0.25, 0.5, 1, or 2 ␮M, or ponasterone A at 1 ␮M for 12, 24, 36, or 48 h. The myc-tagged BACE1 protein levels were analyzed by immunoblotting with anti-myc monoclonal antibody 9E10. ponasterone A, an ecdysone analog, binds to the ecdysone receptor and activates BACE1 gene transcription. Without an inducer there was little BACE1 expression in E2BP1 and N2ABP1 stable inducible cells. However, ponasterone A treatment resulted in a linear increase in BACE1 protein production in both stable cell lines (Fig. 2). The proteins encoded by the inducible BACE1 gene were induced at 0.25 ␮M of ponasterone A treatment and the highest expression occurred at 2 ␮M in E2BP1 cells (R2⫽0.9851) (Fig. 2A, B) and N2ABP1 cells (R2⫽0.9943) (Fig. 2E, F). The expression of BACE1 protein was detected after 12 h and increased to maximal levels at 48 h after ponasterone treatment in E2BP1 cells (R2⫽0.9960) (Fig. 2C, D) and N2ABP1 (R2⫽0.9688) (Fig. 2G, H). Ponasterone A treatment had no effect on ␤-actin protein levels. These results demonstrate that the expression level of BACE1 is induced by ponasterone A in a dose- and time-dependent manner in these two stable BACE1-inducible cell lines.

Western blot analysis

A linear relation between the levels in the BACE1 expression and ␤-secretase activity

Cells were lysed in RIPA-DOC buffer plus protease inhibitor cocktail (Roche) by sonication using Sonic Dismembrator 500 (Fisher, Fairlawn, NJ, USA). The cell lysates were centri-

To examine the effect of the BACE1 protein levels on ␤-secretase activity, the production of APP C-terminal

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on the expression level of BACE1 proteins in a linear relationship. A␤ production in the BACE1-inducible cells

Figure 2. Dose- and time-dependent inducible expression of BACE1 in E2BP1 cells and N2ABP1 cells. E2BP1 and N2ABP1 BACE1-inducible stable cells were treated with vehicle solution control or with ponasterone A for 24 h at 0, 0.25, 0.5, 1.0, or 2.0 ␮M for the dosage-dependent assay (A, E), or with ponasterone A at 1.0 ␮M for 0 to 48 h for the time course assay (C, G). Cells were harvested at the same endpoint of the treatment and were lysed in RIPA-DOC buffer. Cell lysates were analyzed by 12% Tris-glycine gel with 9E10 antibody to detect BACE1. Monoclonal anti ␤-actin antibody (AC-15) was used to detect ␤-actin. The left panel is representative of Western blot gels and the right panel represents quantitative analysis of the Western blots by Kodak Image Analysis. The values of the dots on the plots are mean ⫾ se from 3 independent experiments. ␤-Actin was not affected by the inducer ponasterone A.

fragments (CTF␤) in the BACE1-inducible cell lines was assayed by Western blot analysis. APP C99, the major ␤-secretase cleavage product and C89, the minor cleavage product, were detected with APP C-terminal antibody C20 (42). As shown in Fig. 3A, C, inducer ponasterone A significantly increased ␤-secretases activity, resulting in markedly elevated levels of APP CTF␤ in N2ABP1 cells, relative to control cells. Quantitative analysis showed that inducer ponasterone A treatment increased CTF␤ production in a dosage- and timedependent manner. The levels of CTF␤ production were elevated by 3.69 ⫾ 0.14, 6.86 ⫾ 0.33, 12.60 ⫾ 0.67, and 18.39 ⫾ 0.65-fold in N2ABP1 treated with ponasterone A at 0.25, 0.5, 1, and 2 mM, respectively (R2⫽0.9829) (Fig. 3A, B), and 3.63 ⫾ 0.11, 5.86 ⫾ 0.25, 11.97 ⫾ 0.36, and 17.08 ⫾ 0.55-fold for 12, 24, 36, and 48 h, respectively (R2⫽0.9532) (Fig. 3C, D). There was no change in ␤-actin protein level between the inducertreated cells and control cells. These results indicate that the enzymatic activity of ␤-secretase is dependent 288

