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(The FASEB Journal. 2006;20:1369-1376.)
© 2006 FASEB

BACE2, as a novel APP {theta}-secretase, is not responsible for the pathogenesis of Alzheimer’s disease in Down syndrome

Xiulian Sun*,{dagger}, Guiqiong He*,# and Weihong Song*,{dagger},1

* Department of Psychiatry, Brain Research Center,

{dagger} Graduate Program in Neuroscience, The University of British Columbia, Vancouver, British Columbia, Canada; and

# Department of Human Anatomy, Chongqing University of Medical Sciences, Chongqing, China

1 Correspondence: Department of Psychiatry, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada. E-mail: weihong{at}interchange.ubc.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amyloid ß protein (Aß), the major component of neuritic plaques in Alzheimer’s disease (AD), is derived from APP by sequential cleavages of ß- and {gamma}-secretases. Beta-site APP cleaving enzyme 1 (BACE1) is the major ß-secretase in vivo. Beta-site APP cleaving enzyme 2 (BACE2) is the homologue of BACE1. The majority of people with Down syndrome (DS), also called Trisomy 21 syndrome, will develop AD neuropathology after middle age. We and others have shown that APP C99, the major ß-secretase product, and Aß are markedly increased in DS. Since BACE2 is located on chromosome 21, it is speculated that BACE2 may play a role in AD pathogenesis in DS. In this report we found that BACE2 cleaves APP at a novel {theta} site downstream of the {alpha} site, abolishing Aß production. Overexpression of BACE2 by lentivirus markedly reduced Aß production in primary neurons derived from Swedish mutant APP transgenic mice. Despite an extra copy of the BACE2 gene in DS and the increase of its transcription, BACE2 protein levels are unchanged. Our data clearly demonstrate that BACE2, as a novel {theta}-secretase to cleave APP within the Aß domain, is not involved in the AD pathogenesis of DS patients; instead, therapeutic interventions that potentiate BACE2 may prevent AD pathogenesis.—Sun, X., He, G., Song, W. BACE2, as a novel APP {theta}-secretase, is not responsible for the pathogenesis of Alzheimer’s disease in Down syndrome.


Key Words: BACE2 • {theta}-secretase • Alzheimer • Down syndrome • Trisomy 21


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DOWN SYNDROME (DS) is a frequent genetic disorder occurring at a rate of 1 in 700-1000 live births. DS was first described by Dr. John Langdon Down in 1866 (1) , but it was not until 1959 that Dr. Jerome Lejeune and Patricia Jacobs, working independently, discovered that DS was caused by an extra copy of chromosome 21 (2 , 3) . Individuals with DS inevitably develop characteristic AD, including neuritic plaques and neurofibrillary tangles, after middle age (4 5 6) . Aß, the major component of neuritic plaques (5) , is derived from ß-amyloid precursor protein (APP) by the sequential cleavages of ß- and {gamma}-secretase at the ß- and {gamma}-site, respectively. Beta-site APP cleaving enzyme 1 (BACE1) is the major ß-secretase in vivo (7 8 9 10 11 12 13) . The levels of APP C-terminal fragment C99, the major ß-secretase product, and Aß are increased in DS (14 , 15) , suggesting increased ß-secretase activity in these patients. Beta-site APP cleaving enzyme 2 (BACE2) is a homologue of BACE1 (13 , 16 17 18 19) , and he APP and BACE2 genes are both located on chromosome 21. The extra copy of APP and BACE2 may play a role in the abnormal processing of APP in DS; however, the mechanism by which AD neuropathology develops in DS remains elusive.

Proteolytic processing of APP at the ß site is essential to generate Aß. BACE1 has been identified as a type 1 membrane–associated aspartyl protease of 501 amino acids (8 , 11 12 13) . BACE1 cleaves APP at the major ß site to generate C99, and at a minor glu-11 site to release a lower level of the C89 fragment. The BACE1-cleaved C99 fragments (CTFß) are subsequently cleaved by presenilin (PS) -dependent {gamma}-secretase to generate Aß (20 , 21) .

