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Originally published In Press as doi:10.1074/jbc.M608856200 on February 15, 2007

J. Biol. Chem., Vol. 282, Issue 15, 10873-10880, April 13, 2007
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Hypoxia-inducible Factor 1{alpha} (HIF-1{alpha})-mediated Hypoxia Increases BACE1 Expression and beta-Amyloid Generation*

Xian Zhang{ddagger}§1, Kun Zhou§1, Ruishan Wang{ddagger}§, Jiankun Cui§, Stuart A. Lipton§, Francesca-Fang Liao§, Huaxi Xu{ddagger}§2, and Yun-wu Zhang{ddagger}§3

From the {ddagger}Institute for Biomedical Research and School of Life Sciences, Xiamen University, Xiamen 361005, China, the Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China, and the §Burnham Institute for Medical Research, La Jolla, California 92037

Received for publication, September 14, 2006 , and in revised form, January 22, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The incidence of Alzheimer disease (AD) and vascular dementia is greatly increased following cerebral ischemia and stroke in which hypoxic conditions occur in affected brain areas. beta-Amyloid peptide (Abeta), which is derived from the beta-amyloid precursor protein (APP) by sequential proteolytic cleavages from beta-secretase (BACE1) and presenilin-1 (PS1)/{gamma}-secretase, is widely believed to trigger a cascade of pathological events culminating in AD and vascular dementia. However, a direct molecular link between hypoxic insults and APP processing has yet to be established. Here, we demonstrate that acute hypoxia increases the expression and the enzymatic activity of BACE1 by up-regulating the level of BACE1 mRNA, resulting in increases in the APP C-terminal fragment-beta (betaCTF) and Abeta. Hypoxia has no effect on the level of PS1, APP, and tumor necrosis factor-{alpha}-converting enzyme (TACE, an enzyme known to cleave APP at the {alpha}-secretase cleavage site). Sequence analysis, mutagenesis, and gel shift studies revealed binding of HIF-1 to the BACE1 promoter. Overexpression of HIF-1{alpha} increases BACE1 mRNA and protein level, whereas down-regulation of HIF-1{alpha} reduced the level of BACE1. Hypoxic treatment fails to further potentiate the stimulatory effect of HIF-1{alpha} overexpression on BACE1 expression, suggesting that hypoxic induction of BACE1 expression is primarily mediated by HIF-1{alpha}. Finally, we observed significant reduction in BACE1 protein levels in the hippocampus and the cortex of HIF-1{alpha} conditional knock-out mice. Our results demonstrate an important role for hypoxia/HIF-1{alpha} in modulating the amyloidogenic processing of APP and provide a molecular mechanism for increased incidence of AD following cerebral ischemic and stroke injuries.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
An important pathologic feature of Alzheimer disease (AD)4 is formation of extracellular senile plaques in the brain, whose major components are small peptides called beta-amyloid (Abeta) derived from beta-amyloid precursor protein (APP). APP is sequentially cleaved first by the beta-secretase (beta-site amyloid precursor protein cleaving enzyme, BACE) and then by the {gamma}-secretase complex (including presenilin, nicastrin, APH-1, and PEN-2) to generate the heterogeneous Abeta species, mostly Abeta40 but also the more deleterious Abeta42. Alternatively, APP can be cleaved by {alpha}-secretase within the Abeta domain to generate non-amyloidogenic soluble APP{alpha} (sAPP{alpha}) (1–3). The exact {alpha}-secretase is not known, but a disintegrin and metalloprotease domain 10 (ADAM10) and TNF-{alpha}-converting enzyme (TACE) are two likely candidates (4, 5). It is widely believed that Abeta overproduction directly or indirectly initiates a cascade of neurodegenerative steps resulting in formation of senile plaques, neurofibrillary tangles, and neuronal loss, which characterize AD (6). Hence analysis of cellular regulation affecting Abeta generation, including identification of factors regulating the level/activity of APP cleavage enzymes, should provide invaluable information for AD therapeutic intervention.

