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* 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
2 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: weihong{at}interchange.ubc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: BACE1 • secretase • transcription • APP processing • amyloid ß • Alzheimer’s disease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-secretases. Beta-secretase, a ß-site APP cleaving enzyme 1 (BACE1), is essential for Aß biosynthesis. BACE1 is a type 1 membrane-associated aspartyl protease of 501 amino acids (1
2
3
4)
. BACE1 cleaves APP at the major Asp+1 site of Aß 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 ß-secretase that processes APP in vivo (5
6
7)
. In addition to APP, BACE1 substrates include several other proteins: the low density lipoprotein receptor-related protein (LRP) (8)
, ß 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
14
15
16
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 ß-site, and the shedding seems to have no major physiological significance in Aß 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 Aß generation (27)
. Although genetic analysis has failed to uncover any BACE1 coding sequence mutations in patients with familial AD (28
, 29)
, increased ß-secretase activity was reported in some FAD brains (30)
and greater expression levels of BACE1 were found in the cortex of sporadic AD patients vs. age-matched controls (31
32
33
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 ß-secretase activity (35
, 36)
. siRNA suppression of BACE1 reduced CTFß and Aß production in neurons derived from both wild-type and the Swedish APP mutant transgenic mice (37)
. BACE1-KO mice, without developmental deficits, have abolished Aß generation (5
6
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 ß-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 Aß 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 ß-secretase activity level. However, a slight increase in BACE1 induces a dramatic elevation in Aß production and Aß generation quickly reached a plateau despite a further increase in ß-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 Aß production in the normal conditions and a slight increase of BACE1 can induce a dramatic elevation in Aß production and can potentially facilitate neuritic plaque formation in the pathological condition.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A 2.94 kbp fragment containing the human APP promoter region was 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 µg/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 µg plasmid DNA/35 mm plate using 4 µL 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 µg/mL of zeocin (Invitrogen) and 1000 µg/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 µM 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.
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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 µM for 24 h, or at 1 µM 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 Aß ELISA assay.
Luciferase assay
The luciferase assay was performed according to the manufacturer’s protocol (Promega, Madison, WI, USA). Cells were harvested 48 h after transfection and lysed in 200 µL of 1x Reporter Lysis Buffer for Dual Luciferase Assay (Promega). 2 µL 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® Reagent (10 µL) 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 ß-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.
Western blot analysis
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 centrifuged 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.
Aß40/42 Sandwich ELISA assay
Conditioned media was collected from cells. Protein inhibitors and AEBSF were added to the media to prevent degradation of Aß protein. The concentration of Aß40/42 was detected by ß-amyloid 1-40 or 1-42 Colorimetric ELISA kit (Biosource International, Inc., Camarillo, CA, USA) according to the manufacturer’s instructions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The proteins encoded by the inducible BACE1 gene were induced at 0.25 µM of ponasterone A treatment and the highest expression occurred at 2 µM 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 ß-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.
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A linear relation between the levels in the BACE1 expression and ß-secretase activity
To examine the effect of the BACE1 protein levels on ß-secretase activity, the production of APP C-terminal fragments (CTFß) in the BACE1-inducible cell lines was assayed by Western blot analysis. APP C99, the major ß-secretase cleavage product and C89, the minor cleavage product, were detected with APP C-terminal antibody C20 (42)
. As shown in Fig. 3
A, C, inducer ponasterone A significantly increased ß-secretases activity, resulting in markedly elevated levels of APP CTFß in N2ABP1 cells, relative to control cells. Quantitative analysis showed that inducer ponasterone A treatment increased CTFß production in a dosage- and time-dependent manner. The levels of CTFß 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 ß-actin protein level between the inducer-treated cells and control cells. These results indicate that the enzymatic activity of ß-secretase is dependent on the expression level of BACE1 proteins in a linear relationship.
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Aß production in the BACE1-inducible cells
ß-Secretase cleavage of APP is essential for Aß production. To examine whether up-regulating BACE1 expression also facilitates Aß production in a linear manner, the Aß level in the BACE1-inducible N2ABP1 cells was analyzed. The concentration of Aß40 and Aß42 was measured by using the ß-amyloid 1-40 or 1-42 Colorimetric ELISA kit. As shown in Fig. 4
A, C, compared with control, Aß40 and Aß42 productions were dramatically increased after treatment with ponasterone A at 0.25 µM (27.12±0.01 and 22.55±0.01-fold), and reached near plateau levels at 0.5 µM (34.35±0.10 and 31.42±0.01-fold). An increase in the ponasterone A dose to 1 and 2 µM had no additional effect on increases in Aß production, and the levels of Aß40 and Aß42 were 37.30 ± 0.12 and 37.77 ± 0.01 at 1 µM (P>0.05), and 33.57 ± 0.01 and 33.91 ± 0.01 at 2 µM (P>0.05), respectively. The generation of Aß40 and Aß42 also quickly reached the plateau after a short induction of BACE1 gene expression by ponasterone A (Fig. 4B, D
). The Aß 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 Aß40 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 Aß42 production. The levels of Aß42 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 Aß42 production among 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 Aß production and further up-regulating BACE1 expression has little additional effect on Aß production.
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BACE1 gene had significantly lower transcription levels than APP gene
APP is processed by 
, ß, and secretases. Aß is generated from APP by the ß and secretase in an amyloidogenic pathway. However, the majority of APP protein undergoes -secretase pathway, a non-amyloidogenic pathway, which precludes Aß production. To examine if the BACE1 transcription is partially responsible for the minority of APP proteins undergoing amyloidogenic pathway to generate Aß, 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. 5
A). 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.
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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 pGL3-promoter 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 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-secretases. BACE1 is the ß-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 ß-secretase, but processes APP within the Aß 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 Aß production occurs (2
3
4)
. Aß generation is abolished in BACE1 knockout mice (5
6
7)
, whereas Aß 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
32
33
34)
. These results suggest that inhibition of BACE1 is a valid therapeutic target for AD.
ß-secretase (BACE1) triggers the amyloidogenic processing of APP, resulting in the deposition of Aß, the key component of senile plaques in Alzheimer’s disease. Cleavage of APP by ß-secretase is the first step in a process that is essential for Aß generation. To investigate how BACE1 expression controls the dynamics of ß-secretase in processing APP to generate Aß, we established tightly controlled BACE1-inducible cell systems. Our data show that ß-secretase activity, measured by the amount of CTFß generation in cells, is dependent on the protein level of BACE1 expression. BACE1 facilitates ß-secretase activity and CTFß generation in a linear manner, i.e., the more BACE1 protein, the higher the ß-secretase activity. The generation of APP CTFß, the ß-secretase product, was proportionally elevated with the level of BACE1 protein expression. However, such a linear relationship between the BACE1 protein level and ß-secretase activity cannot be translated into Aß production. Aß40/42 production was sharply increased to plateau levels with a relatively small increase in BACE1 expression. Although further increasing BACE1 expression continuously increased ß-secretase activity, resulting in more Aß substrate C99 production, it had no additional effect on Aß production. These results suggest that despite excessive substrate C99 generation by up-regulated BACE1 expression, -secretase activity might limit additional Aß 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 up-regulated 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 Aß generation in the BACE1-inducible cells. Our study demonstrates that any factors causing a slight increase in BACE1 can induce a dramatic elevation in Aß 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 ß-secretase and -secretases accounts for the minority of APP processing, resulting in a very small amount of Aß 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 expression 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 Aß 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 24, 2005. Accepted for publication October 4, 2005.
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