BACE1 undergoes a complex set of post-translational modifications during its maturation, including removal of the pro-peptide (22 23 24 25 26) , phosphorylation (24 , 27 , 28) , and glycosylation (24 , 27 , 29 , 30) . BACE1 is ubiquitinated and degraded by the ubiquitin proteasome pathway (31) . ß-Secretase activity is dependent on protein levels of BACE1 (32) . BACE1 gene expression is relatively low under normal conditions due to weak promoter activation during transcription (32) and the effect of its uAUG on leaky scanning and reinitiation during translation (33) . BACE1 abnormalities have been suggested in the pathogenesis of some sporadic AD cases. The AD-associated Swedish mutant APP contains double mutations close to the major ß-secretase cleavage site (Lys595-Met596 to Asn595-Leu596 that are associated with increased ß-secretase activity (34 , 35) . Although genetic analysis has failed to uncover any BACE1 coding sequence mutations in patients with familial AD (FAD) (36 , 37) , increased ß-secretase activity has been reported in some FAD brains (38) , and increased BACE1 expression levels were found in the cortex of sporadic AD patients compared to age-matched controls (39 40 41 42) . Our recent finding suggests that abnormal BACE1 trafficking and maturation contribute to the AD pathogenesis in Down syndrome (15) .

BACE2 is a type I transmembrane aspartyl protease of the A1 family with 518 amino acids. BACE2 is a homologue of BACE1 and is mapped to Down syndrome critical region on chromosome 21 (21q22.3) (13 , 16 17 18 19) . The amino acid sequences of human BACE1 and BACE2 are ~ 45% identical and 75% homologous (43) . Similar to BACE1, its C-terminal domain is significantly larger than that of other family members. BACE2 contains a 20-residue signal peptide and two putative N-glycosylation sites at Asn 170 and 366 (19) , and its structure contains typical features of A1 aspartic proteases. The crystal structures of BACE2 and BACE1 show differences in the substrate pockets of the S2, S3, S1', and S2' active sites (44) . In contrast to BACE1, pro-BACE2 requires autocatalytic pro-domain processing for enzymatic activation (45) . Analysis of the BACE2 and BACE1 sequences shows that despite 75% homology in the coding sequence, there is no similarity in the promoter regions and there are different putative transcription factor binding sites in these two promoters (43) , which could explain why BACE1 and BACE2 have distinct expression patterns. BACE1 mRNA is highly expressed in neurons of most brain regions. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in the colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA (17) . Furthermore, the BACE2 promoter has higher activity in HEK293 cells, whereas the BACE1 promoter has higher activity in neuronal cells (43) .

Similar to BACE1, BACE2 also has two D T/S G sites and a transmembrane domain (19) . While it is known that BACE1 cleaves APP at the major Asp+1 site and a minor Glu+11 of Aß, the function of BACE2 as either a ß-secretase or {alpha}–secretase has not been fully defined; however, several studies have shown that BACE2 can cleave APP at the ß-secretase cleavage site in vitro (46 , 47) . BACE2, but not BACE1, was shown to be responsible for the production of Aß in Flemish mutant APP transfected cells (46) ; however, other studies have shown that BACE2 cleaves APP at the Phe+19 and Phe+20 sites adjacent to the {alpha}-secretase cleavage site and that BACE2 functions as an alternative {alpha}-secretase and as antagonist of BACE1 (48 , 49) . There is no compensatory up-regulation of BACE2 in BACE1 knockout mice, which have a deficiency in generating Aß (50) , and knockout of BACE2 gene by deletion of exon 6 in mice did not show any phenotypic alterations (51) . Our recent study shows that BACE2 and BACE1 have distinct transcriptional regulation and function and that BACE2 functions not as a ß-secretase (43) , but rather processes APP within the Aß domain at a site downstream of {alpha}-secretase cleavage (43) .