BACE is a membrane-bound aspartic protease whose activity is the rate-limiting step in Abeta production from APP. Among two BACE homologs, BACE1 and BACE2, BACE1 is the major protease for beta-cleavage of APP (7–12). Mice deficient in BACE1 are viable and show an almost total loss of Abeta (13–15). These findings, together with other biochemical studies, indicate that BACE1 is an ideal therapeutic target for blocking Abeta production. However, identification of small molecules specifically targeting BACE1 and not other aspartic proteases has been challenging. In addition, delivering inhibitory small molecules across the cell membrane and blood-brain barrier poses another difficulty. Recently, the BACE1 promoter was defined, and several transcription factors regulating BACE1 expression identified, including SP1 (16), PPAR{gamma} (17), and Yin Yang 1 (YY1) (18). Interestingly, protein levels of BACE1 and some of these transcription factors have been found altered in AD brain regions containing Abeta plaques, suggesting that increased BACE1 expression resulting from altered levels of its transcriptional activators may contribute in part to AD pathogenesis (17–20).


Figure 1
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  		FIGURE 1.
Acute hypoxia increases protein levels and activity of BACE1 and secretion of Abeta. N2a-APP cells were treated with 1% O2 for 2, 4, or 8 h. A, equal amounts of protein from lysates were analyzed and immunoblotted with antibodies against HIF-1{alpha}, BACE1, PS1-NTF, TACE, APP, and {alpha}-tubulin (as a loading control). B, lysates of hypoxia-treated and untreated N2a-APP cells were assayed for beta-secretase activity using BACE1 activity assay kit. C, lysates of N2a-APP cells were immunoblotted with the 6E10 antibody, which detects human APP betaCTF (upper panel). Conditioned media were assayed for Abeta by immunoprecipitation with the 4G8 antibody, followed by Western blot analysis using 6E10 (lower panel). D, conditioned media were collected and secreted Abeta40 and Abeta42 were quantified using commercial ELISA kits. All data represent means ± S.E. of levels relative to that of control from three independent experiments. *, p < 0.05, hypoxia-treated versus untreated samples at the indicated time. C, control; H, hypoxia.

 
Hypoxia-inducible factor 1 (HIF-1) is a major transcription factor that responds to cellular oxygen reduction. HIF-1 has two subunits, HIF-1{alpha} and HIF-1beta. HIF-1{alpha} levels are low under physiological conditions but increase dramatically under hypoxia. HIF-1{alpha} binds to HIF-1beta to form functional HIF-1 heterodimers, consequently regulating a series of transcriptional events (21). HIF-1{alpha} is essential for adaptation to low oxygen levels, and a complete deficiency in HIF-1{alpha} leads to embryonic lethality (22). Cerebral ischemia results from an insufficient oxygen supply to the brain and causes neuronal damage in vulnerable brain areas (23). Accumulating evidence indicates that cerebral ischemia/stroke significantly increases AD risk (24–26). Moreover, APP expression is elevated in post-ischemic brain, and cleavage of APP leading to amyloidogenic Abeta peptides may hence be increased by ischemia (27–29). Although it is likely that cellular stresses such as ischemia or hypoxia facilitate or worsen the pathogenesis of AD, molecular links between these conditions have yet to be established. We recently characterized the promoter of the APH-1a gene, which encodes a key component of the PS1/{gamma}-secretase complex, and identified a functional HIF-1 binding element. We further demonstrated that activation of HIF-1 by short-term hypoxia increases expression of APH-1a mRNA and protein, leading to increased {gamma}-cleavage of APP and the Notch receptor (30). Sequence analysis of the mouse BACE1 promoter revealed a putative HIF-1 binding element, although there are no reports of regulation of BACE1 mRNA expression by HIF-1 or hypoxia. Given that BACE1 cleavage of APP is a prerequisite for {gamma}-cleavage and thus widely viewed as the key regulatory step in inhibiting Abeta production, we investigated the effects of acute hypoxia (up to 8 h) and HIF-1 on BACE1 expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Transfection, and Hypoxic Treatment—Mouse neuroblastoma N2a cells stably expressing human APP695 (N2a-APP) were maintained in cell culture medium containing 50% DME high glucose medium (Hyclone, Logan, CT), 50% OptiMEM medium (Invitrogen, Carlsbad, CA), 5% fetal bovine serum (Hyclone, Logan, CT), and 0.2 mg/ml G418 (Omega Scientific, Tarzana, CA). N2a-APP cells were transiently transfected with HIF-1{alpha} cDNA or pcDNA (as control) using FuGENE 6 (Roche Applied Science, Indianapolis, IN), following the manufacturer's instructions. Cells were harvested 48 h after transfection. For hypoxic treatment, N2a-APP cells were incubated in a 37 °C chamber containing 1% O2 and 5% CO2 for 2, 4, and 8 h.