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␤-Secretase cleavage of APP is essential for A␤ production. To examine whether up-regulating BACE1 expression also facilitates A␤ production in a linear manner, the A␤ level in the BACE1-inducible N2ABP1 cells was analyzed. The concentration of A␤40 and A␤42 was measured by using the ␤-amyloid 1-40 or 1-42 Colorimetric ELISA kit. As shown in Fig. 4A, C, compared with control, A␤40 and A␤42 productions were dramatically increased after treatment with ponasterone A at 0.25 ␮M (27.12⫾0.01 and 22.55⫾0.01-fold), and reached near plateau levels at 0.5 ␮M (34.35⫾0.10 and 31.42⫾0.01-fold). An increase in the ponasterone A dose to 1 and 2 ␮M had no additional effect on increases in A␤ production, and the levels of A␤40 and A␤42 were 37.30 ⫾ 0.12 and 37.77 ⫾ 0.01 at 1 ␮M (P⬎0.05), and 33.57 ⫾ 0.01 and 33.91 ⫾ 0.01 at 2 ␮M (P⬎0.05), respectively. The generation of A␤40 and A␤42 also quickly reached the plateau after a short induction of BACE1 gene expression by ponasterone A (Fig. 4B, D). The A␤ generation in the N2ABP1 cells was drastically induced by ponasterone A at 12 h (27.77⫾0.02-fold) and 24 h (32.16⫾0.02-fold). There was no difference in A␤40 production among 24, 36, and 48 h induction (32.16⫾0.02, 32.53⫾0.07, and 33.30⫾0.04-fold, respectively) (P⬎0.05). Similar results were obtained for the A␤42 production. The levels of A␤42 in N2ABP1 cells treated with ponasterone A were 23.39 ⫾ 0.36, 26.23 ⫾ 0.35, 28.02 ⫾ 0.03, and 28.38 ⫾ 0.03-fold for 12, 24, 36, and 48 h inductions. There was no significant difference in A␤42 production among

Figure 3. Increase in ␤-secretase activity by BACE1 protein level in a linear manner. N2ABP1 cells were treated with ponasterone A at 0, 0.25, 0.5, 1.0, and 2.0 ␮M for 24 h for dosage-dependent assay (A, B), or with ponasterone A at 1.0 ␮M for 0, 12, 24, 36, and 48 h for the time-dependent assay (C, D). Cell lysates were analyzed by 16% Tris-Tricine gel with antibody C20 to detect APP CTF␤ (C99 and C89) and AC-15 to detect ␤-actin. Western blots were quantitated by Kodak Image Analysis. The values of the dots on the plots are mean ⫾ se from 3 independent experiments. The generation of APP CTF␤, the ␤-secretase product, was proportionally elevated with the level of BACE1 protein expression.

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Figure 4. Increased production of A␤40 and A␤42 in BACE1inducible cell lines. N2ABP1 cells were treated with ponasterone A at 0, 0.25, 0.5, 1.0, and 2.0 ␮M for 24 h (A, C), or with ponasterone A at 1.0 ␮M for 0, 12, 24, 36, and 48 h for the time course assay (B, D). The conditioned media of cells was collected. The concentration of A␤40 (A, B) and A␤42 (C, D) were measured by ␤-amyloid 1-40 or 1-42 Colorimetric ELISA kit. The values represent mean ⫾ se, n ⫽ 3.

To examine if the difference in the mRNA levels was due to differences in promoter strength in regulating the transcriptional activation of these two genes, the human APP and BACE1 gene promoter activity in the different cell lines was measured. Plasmid pB1P-A and pAPP-Luc contain the human BACE1 and APP gene promoter in a promoterless plasmid vector pGL3-basic, respectively. The pGL3-basic vector lacks eukaryotic promoter and enhancer sequences upstream of a reporter luciferase gene. Expression of luciferase activity in cells transfected with this plasmid depends on proper insertion and orientation of a functional promoter upstream from the luciferase gene. The pGL3promoter plasmid contains a minimal SV40 promoter upstream of the luciferase gene without any enhancer elements. This plasmid lacking cell-specific expression was used as an internal control. The pGL3-promoter, BACE1 and APP gene promoter constructs were introduced into cells. The transcriptional activation ratios of the BACE1 or APP promoter to the pGL3-promoter control in HEK293 cells and neuroblastoma SH-SY5Y cells were determined (Fig. 5C). Transfection of pB1P-A resulted in robust luciferase expression in HEK293 and SH-SY5Y cells, and the BACE1 promoter