In this report, we identified BACE2 as a novel {theta}-secretase tht cleaves APP at a novel {theta}-site downstream of the {alpha} site to generate APP C80 fragments. Cleavage of APP at the {theta}-site by BACE2 abolished Aß production. Even though BACE2 mRNA levels were increased by ~1.5-fold in DS patients relative to controls, immunoblotting analysis showed that BACE2 protein expression is unchanged in DS. Overexpression of BACE2 by lentivirus markedly reduced Aß production in primary neurons derived from Swedish mutant APP transgenic mice. Our data exclude the involvement of BACE2 in AD pathogenesis in DS and support the therapeutic potential of overexpressing BACE2 for AD and DS therapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids, transfection, cell culture, and stable cell lines
The 20E2 cell line is a Swedish mutant APP stable HEK293 cell line. pBACE1-mycHis was stably transfected into 20E2 to generate 2EB2 (52) . 4EB2 cell line stably expressing BACE2 and Swedish APP695 was established by transfecting plasmid pZ-BACE2mycHis into 20E2 cells under selection of Zeocin (43) . The stable cell lines were maintained in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 µg/ml Zeocin. APP C99 and C83 cDNA were amplified by polymerase chain reaction (PCR), then cloned into pcDNA3 vector (Invitrogen, San Diego, CA, USA) to generate mammalian expression plasmid pAPP-C99 and pAPP-C83. pAPP-C99 and pAPP-C83 were transfected into cells and the expressed fragments were used as the APP CTF protein markers. pAPP-C99Flag was generated by PCR with C terminus of C99 fragment tagged with Flag epitope. HEK293 cells were cultured in DMEM containing 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 U/ml penicillin G sodium, and 50 µg/ml streptomycin sulfate (Invitrogen). All cells were maintained at 37°C in an incubator containing 5% CO2. For transient transfection, cells were grown to ~70% confluency and transfected with plasmids using LipofectAMINE2000 (Invitrogen) according to the manufacturer’s instructions. The cells were harvested 48–72 h following transfection.

Immunoblotting
Frozen brain tissues were homogenized in radio-immuno-precipitation assay (RIPA) lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.05M Tris-HCl, pH 7.2) supplemented with Complete (Roche, Nutley, NJ, USA) protease inhibitors cocktail and sonicated. Tissue lysates were resolved by 12% Tris-glycine or 16% Tris-tricine gels. Immunoblotting was performed as described (52) . BACE2 was detected by 210 antibody (Ab), which was raised against its C-terminal amino acid sequence PRDPEVVNDESSLVRH (43) . APP was detected by the C20 Ab against the last 20 amino acids of its C terminus (32) . Internal control ß-actin expression was analyzed using monoclonal anti-ß-actin Ab AC-15 (Sigma, St. Louis, MO, USA). Anti-Flag Ab M2 was from Sigma.

Quantitative RT-PCR
Total RNA was isolated from frozen brain tissue using TRI-Reagent (Sigma). PowerScriptTM reverse transcriptase (Invitrogen) was used to synthesize first strand cDNA from an equal amount of RNA sample, as per the manufacturer’s instruction. The newly synthesized cDNA templates were further amplified by Platinum Tag DNA polymerase (Invitrogen) in a 50 µl reaction. Twenty-five to 35 PCR reaction cycles were used to cover the linear range of PCR amplification. BACE2 gene-specific primers 5'-gtataacgcagacaaggc-3' and 5'-tcatttccagcgatgtctg-3' were used to amplify a 675bp fragment of the BACE2 coding region. A pair of gene-specific primers 5'-ggacttcgagcaagagatgg-3' and 5'-gaagcatttgcggtggag-3' were used to amplify a 462 bp fragment of ß-actin. The samples were further analyzed on a 1% agarose gel and data were analyzed using Kodak Image Station 1000 software (Perkin Elmer, Norwalk, CT, USA).

N-terminal sequencing
pZ-BACE2mychis was stably transfected into HEK293 cells to establish the stable cell line 4B25. Stable cell lines were maintained in complete DMEM supplemented with 100 µg/ml Zeocin. Transfection was performed using Lipofectamine 2000 as described (52) . 4B25 cells were transfected with pAPP-C99Flag and harvested 72 h after transfection. The cell lysates were immunoprecipitated with monoclonal M2 Flag Ab (Sigma), and immunoprecipitates were resolved by 16% Tris-tricine gel and stained with Gelcode blue stain reagent (Pierce, Rockford, IL, USA). The C80 band, confirmed by C20 Ab detection, was subjected to N-terminal sequencing using Applied Biosystems Precise Sequencer (UBC NAPS). The N-terminal sequence of the sequenced peptide was mapped to the APP sequence to identify the BACE2 cleavage site.