Immunoblotting and Antibodies—Treated cells were lysed in RIPA (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, supplemented with a protease inhibitor mixture). Equal amounts of lysates were analyzed and immunoblotted as indicated. Mouse anti-HIF-1{alpha} antibody was from Novus Biologicals (Littleton, CO). Rabbit anti-BACE1 antibody B690, rabbit antibody 369 against the APP C terminus, and rabbit antibody Ab14 against the PS1 N terminus were developed in our laboratory (31–34). Rabbit anti-TACE antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-{alpha}-tubulin antibody was from Sigma. Monoclonal antibodies 6E10 and 4G8 (Signet Laboratories, Dedham, MA) that recognize amino acids 1–17 and 17–24 of human Abeta peptide, respectively, were used to detect Abeta and APP betaCTFs.

Abeta ELISA Assay—Conditioned media from hypoxia-treated and untreated cells were collected. The concentrations of Abeta40 and Abeta42 were quantified using commercial ELISA kits (Invitrogen, Carlsbad, CA), following the manufacturer's protocols.

In Vitro beta-Secretase Activity Assay—Lysates of hypoxia-treated or untreated cells were assayed for beta-secretase activity using a kit from Calbiochem (San Diego, CA), following the manufacturer's protocol.

Pulse-Chase Experiments—To assay the kinetics of BACE1 metabolism, 4-h hypoxia-treated and untreated N2a-APP cells were labeled with [35S]methionine (500 µCi/ml) for 5 or 10 min at 37 °C and collected for analysis. In some experiments, N2a-APP cells were pretreated for 4 h with hypoxia and then subjected to 15 min of pulse-labeling followed by chase for 15, 30, 60, and 120 min under hypoxic conditions. Cell lysates were immunoprecipitated with anti-BACE1 antibody B690, followed by SDS-PAGE analysis and autoradiography. Data were quantified using Scion Image (Scion Corp., Frederick, MD). The level of labeled BACE1 after 5 min of labeling of untreated cells was defined as one arbitrary unit.

Quantitative Real-time Polymerase Chain Reaction (RT-PCR)—Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). SuperScript First-Strand kit (Invitrogen) was used to synthesize first strand cDNA from samples with an equal amount of RNA, according to the manufacturer's instruction. Synthesized cDNAs were amplified using IQTM SYBR green supermix and ICycler from Bio-Rad; data were analyzed using Bio-Rad MyIQ 2.0. Primers used for BACE1 amplification were: BACE1–5, 5'-GATGGTGGACAACCTGAG-3' and BACE1–3, 5'-CTGGTAGTAGCGATGCAG-3'. Primers used for beta-actin amplification were: actin-5, 5'-AGCCATGTACGTAGCCATCC-3' and actin-3, 5'-CTCTCAGCTGTGGTGGTGAA-3'. BACE1 mRNA levels were normalized to levels of beta-actin. Three independent experiments were performed, and statistical analysis was carried out using the Student's t test.

Construction of Luciferase Reporter Plasmids, Site-directed Mutagenesis, and Electrophoretic Mobility Shift Assay (EMSA)—PCR was performed to amplify a 5'-flanking region of the BACE1 gene by using genomic DNA from mouse N2a cells as template. Primers used were BACE primer5, 5'-GGCTGGCATGCATGACAGGGTGCGCACGGGGGTGTG-3' and BACE primer3, 5'-CAGCACCTAGGCAGGCTGGGGAGGCGGAAAGGCTTG-3'. The mutBACE primer5, 5'-GGCTGGCATGCATGACAGGGTGCGTCACGGGGTGTG-3', was paired with BACE primer3 for PCR amplification to introduce mutations (boldface and underlined) into the potential HIF-1 binding site. After amplification, PCR products were inserted into the pCR2.1-TOPO vector (Invitrogen) for sequencing. After cleavage of the vectors with KpnI and XhoI, released fragments were introduced into the pGL3-enhancer vector containing the firefly luciferase gene (Promega, Madison, WI). Firefly luciferase vectors were co-transfected with phRL-SV40 containing the Renilla luciferase gene (Promega) into N2a cells, and the cells were treated with or without hypoxia for 4 h. Firefly luciferase activities were assayed and normalized to those of Renilla luciferase.