24, 36, and 48 h induction (P⬎0.05). These results demonstrate that a slight increase in BACE1 expression can induce a dramatic elevation in A␤ production and further up-regulating BACE1 expression has little additional effect on A␤ production. BACE1 gene had significantly lower transcription levels than APP gene APP is processed by ␣, ␤, and ␥ secretases. A␤ is generated from APP by the ␤ and ␥ secretase in an amyloidogenic pathway. However, the majority of APP protein undergoes ␣-secretase pathway, a non-amyloidogenic pathway, which precludes A␤ production. To examine if the BACE1 transcription is partially responsible for the minority of APP proteins undergoing amyloidogenic pathway to generate A␤, we examined human BACE1 and APP gene transcription in neuronal and non-neuronal cells. Quantitative RT-PCR experiments were performed to assay the endogenous mRNA levels of the human BACE1 and APP gene (Fig. 5A). There was significantly higher level of BACE1 mRNA in SH-SY5Y cells (3.40⫾0.11-fold) than in HEK293 cells (P⬍0.0001). The APP mRNA level in SH-SY5Y cells was similar to the level in HEK293 cells, 31.17 ⫾ 2.412-fold and 31.58 ⫾ 1.85-fold, respectively (P⬎0.05) (Fig. 5B). The endogenous mRNA levels of the human BACE1 gene were significantly lower than that of the human APP gene in both SH-SY5Y and HEK293 cells (P⬍0.001). These data suggest that the human BACE1 gene exhibits lower transcription than the human APP gene. REGULATION OF APP PROCESSING BY BACE1

Figure 5. BACE1 mRNA level and promoter activity were significantly lower than that of APP. Total RNA was isolated from cells. Quantitative RT-PCR was performed to measure the endogenous level of the BACE1 and APP mRNA. Specific BACE1, APP, and ␤-actin coding sequence primers were used to amplify the BACE1, APP, and ␤-actin cDNA, as described in Materials and Methods. A) Different cycles and amounts of PCR products were analyzed, and the DNA gel represents RT-PCR products on 1.2% agarose gel. B) The ratio of BACE1 or APP to ␤-actin gene transcription in the cells was quantitated by Kodak Image Analysis. Shown are mans ⫾ se, (n⫽3). *P ⬍0.001 by Student’s t test. The endogenous BACE1 mRNA level was significantly lower than the APP mRNA level in HEK293 and SH-SY5Y cells. C) Plasmid pGL3-basic, pGL3promoter, human BACE1 promoter plasmid pB1P-A and human APP promoter construct pAPP-Luc were transfected into HEK293 and SH-SY5Y cells. Plasmid pCMV-Rluc was cotransfected to normalize transfection efficiency. Cells were harvested at 48 h post transfection and dual luciferase assay was performed to measure luciferase activity. The values represent means ⫾ se (n⫽3). *P ⬍ 0.0001 by Student’s t test. 289

activity in SH-SY5Y cells was significantly higher than in HEK293 cells by a factor of 1.83 ⫾ 0.07 (P⬍0.01). These data are consistent with our previous report that BACE1 promoter activity was increased in neuronal cells relative to non-neuronal cells (42). However, there was no difference in the APP promoter activity between HEK293 and SH-SY5Y cells, 10.66 ⫾ 1.48 and 9.08 ⫾ 1.74-fold, respectively (P⬎0.05). Compared with pB1P-A, pAPP-Luc had a much stronger promoter activity in both HEK293 and SH-SY5Y cells (P⬍0.0001). These results indicate that the human BACE1 gene promoter has much weaker transcriptional activity than the human APP gene promoter in neuronal and non-neuronal cells.

DISCUSSION A␤ is generated from APP by sequential cleavages by ␤and ␥-secretases. BACE1 is the ␤-secretase in vivo. BACE2 is the homologue of BACE1 (1, 43, 44). Despite being homologous in amino acid sequence, BACE2 and BACE1 have distinct functions and transcriptional regulation. BACE2 is not a ␤-secretase, but processes APP within the A␤ domain at a site downstream of the ␣-secretase cleavage site (42). BACE1 is a type I transmembrane aspartyl protease. It is predominantly expressed in neuronal cells, and localizes to acidic compartments in the secretory pathway where A␤ production occurs (2– 4). A␤ generation is abolished in BACE1 knockout mice (5–7), whereas A␤ formation is increased by the overexpression of BACE1 in APP transgenic mice (45). Increased BACE1 expression has been implicated in the pathogenesis of AD (31–34). These results suggest that inhibition of BACE1 is a valid therapeutic target for AD. ␤-secretase (BACE1) triggers the amyloidogenic processing of APP, resulting in the deposition of A␤, the key component of senile plaques in Alzheimer's disease. Cleavage of APP by ␤-secretase is the first step in a process that is essential for A␤ generation. To investigate how BACE1 expression controls the dynamics of ␤-secretase in processing APP to generate A␤, we established tightly controlled BACE1-inducible cell systems. Our data show that ␤-secretase activity, measured by the amount of CTF␤ generation in cells, is dependent on the protein level of BACE1 expression. BACE1 facilitates ␤-secretase activity and CTF␤ generation in a linear manner, i.e., the more BACE1 protein, the higher the ␤-secretase activity. The generation of APP CTF␤, the ␤-secretase product, was proportionally elevated with the level of BACE1 protein expression. However, such a linear relationship between the BACE1 protein level and ␤-secretase activity cannot be translated into A␤ production. A␤40/42 production was sharply increased to plateau levels with a relatively small increase in BACE1 expression. Although further increasing BACE1 expression continuously increased ␤-secretase activity, resulting in more A␤ substrate C99 production, it had no additional effect on A␤ production. These results suggest that despite excessive sub290