Primary neuronal culture
APP23 transgenic mice were obtained from Novartis (Basle, Switzerland). Mice were genotyped using Thy1E2 primer 5'-caccacagaatccaagtcgg and APP1082r primer 5'-cttgacgttctgcctcttcc by PCR. ß-Actin was amplified by PCR using primers actin-1F 5'-gacaggatgcagaaggagat and Actin-1R 5'-ttgctgatccacatctgctg and used as an internal control. Hippocampal and neocortical tissues were removed from APP23 newborn mice at postnatal day 1 and gently digested with trypsin (0.025% EDTA; Life Technologies, Inc., Grand Island, NY, USA). The cells were suspended in neurobasal medium supplemented with B27 (Life Technologies) and plated on 35 mM plates coated with poly-D-lysine (0.01 mg/ml; Sigma) at a density of 2 x 106 cells per plate. Genomic DNA was isolated from the tails of the newborn mice for genotyping. Cultures were maintained at 37°C in a humidified incubator containing 5% CO2.

Lentivirus generation and infection
Enhanced GFP (EGFP)-BACE2 fusion cDNA fragment was cut from pEGFP-BACE2 plasmid and subcloned into L26FSGW lentivirus vector at BamH I and XbaI. The new construct was cotransfected with helper plasmids VSVG and {delta}8.87 into HEK293T cells by the calcium-phosphate method. Forty-eight hours after transfection, the conditioned media were harvested, filtered at a 0.45 µm pore size, and ultracentrifuged with MLS50 at 45,000 for 2 h. The pellet was resuspended in 100 µl of PBS. Primary neuronal cultures in 35 mM plates were infected with 10 µl virus solution and the media was changed 6 h after infection. 36 h after infection, the conditioned media was harvested for Aß ELISA.

Aß40/42 sandwich ELISA assay
ELISA assay was performed as described previously (32) . In brief, protein inhibitors and AEBSF (Sigma) were added to conditioned media to prevent degradation of Aß. The concentration of Aß40/42 was measured using the ß-amyloid 1–40 and 1–42 Colorimetric ELISA kit (Biosource International, Inc., Camarillo, CA, USA) according to the manufacturer’s protocol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
BACE2 processes APP at a site downstream of {alpha}-secretase cleavage
To determine the role of BACE1 and BACE2 in DS, we compared the function of BACE1 and BACE2 in APP processing. The 2EB2 cell line was established to stably express human BACE1 and Swedish mutant APP695 (52) , while the 4EB2 cell line was established to stably express human BACE2 and Swedish mutant APP695 (43) . Overexpression of BACE1 significantly increased ß-secretase activity, resulting in the elevation of C99 and C89 levels in 2EB2 cells (Fig. 1 A). In contrast, overexpression of BACE2 had no effect on ß-secretase cleavage of APP, but resulted in the generation of a new APP C-terminal fragment with lower molecular weight than the {alpha}-secretase product C83 fragment (Fig. 1B ). These results indicate that BACE2 is not a ß- or {alpha}-secretase, but cleaves APP at a site downstream of {alpha}-secretase cleavage.


Figure 1
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  		Figure 1. Cleavage of APP by BACE1 and BACE2. A) APP C83 is the major APP CTF in 20E2 cells expressing Swedish mutant APP695. 2EB2 cells stably express Swedish mutant APP695 and BACE1. BACE1 overexpression results in a significant increase in ß-secretase activity, leading to a marked increase in C99 and C89. Plasmid pAPP-C99 (pC99) was transfected into HEK293 cells and the cell lysate was used as the C99 protein marker. B) 4EB2 is the BACE2 and Swedish mutant APP695 stable cell line. Overexpression of BACE2 induces APP C80 generation and no increase in the ß-secretase product, C99.