EMSA was performed as previously described (30, 35). Briefly, nuclear extracts from N2a-APP cells treated under hypoxia for 4 h were prepared using a nuclear extract kit (Activemotif, Carlsbad, CA). Oligonucleotides were synthesized and annealed with their respective reverse complements to generate the following double-stranded oligonucleotides: wild-type BACE1 HIF-1 containing the potential HIF-1 binding site in the mouse BACE1 promoter (5'-CAGGGTGCGCACGGGGGTGTGGG-3'); mutant BACE1 HIF-1 with an CACG -> TCAC mutation (boldface and underlined) (5'-CAGGGTGCGTCACGGGGTGTGGG-3'); and a HIF-1 consensus probe (5'-ACCGGCCCTACGTGCTGTCTCAC-3') (37). The SP1 consensus probe was provided in the Gel Shift Assay Core System (Promega). Wild-type BACE1 HIF-1 and putative HIF-1 probes were labeled with [{gamma}-32P]ATP and incubated with N2a nuclear extracts with or without unlabeled BACE1 HIF-1 competitor (wild type or mutant form). Samples were analyzed by non-denaturing PAGE and autoradiography.


Figure 2
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  		FIGURE 2.
Acute hypoxia increases biogenesis of BACE1 by up-regulating transcription. A, N2a-APP cells were treated with hypoxia for 4 h and then pulse-labeled with [35S]methionine (500 µCi/ml) for 5 or 10 min. In some experiments, 4-h hypoxia-treated cells were first pulse-labeled with [35S]methionine for 15 min and then chased for various time periods under hypoxic conditions. Equal amounts of protein in cell lysates were immunoprecipitated with an anti-BACE1 antibody, followed by SDS-PAGE analysis and autoradiography. BACE1 levels were quantified (by densitometry of autoradiographic bands), and the level of labeled BACE1 after 5 min labeling of untreated cells was defined as one arbitrary unit. B, total RNA was extracted from hypoxia-treated or untreated cells and reverse-transcribed for quantitative real time-PCR. BACE1 mRNA levels were normalized to beta-actin and compared with untreated controls (defined as one arbitrary unit). Data represent means ± S.E. from three independent experiments. *, p < 0.05, hypoxia-treated versus untreated samples at indicated time point.

 
HIF-1{alpha} RNA Interference—The mouse HIF-1{alpha} siRNA sequence used was 5'-AAGCAUUUCUCUCAUUUCCUCAUGG-3' (38). The control siRNA sequence was from Invitrogen. siRNA was transfected into cells using Lipofectamine RNAiMAX reagent (Invitrogen), following the manufacturer's protocol.

Tissue Isolation from HIF-1{alpha} Conditional Knock-out Mice—The hippocampus and cortex were dissected from HIF-1{alpha} conditional knock-out mice (in which Cre is driven by the calcium/calmodulin-dependent kinase CaMKII{alpha} promoter) and littermates at 2 months of age (39). Samples were either lysed in RIPA for immunoblotting or used for RNA extraction and quantitative RT-PCR.