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strate C99 generation by up-regulated BACE1 expression, ␥-secretase activity might limit additional A␤ generation (Fig. 6). We have preliminary data showing that inhibition of ␥-secretase by disruption of PS genes has no effect on the linear increase in C99 by upregulated BACE1 expression, indicating that the limiting factor might not be the ␣-secretase. Future studies will determine if increases in ␥-secretase activity, such as by FAD-associated PS mutations, will further potentiate A␤ generation in the BACE1-inducible cells. Our study demonstrates that any factors causing a slight increase in BACE1 can induce a dramatic elevation in A␤ production and the increase in BACE1 can potentially facilitate neuritic plaque formation in the pathological condition. APP undergoes amyloidogenic and non-amyloidogenic pathways. The non-amyloidogenic pathway via ␣-secretase is predominant under normal conditions. The amyloidogenic pathway via ␤-secretase and ␥-secretases accounts for the minority of APP processing, resulting in a very small amount of A␤ generation in the normal brain. The molecular mechanism behind this phenomenon has not yet been fully defined. BACE1 gene expression is tightly controlled at the transcriptional level and Sp1 and oxidative stress can facilitate BACE1 gene expression (41, 46). The human BACE1 promoter has higher activity in neuronal cells than in non-neuronal cells (42). Our data show that BACE1 gene transcription is much lower than APP gene transcription both in neuronal and non-neuronal cells. Although the APP gene is highly expressed in neuronal and non-neuronal cells, the expression of the BACE1 gene is relatively low. Quantitative RT-PCR assays show that BACE1 mRNA levels are markedly lower than the APP mRNA level in both neuronal and non-neuronal cells. A promoter assay was performed to further examine the molecular mechanism by which BACE1 expres-

Figure 6. Regulation of APP processing and A␤ generation by BACE1 enzymatic activity and transcription. The minority of APP protein undergoes ␤- and ␥-secretase cleavages to generate A␤ in the amyloidogenic pathway. ␤-Secretase activity is dependent on the protein level of BACE1 expression. However, a slight increase in BACE1 can induce a dramatic elevation in A␤ production and ␥-secretase activity might limit further A␤ generation. Furthermore, lower BACE transcription is responsible for the minority of APP undergoing the amyloidogenic pathway and relatively lower A␤ production under normal conditions.

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sion is lower than APP expression. The promoter assay reveals that BACE1 promoter activity is significantly lower than APP promoter activity, which is consistent with the differences found in endogenous mRNA levels. This indicates that the lower BACE1 gene expression resulted from the weaker BACE1 gene promoter, relative to the APP gene promoter. Our data suggest that lower BACE1 transcription is responsible for the minority of APP undergoing the amyloidogenic pathway and relatively lower A␤ production in the normal conditions (Fig. 6). Further studies may be warranted to investigate the transcription factors limiting BACE1 gene transcription and its pharmaceutical potentials. We thank Hong Qing, Odysseus Zis, and Diane Parsons for their technical assistance and helpful comments. We thank Dr. Debomoy Lahiri for providing pUCR5r plasmid. This work was supported by Canadian Institutes of Health Research (CIHR), Jack Brown and Family Alzheimer's Research Foundation, and Michael Smith Foundation for Health Research (W.S.). W.S. is the holder of the Canada Research Chair in Alzheimer's disease. Y.L. and G.H. were supported by the Chinese Scholarship Council award. W.Z. was the recipient of the Arthur and June Willms Fellowships and Y.T. was the recipient of the Michael Smith Foundation for Health Research Postdoctoral Fellowship.

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Received for publication August 24, 2005. Accepted for publication October 4, 2005.

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