N-terminal sequencing analysis shows that BACE2 cleaves APP at a novel {theta} site
To determine the identity of the new fragment and the APP cleavage site of the BACE2 protease, pAPP-C99Flag expression plasmid was transfected into 4B25 cells, which stably express BACE2. APP CTFs in the transfected cells were immunoprecipitated with anti-Flag Ab M2, resolved on SDS-PAGE gels, and stained by Gelcode blue (Fig. 2 A). The APP CTFs were confirmed by C20 Ab immunoblotting. The lower molecular weight band generated by BACE2 was subjected to N-terminal sequencing for identification of the first eight amino acids (Fig. 2B ), which revealed that the cleavage product sequence is FAEDVGSN (Fig. 2B, C ). The sequencing data clearly demonstrate that BACE2 cleaved APP between Phe+19 and Phe+20 of the Aß domain and that this site is distinct from both the ß- or {alpha}-secretase cleavage sites (Fig. 2E ).


Figure 2
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  		Figure 2. N-terminal sequencing of the APP C-terminal cleaved by BACE2 showed that BACE2 cleaves APP at a novel {theta} site. A) For identification of the APP cleavage site by BACE2, BACE2 stably transfected 4B25 cells were transfected with pAPP-C99Flag and harvested 72 h after transfection. Anti-Flag M2 Ab was used to immunoprecipitate APP CTFs and the samples were resolved by 16% Tris-tricine gel. The C80 band was subjected to N-terminal sequencing (Applied Biosystems Precise Sequencer, UBC NAPS). B) The N-terminal sequencing result of the first six residues of the C80 fragment. Each panel represents an amino acid residue, denoted by the letter above the peak signal of the amino acid. C) The sequence of the first eight amino acids of the C80 NH2 terminus sequencing. D) Plasmid pAPP-C99Flag was transfected into HEK293 control and BACE2 stably transfected cells 4B25. Flag-tagged C99 protein and its derivatives were detected with anti-Flag M2 Ab. Overexpression of BACE2 induces a cleavage within the C99 fragment, resulting in a decrease in C99 levels and the generation of C80 proteins. E) The schematic diagram shows that BACE2 has a distinct APP cleavage site compared with BACE1 and {alpha}-secretase. BACE2 cleaves APP between Phe+19 and Phe+20 of Aß at the {theta} site. BACE1 processes APP at the major ß-secretase site at Asp+1 and the minor ß-secretase site at Glu+11 of the Aß domain. The number represents the position of amino acids in Aß. The arrows point to the protease cleavage sites.

To further confirm BACE2’s role as a novel site secretase in APP processing, a C99 flag expression plasmid was transfected into the BACE2 stable 4B25 cells. Overexpression of BACE2 in these cells markedly increased its secretase activity, resulting in decreased C99 flag production and the generation of a new C80 flag fragment (Fig. 2D ), indicating that BACE2 can process APP at a site downstream of the ß-secretase site and can also cleave the ß-secretase product in cells. We designate this novel site as the APP {theta}-secretase cleavage site. BACE2, as a {theta}-secretase, cleaves APP at the {theta}-site within Aß domain, precluding Aß production and generating the APP C80 fragment (Fig. 1E ). These data suggest that potentiation of BACE2 {theta}-secretase activity may be a potential pharmaceutical strategy for preventing overgeneration of Aß leading to Aß deposition in AD.

Overexpression of BACE2 markedly reduced Aß generation in primary neurons from AD transgenic mice
Lentivirus is a type of retrovirus that can infect both dividing and nondividing cells. When the Synapsin I promoter is located in the lentivirus vector, it can direct expression of the target gene such that it is specifically expressed in neurons (53 , 54) . APP23 mice carry the human Swedish mutant APP751 transgene driven by the Thy1.2 promoter, which can drive the transgene to be specifically expressed in neurons, making APP23 a valued AD mice model. Neuritic plaques can be detected in the brains of APP23 mice at 6 mo of age and memory deficits can be detected as early as 3 mo (55 , 56) . We dissected primary neurons from day 1 newborn APP23 mice and infected them with empty vector or EGFP-BACE2 lentivirus. Infection with BACE2-containing lentivirus resulted in a significant 75 ± 0.5% decrease in Aß40 and 53 ± 11% decrease in Aß42 production in neurons derived from APP23 transgenic mice, relative to controls (P<0.001) (Fig. 3 ). Our results clearly demonstrate that BACE2 overexpression can reduce Aß production in primary APP23 neuronal cultures. These results are consistent with our previous report using BACE2 and Swedish APP double stable cell line showing that BACE2 overexpression drastically inhibited Aß generation (43) .