Figure 3
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  		FIGURE 3.
Identification of a functional HIF-1 binding site in the mouse BACE1 promoter. A, a potential HIF-1 binding site identified on the minus strand of the mouse BACE1 promoter. Numbering is relative to the translation start site (defined as +1). The HIF-1 binding site is underlined and in boldface. Small capitals represent sites mutated to block DNA binding. B, luciferase reporter plasmids carrying the wild type or mutant mouse BACE1 promoter regions were co-transfected with the Renilla luciferase reporter plasmid into N2a-APP cells; and the cells were treated with or without hypoxia for 4 h. Relative luciferase activity was determined in triplicate, and data were normalized to Renilla luciferase activity. Data represent means ± S.E. *, p < 0.05 versus the wild type. C, gel shift assays of the HIF-1 binding site in the BACE1 promoter. The consensus HIF-1 binding probe (P, lane 1) and the BACE1 HIF-1 binding probe (lane 2) were labeled with [{gamma}-32P]ATP and incubated with nuclear extracts from N2a-APP cells treated under hypoxia for 4 h. Competition assays were performed with unlabeled HIF-1 consensus probe (lane 3), BACE1 HIF-1 wild-type probe (lane 4), BACE1 HIF-1 mutant probe (lane 5), or SP1 probe (lane 6).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Acute Hypoxia Increases the Protein Level and Activity of BACE1 by Up-regulating Its Transcription—To analyze effects of acute hypoxia on key proteins involved in Abeta generation, we treated mouse neuroblastoma N2a cells stably expressing human APP695 with hypoxic conditions (1% O2) for 2, 4, and 8 h. As shown in Fig. 1A, hypoxia significantly increased the protein level of BACE1, accompanied by a concomitant increase in HIF-1{alpha} protein levels, as expected. The levels of TACE, the N terminus of PS1 (PS1-NTF) and full-length APP were not affected by hypoxic treatment. A modest but significant increase in total BACE1 activity was observed, reflecting increased cellular protein levels (Fig. 1B). Consistent with our previous observation (30), hypoxic treatment led to a marked increase in overall levels of secreted Abeta (both Abeta40 and Abeta42) (Fig. 1, C and D). While we previously observed a modest decrease in the total APP CTFs in HeLa cells by chemical hypoxia, the current study showed a slight increase in APP betaCTF, an immediate cleavage product of BACE1, by low oxygen hypoxic treatments in N2a cells (Fig. 1C).

The increased steady state level of BACE1 may result from increased biogenesis, decreased degradation, or both. To determine at which step(s) hypoxia affects BACE1 expression, we undertook pulsechase experiments. Cells pretreated with hypoxia were pulse-labeled with [35S]methionine for 5 or 10 min without chase, or 15 min followed by chase periods up to 120 min under hypoxia. Biosynthesis of nascent BACE1 was significantly higher in cells treated with hypoxia than in untreated cells, whereas turnover curves for both hypoxia-treated and untreated cells were similar, suggesting that hypoxia mainly affects biosynthesis rather than degradation of BACE1 protein (Fig. 2A). Quantitative real-time PCR analysis showed 1.5–2-fold increases in BACE1 mRNA following hypoxic treatment, confirming the effect of hypoxia on BACE1 transcription (Fig. 2B).

Identification of a Functional HIF-1{alpha} Binding Element in the BACE1 Promoter—Given that HIF-1 is a critical transcription factor activated in response to hypoxic stresses, we looked for a potential HIF-1 binding element in the BACE1 promoter region and asked whether binding by HIF-1 could be responsible for up-regulation of BACE1 transcription under hypoxic conditions. Indeed, sequence analysis of the 5'-flanking sequence of the mouse BACE1 gene revealed a potential HIF-1 binding site on the minus strand at –835 to –821 (acgcGTGCccccaca) upstream of the BACE1 start ATG (defined as +1) (Fig. 3A). To determine whether this site was important for promoter activity, we constructed luciferase reporter vectors containing this region or a mutant form that lacks HIF-1 binding ability and transfected both constructs into N2a-APP cells. As expected, the promoter activity of this BACE1 promoter region was significantly higher in cells treated under hypoxia than in untreated cells (Fig. 3B). Moreover, mutation of the putative HIF-1 binding site resulted in a significant reduction in promoter activity in untreated cells and hypoxia did not significantly potentiate the promoter activity of this mutant (Fig. 3B). These results indicate the functionality of this HIF-1 site. To further investigate the interaction between this HIF-1 binding site and HIF-1 protein, we performed gel shift analysis. Such an analysis revealed a DNA-protein complex forming after incubation of this probe with nuclear extracts of N2a-APP cells treated under hypoxia for 4 h (Fig. 3C, lane 2). This complex migrated at the same position as a HIF-1 consensus probe-protein complex (positive control, Fig. 3C, lane 1). Formation of the BACE1 HIF-1 probe-protein complex was abolished by competitive binding of unlabeled HIF-1 consensus probe and BACE1 HIF-1 probe (Fig. 3C, lanes 3 and 4) but was not affected in the presence of unlabeled BACE1 HIF-1 mutant probe (Fig. 3C, lane 5) or SP1 probe (Fig. 3C, lane 6). These data support the idea that the identified BACE1 HIF-1 binding element can form a complex with nuclear HIF-1 protein.