Figure 3
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  		Figure 3. Overexpression of BACE2 by lentivirus decreased Aß production in primary transgenic neurons. Primary APP23 neurons were infected with EGFP-BACE2 or empty vector lentivirus. Aß40 and 42 ELISA was performed using media 36 h after infection. EGFP-BACE2 lentivirus infection significantly decreased Aß40 (A) and 42 (B) production by ~75% and 53% (P<0.01). Values are means ± SE (n=3). *P < 0.01 by Student’s t test.

BACE2 transcription is elevated in DS patients.
The BACE2 gene is located in Down syndrome critical region on chromosome 21; thus, we examined whether the transcription of BACE2 was increased in DS due to its triplication. Fetal cortical tissues from Trisomy-21 and gestation age-matched control brains were homogenized for RNA extraction, and total RNA was isolated from frozen brain tissue using TRI-Reagent. Quantitative RT-polymerase chain reaction was used to measure BACE2 mRNA levels in both DS brain tissues and normal controls (Fig. 4 A). BACE2 mRNA levels were increased by 147.7 ± 11.46% in DS brain tissues relative to controls (P<0.05) (Fig. 4B ). Such increases may be due to an extra copy of the BACE2 gene on chromosome 21 in DS.


Figure 4
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  		Figure 4. Increased BACE2 mRNA levels in DS patients. A) A representative blot of the quantitative RT-polymerase chain reaction products. RNA was isolated from the cerebral cortical tissues of 16 to 20 wk gestational fetal abortuses in 7 DS and 7 age-matched controls. Quantitative RT-PCR was performed to measure the endogenous levels of BACE2 mRNA. Specific BACE2 and ß-actin coding sequence primers were used to amplify the cDNA. Different cycles and amounts of PCR products were analyzed, and the DNA gel represents 25 cycles of RT-PCR products separated on 1% agarose gel. B) The ratio of BACE2 to ß-actin mRNA in DS and age-matched controls were quantitated by Kodak image analysis. Endogenous BACE2 mRNA levels were increased by ~1.5-fold in DS brain tissues relative to controls. *P < 0.05 relative to controls by Student’s t test. Shown are the mean ± SE (n=7).

BACE2 protein level remains unchanged in DS patients
Trisomy-21 and gestation age-matched control brains were homogenized for protein extraction. BACE2 levels in DS brain tissue lysates were assayed by Western blot with BACE2 C terminus Ab 210 (Fig. 5 A) (43) . BACE2 protein levels were not significantly increased in DS brains relative to controls (103.10±1.24%, >0.05) (Fig. 5B ). These data indicate that despite an extra copy of the BACE2 gene in DS and the increase in its transcription, BACE2 has little effect on APP processing in DS brains and cannot account for increased C99 production. These data are also consistent with our results demonstrating that BACE2 is a {theta}-secretase that cleaves APP within the Aß domain. Increased BACE2 protein level would have elevated {theta}-secretase activity and prevented Aß overproduction and neuritic plaque formation in DS.


Figure 5
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  		Figure 5. Protein concentration of BACE2 remains unchanged in DS. A) A representative blot of BACE2 detection by 210 Ab. Cerebral cortical tissues of 16 to 20 wk gestational fetal abortuses in 7 DS and 7 age-matched controls were lysed in RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.05M Tris-HCl, pH 7.2) supplemented with Complete (Roche) protease inhibitors cocktail. 150 µg of the brain tissue lysates was separated on a 12% Tris-Glysine gel. ß-actin was detected by AC-15 was used as the loading control. B) Quantitative analysis of BACE2 proteins by Kodak image analysis. Values are means ± SE (n=7). Protein levels are expressed as a percentage of control levels. There was no significant difference in BACE2 protein levels between DS and control (P>0.05 by Student’s t test).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Since the APP and BACE2 genes are located on chromosome 21 and there are extra copies of these two genes in DS, it was speculated that the extra gene copies may play a role in abnormal processing of APP in DS. While the additional copy of the APP gene is seen in 99% of DS patients, the gene dosage effect cannot fully account for the occurrence of AD in DS (57) . We and others have shown that APP C99, the major ß-secretase products, and Aß are significantly increased in DS fetus tissues (14 , 15) . Our recent study indicates that increased BACE1 protein levels, particularly the higher levels of N-glycosylated mature BACE1 proteins, result in up-regulated ß-secretase activity, leading to higher C99 production and Aß generation in DS (15) . In this report, our data show that BACE2 gene transcription is indeed increased in DS, but BACE2 protein levels are not significantly changed. Our studies suggest that BACE1, but not BACE2, contributes to this increased ß-secretase activity, leading to the AD pathogenesis of DS.

A previous study using Swedish APP stable cells transiently transfected with BACE2 cDNA showed that purified BACE2 cleaves APP sequences at the ß-secretase site and near the {alpha}-secretase site, mainly between Aß-Phe+20 and Aß-Ala+21, and also between Aß-Phe+19 and Aß-Phe+20 (49) . The report suggests that BACE2 more likely serves as an alternative {alpha}-secretase (49) . Using the cell line stably expressing both human BACE2 and Swedish mutant APP genes, our experiments show that the cleavage product of BACE2 is C80, not C83 as is generated by {alpha}-secretase, and N-terminal sequencing clearly demonstrates that the BACE2 cleavage site is located between Aß-Phe+19 and Aß-Phe+20. Our data suggest that BACE2 is a new class of APP cleaving enzyme, neither a ß-secretase nor an alternative {alpha}-secretase, and that BACE2 functions as a novel {theta}-secretase to cleave APP within the Aß domain, thus precluding Aß generation (Fig. 6 ). This finding indicates that BACE2, despite its extra gene copy in DS, cannot account for the increased Aß production in the AD pathogenesis of DS; instead, BACE2, as an APP {theta}-secretase inhibiting Aß generation, may be a potential drug target, and up-regulation of BACE2 activity may be a useful pharmaceutical strategy for AD therapy. A recent study shows that BACE1 knockout mice do not acquire compensatory BACE2 up-regulation (50) , which is consistent with our result that BACE2 and BACE1 have distinct functions (43) . The crystal structures of BACE2 and BACE1 reveal some key differences between these two enzymes, which may allow for the design of selective BACE1 or BACE2 inhibitors (44 , 58 , 59) . As BACE1 is the major ß-secretase and an important drug target, inhibitors that can specifically inhibit BACE1 without affecting BACE2 should be more selectively potent in preventing Aß production.


Figure 6
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  		Figure 6. BACE2, as a novel APP {theta}-secretase, is not responsible for Alzheimer’s disease pathogenesis in Down syndrome.

Previous linkage studies suggest that chromosome 21q may harbor a susceptibility gene for late-onset AD (60 61 62 63 64 65) . Recently, in a high-resolution genome screen, a maximum linkage peak was found with marker D21S1440, which is mapped between the APP and BACE2 genes, 5 Mb from BACE2, and 12 Mb from APP (66) . BACE2 haplotype analyses showed that a haplotype H5 was associated with AD and a second haplotype H7 was associated with protection from AD, providing further evidence for an AD susceptibility locus on chromosome 21q within or close to BACE2 (64) . These linkage studies are consistent with our results, which show that the expression of BACE2 by lentivirus in primary APP23 neurons can significantly reduce Aß production. Further studies are needed to determine whether overexpression of BACE2 in vivo can decrease plaque formation and ameliorate the memory deficits seen in APP23 mice. Our study suggests that potentiation of BACE2 in the elderly may protect against AD pathogenesis.


   ACKNOWLEDGMENTS
 
We thank the University of Maryland Brain and Tissue Bank for Developmental Disorders for Trisomy-21 and control brain tissues. We thank Dr. Matthias Staufenbiel for providing us APP23 mice. We thank Weihui Zhou and Jane Wang for their technical assistance. We also thank Steven R. Vincent, Anthony G. Phillips, Kelley Bromley, and Diane Parsons for helpful discussions. This work was supported by Canadian Institutes of Health Research (CIHR), Jack Brown and Family Alzheimer’s Research Foundation, and the Michael Smith Foundation for Health Research (to W.S.). W.S. is the Holder of Canada Research Chair in Alzheimer’s disease. G.H. was supported by the Chinese Scholarship Council award.

Received for publication December 14, 2005. Accepted for publication March 20, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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