Figure 4
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  		FIGURE 4.
HIF-1{alpha} regulates BACE1 expression. A, N2a-APP cells were transfected with 5 µg of pcDNA (as Control), or 2 or 5 µg of HIF-1{alpha} expression vector for 48 h. Equal amounts of lysate proteins were analyzed and immunoblotted with antibodies against HIF-1{alpha}, BACE1, PS1-NTF, TACE, or {alpha}-tubulin as a control. B, N2a-APP cells were transfected with 5 µg of pcDNA or HIF-1{alpha} expression vector for 48 h. Total RNA was extracted, reverse transcribed, and subjected to quantitative RT-PCR. BACE1 mRNA levels were normalized to beta-actin and compared with that of the pcDNA vector control (defined as one arbitrary unit). C, cells were transfected with HIF-1{alpha} siRNA or control siRNA for 24–48 h and then treated with or without hypoxia for 4 h. Cells were lysed, and the lysates were subjected to immunoblotting with antibodies against HIF-1{alpha}, BACE1, or {alpha}-tubulin. C, control; H, hypoxia. Data represent means ± S.E. from three independent experiments. *, p < 0.05.

 
HIF-1{alpha} Regulates the Level of BACE1—To determine whether HIF-1{alpha} overexpression mediates effects on BACE1 expression similar to those of hypoxia, we transfected N2a-APP cells with various doses (0, 0.5, 1, 2, 5, and 10 µg; only 0, 2, and 5 µg were shown here) of HIF-1{alpha} cDNA. Following HIF-1{alpha} overexpression we observed a maximum of an ~2.5-fold increase in BACE1 protein level at 2 µg of HIF-1{alpha} DNA. We also observed a 2-fold increase in BACE1 mRNA level, along with minimal changes in levels of PS1 NTF or TACE (Fig. 4, A and B). The observation that a higher dose (5 µg) of HIF-1{alpha} expression failed to further up-regulate BACE1 expression suggests that the regulatory effect of HIF-1 on BACE1 expression is saturable.

On the other hand, we tested whether down-regulating HIF-1{alpha} could reduce the level of BACE1. As shown in Fig. 4C, when the level of HIF-1{alpha} was down-regulated (by ~80%) by siRNA treatment, the level of BACE1 was also reduced in both hypoxia-treated and untreated cells. However, hypoxic treatment still stimulated, but to a much lesser extent, BACE1 expression in HIF-1{alpha}-down-regulated cells (lane 3 versus lane 4), compared with that in control siRNA-transfected cells (lane 1 versus lane 2); and this could be attributed to the incomplete knock-out of HIF-1{alpha}. This result indicates a direct involvement of HIF-1{alpha} in hypoxia-mediated BACE1 up-regulation.

The Hypoxic Effect Is Mediated Mainly by HIF-1—To determine whether effects of HIF-1{alpha} overexpression and hypoxia on BACE1 expression are additive, we treated HIF-1{alpha} overexpressing N2a cells with hypoxic conditions for various time periods. As shown in Fig. 5, while cellular HIF-1{alpha} levels were further elevated by hypoxia, the levels of BACE1 mRNA did not increase. Similarly, combining hypoxic treatment with overexpression of HIF-1{alpha} did not further increase Abeta levels (Fig. 5A). Together with the siRNA knockdown data shown in Fig. 4C, these results strongly suggest that the hypoxic effect of BACE1 expression and Abeta generation are largely mediated by HIF-1.

BACE1 Protein Levels Are Reduced in Brains of HIF-1{alpha} Conditional Knock-out Mice—Because BACE1 activity is higher in neuronal than in non-neuronal tissues, and neuronal Abeta in particular is thought to be the major contributor to AD pathogenesis, we analyzed the patho-/physiological relevance of HIF-mediated BACE1 expression using a mouse model of neuron-specific HIF-1{alpha} deficiency (39). A total deficiency of HIF-1{alpha} mRNA in isolated tissues was confirmed by RT-PCR (data not shown). We then examined and compared BACE1 protein levels in total brain lysates of conditional knock-out mice with those from lysates from littermate controls and observed no significant differences in BACE1 total protein (data not shown). We then dissected the hippocampus and the cortex, two brain regions most affected in AD, from conditional knock-out and littermate control mice and found that HIF-1{alpha} deficiency led to a marked reduction in BACE1 protein levels in these two brain regions, suggesting an important role for HIF-1 in regulating BACE1 expression (Fig. 6).


Figure 5
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  		FIGURE 5.
Acute hypoxia does not potentiate BACE1 expression in HIF-1{alpha}-overexpressing cells. N2a-APP cells were transfected with a HIF-1{alpha} expression vector for 24 h and then divided into 6 plates and treated with or without hypoxia for 2, 4, or 8 h. A, equal amounts of cell lysate proteins were immunoblotted with antibodies against HIF-1{alpha}, BACE1, and {alpha}-tubulin as a control. Abeta in conditioned media was assayed by immunoprecipitation using the 4G8 antibody followed by Western blot analysis with the 6E10. B, total RNA was extracted and reverse-transcribed for RT-PCR. BACE1 mRNA levels were normalized to beta-actin. Data represent means ± S.E. from three independent experiments. C, control; H, hypoxia.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
BACE1 provides the predominant neuronal beta-secretase activity and is the major protease catalyzing beta-cleavage of APP (7–12). Several studies indicate that protein levels and BACE1 activity are elevated in brain regions affected by AD, suggesting that abnormal BACE1 activity contributes significantly to AD pathogenesis (19, 20). Ischemia or stroke gives rise to hypoxic conditions known to greatly increase the incidence of AD (24–26). These observations indicate a potential link between hypoxia-activated signaling pathways triggering HIF-1 induction and activation of the Abeta generation machinery, which requires up-regulation of BACE1 level/activity.

Although several studies have addressed the effects of ischemia or hypoxia on enzymes involved in APP processing/Abeta production, the results have been contradictory. Wen et al. (40) found that both activity and expression of BACE1 were significantly increased in rats under transient cerebral ischemia. Another study, however, using AD transgenic mice overexpressing mutant APP showed that neither expression nor activity of BACE was significantly affected by ischemic insult; instead ADAM10 was markedly increased in the early stage of ischemic insult and then down-regulated at later stages (41). Other studies report a drastic decrease in ADAM10 and TACE protein levels with unchanged BACE1 levels in human neuroblastoma SH-SY5Y cells subjected to chronic hypoxic treatments (42–44). While it is hard to reconcile these results, differing experimental procedures, such as cells/tissues examined, culture conditions, the percentage of O2 utilized, or duration of hypoxic treatment, could all contribute to discrepant results. In fact, we observed that effects of hypoxia and/or elevated levels of HIF-1{alpha} on BACE1 expression and Abeta generation were gradually reduced when cells were exposed to prolonged periods of hypoxic stress or when high levels of HIF-1{alpha} expression were sustained (data not shown); both outcomes could be due to the cellular ability to adapt to deleterious conditions. Cellular adaptation to a non-physiological condition such as HIF-1{alpha} deficiency is also manifested by the fact that non-neuronal cells and/or cells of brain regions other than the forebrain can compensate for decreases in BACE1 expression.5 Our study provides the first evidence for a link between ischemia/hypoxia and APP processing/Abeta production at the molecular level, which is defined mechanistically by transcriptional regulation of BACE1 by HIF-1. In addition, our observation of reduced BACE1 expression in the hippocampus and cortex of HIF-1{alpha}-deficient mice further emphasizes the patho-physiological relevance of regulation of BACE1 expression and activity by hypoxia/HIF-1 pathways.


Figure 6
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  		FIGURE 6.
Decreased BACE1 expression in the hippocampus and cortex of HIF-1{alpha} conditional knock-out mice. Hippocampus and cortex were dissected from 6 HIF-1{alpha} conditional knock-out mice (HIF-1 KO) and 5 littermates (control) at 2 months of age, and samples were lysed in RIPA buffer. Equal amounts of lysate proteins were analyzed and immunoblotted with antibodies against BACE1, PS1-NTF, TACE, APP/APP CTFs, and {alpha}-tubulin. Data represent means ± S.E. p < 0.05 compared with littermate controls.

 
Although a recent study showed that when BACE1 was overexpressed at very high levels in transgenic mice, the generation of Abeta was reduced because of an altered BACE1 subcellular localization and hence a shift on APP cleavage, modest increase of BACE1 level indeed promoted Abeta deposition (45). In addition, more and more reports have shown that increased endogenous BACE1 levels in cell and animal models and more significantly in AD brains result in an increased amyloidogenic cleavage of APP and may contribute directly to (sporadic) AD pathogenesis (19, 46, 47). Consistent with this, our study also showed that Abeta generation (both Abeta40 and Abeta42) was increased accompanied by an increased endogenous BACE1 level upon hypoxia treatment (Fig. 1). The greater increase of Abeta detected by ELISA (2-fold) than that by the IP-Western method (1.5-fold) reflects the more sensitive and quantitative feature of the ELISA technique which detects more Abeta variants than the IP-Western method. These results suggest that the generation of Abeta is positively correlated with an increase of BACE1 within the patho/physiological range.

Inhibiting BACE1 activity or reducing levels of BACE1 in vivo has been shown to decrease production of Abeta without severe detrimental phenotypes (13–15), suggesting that BACE1 is a valuable candidate for therapeutic targeting. However, given the difficulty of developing specific small-molecule inhibitors, it is critical to explore alternative approaches such as down-regulating BACE1 transcription, particularly in cases of ischemia and stroke in which elevated BACE1 expression/activity may contribute to a higher incidence of AD.


   FOOTNOTES
 
Note Added in Revision—While this manuscript was under review, an article by Sun et al. was published in Proc. Natl. Acad. Sci. U. S. A. (48) reporting a functional hypoxia-responsive element in the BACE1 gene promoter and that hypoxia can increase BACE1 transcription and expression leading to increased APP processing and Abeta generation.

* This work was supported in part by National Institutes of Health Grants R01 AG030197, R01 NS046673, and R01 AG021173 (to H. X.) and R01 NS054880 (to F. F. L.), and grants from the Alzheimer Association (to H. X.), the American Health Assistance Foundation (to H. X.), and the National Natural Science Foundation of China (No. 30672198, to Y.-w. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this study and share first authorship. Back

3 Recipient of National Institutes of Health Training Grant F32 AG024895. To whom correspondence may be addressed: Burnham Institute for Medical Research, La Jolla, CA 92037. Tel.: 858-795-5246; Fax: 858-795-5273; E-mail: yunzhang{at}burnham.org. Back

2 To whom correspondence may be addressed: Burnham Institute for Medical Research, La Jolla, CA 92037. Tel.: 858-795-5246; Fax: 858-795-5273; E-mail: xuh{at}burnham.org.

4 The abbreviations used are: AD, Alzheimer disease; ADAM10, a disintegrin and metalloprotease domain 10; APH-1, anterior pharynx-defective-1; APP, beta-amyloid precursor protein; Abeta, beta-amyloids; BACE, beta-site APP cleaving enzyme; CTF, C-terminal fragment; EMSA, electrophoretic mobility shift assay; HIF-1, hypoxia-inducible factor-1; NTF, N-terminal fragment; N2a-APP, N2a cells stably expressing human APP695; PEN-2, presenilin enhancer-2; PS, presenilin; RT-PCR, real-time polymerase chain reaction; siRNA, small interfering RNA; TACE, tumor necrosis factor-{alpha}-converting enzyme; ELISA, enzyme-linked immunosorbent assay; RIPA, radioimmune precipitation assay buffer. Back

5 X. Zhang, K. Zhou, R. Wang, J. Cui, S. A. Lipton, F. F. Liao, H. Xu, and Y.-W. Zhang, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Ze'ev Ronai for technical help and Dr. Robert Abraham for providing the HIF-1{alpha} cDNA construct.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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