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Keywords:

  • β-secretase;
  • Alzheimer’s disease;
  • BACE1

Abstract

  1. Top of page
  2. Abstract
  3. Identification and validation of BACE1 as the Alzheimer’s β-secretase
  4. BACE1
  5. BACE1 localization
  6. BACE1 knockout mice
  7. BACE1 substrates
  8. Conclusions
  9. Acknowledgements/Conflict of Interests
  10. References

J. Neurochem. (2012) 120 (Suppl. 1), 55–61.

Abstract

Our knowledge of the etiology of Alzheimer’s disease (AD) has advanced tremendously since the discovery of amyloid beta (Aβ) aggregation in diseased brains. Accumulating evidence suggests that Aβ plays a causative role in AD. The β-secretase enzyme, beta-site APP cleaving enzyme-1 (BACE1), is also implicated in AD pathogenesis, given that BACE1 cleavage of amyloid precursor protein is the initiating step in the formation of Aβ. As a result, BACE1 inhibition has been branded as a potential AD therapy. In this study, we review the identification and basic characteristics of BACE1, as well as the progress in our understanding of BACE1 cell biology, substrates, and phenotypes of BACE1 knockout mice that are informative about the physiological functions of BACE1 beyond amyloid precursor protein cleavage. These data are crucial for predicting potential mechanism-based toxicity that would arise from inhibiting BACE1 for the treatment or prevention of AD.

Abbreviations used
AD

Alzheimer’s disease

BACE1

beta-site APP cleaving enzyme-1

ER

endoplasmic reticulum

GGA

Golgi-localized gamma-ear-containing ARF-binding

Substantial evidence points to a role for cerebral aggregation of amyloid beta (Aβ) peptide in Alzheimer’s disease (AD). Aβ is derived from the sequential action of two aspartic proteases, the β- and γ-secretases, on amyloid precursor protein (APP). β-Secretase initiates Aβ formation by cleaving APP to generate the N-terminus of Aβ (Citron et al. 1995). This cleavage produces a secreted ectodomain of APP (APPsβ) and a membrane-tethered C-terminal fragment that is 99 amino acids in length (C99). Subsequently, γ-secretase cleaves within the transmembrane region of C99 to release Aβ that is secreted from the cell. Aβ peptides may vary in length (38–42 amino acids) at the C-terminus because of the imprecise cleavage of the γ-secretase. As Aβ accumulation is implicated in AD pathogenesis, the identity of the β-secretase was highly sought after due to its ideal status as a drug target for lowering cerebral Aβ levels. Herein, this review will discuss the identification and characterization of two aspartic proteases, beta-site APP cleaving enzyme-1 (BACE1) and beta-site APP cleaving enzyme-2 (BACE2), and provide evidence that unequivocally validates BACE1 as the β-secretase. Information regarding BACE1 physiological functions derived from deletion mutants, as well as BACE1 cell biology and substrates, will also be discussed.

Identification and validation of BACE1 as the Alzheimer’s β-secretase

  1. Top of page
  2. Abstract
  3. Identification and validation of BACE1 as the Alzheimer’s β-secretase
  4. BACE1
  5. BACE1 localization
  6. BACE1 knockout mice
  7. BACE1 substrates
  8. Conclusions
  9. Acknowledgements/Conflict of Interests
  10. References

Over a decade ago, five groups reported two unique aspartic proteases that shared 64% amino acid sequence similarity, and that served as potential β-secretase candidates: BACE1 (also termed memapsin 2 and Asp2) (Hussain et al. 1999; Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999; Lin et al. 2000), and BACE2 (also termed Asp1, memapsin 1, and Down region aspartic protease) (Saunders et al. 1999; Yan et al. 1999; Acquati et al. 2000; Bennett et al. 2000a; Lin et al. 2000; Solans et al. 2000). Prior to these reports, β-secretase properties had been well-characterized, a sequence of events that, as it turned out, was instrumental for the identification of the β-secretase. In the discussion below, we evaluate the properties of β-secretase that served as a tool to clearly validate BACE1 as the β-secretase essential for Aβ formation.

Although β-secretase activity is widely expressed, the highest proteolytic activity is observed in the brain (Seubert et al. 1993; Zhao et al. 1996). Consistent with this expression pattern, BACE1 is present in many tissues, but is predominantly expressed within the brain (Vassar et al. 1999; Bennett et al. 2000a; Lin et al. 2000; Marcinkiewicz and Seidah 2000). BACE2, however, is expressed at moderate to low levels across a variety of cell types, but it is low to undetectable in most brain regions. There are a few exceptions, as there is evidence of BACE2 expression in the mammilary bodies, the ventromedial hypothalamus, and other small brain stem nuclei (Bennett et al. 2000a; Marcinkiewicz and Seidah 2000) moreover, BACE2.

The optimal pH for β-secretase activity is within a low pH range (Haass et al. 1993, 1995a; Knops et al. 1995), and as such β-secretase localizes primarily to endosomes and the Golgi apparatus (Koo and Squazzo 1994; Haass et al. 1995b; Thinakaran et al. 1996). Likewise, BACE1 has an acidic pH optimum (Vassar et al. 1999), and resides predominantly within acidic intracellular compartments with its active site in the lumen of the vesicle (Hussain et al. 1999; Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999; Lin et al. 2000; Kinoshita et al. 2003).

APP constructs devoid of the transmembrane domain are not cleaved by β-secretase, which implies that β-secretase specifically targets membrane-bound substrates (Citron et al. 1995). Thus, one may deduce that β-secretase is either tightly associated with a membrane protein, or membrane-bound itself. In both cases, BACE1 and BACE2 contain membrane-spanning segments (Hussain et al. 1999; Saunders et al. 1999; Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999; Acquati et al. 2000; Lin et al. 2000).

Site-directed mutagenesis analysis of the amino acids surrounding the APP cleavage site demonstrates that β-secretase cleavage is highly sequence-specific (Citron et al. 1995). Substitutions at this site and nearby positions decrease β-secretase cleavage of APP. In addition, radiosequencing studies have shown that Aβ isolated from amyloid plaques primarily begins at Asp+1 (Roher et al. 1993), but may also start at Glu+11 (Gouras et al. 1998). The activity of BACE1 on wild-type and mutant APP substrates is consistent with the sequence specificity of β-secretase. BACE1 cleaves APP only at Asp+1 and Glu+11 (Vassar et al. 1999), and cleaves APP with the Swedish familial AD-causing mutation (APPswe) more efficiently than wild-type APP (Citron et al. 1992; Sinha et al. 1999; Vassar et al. 1999). BACE2 does not have the same cleavage specificity for APP as BACE1, cleaving APP not only at Asp+1 (Farzan et al. 2000; Hussain et al. 2000; Yan et al. 2001), but also at two other positions: Phe+19 and Phe+20 (Farzan et al. 2000).

When cells are transfected with BACE1 and either wild-type or mutant APP, Aβ levels are increased (Sinha et al. 1999). Additional credence to BACE1 as the β-secretase comes from experiments using APP-over-expressing cell lines. When BACE1 is transfected into wild-type APP-over-expressing cells, Aβ, APPsβ and C99 are elevated over controls (Hussain et al. 1999; Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999; Lin et al. 2000). Conversely, transfection of BACE1, but not BACE2, antisense oligonucleotides into APP-over-expressing cells decreases Aβ and C99 fragments (Vassar et al. 1999; Yan et al. 1999).

The strongest evidence for BACE1 as the β-secretase in vivo came from analyses of BACE1-deficient mice (BACE1−/−) bred to mice over-expressing APP with the Swedish mutation (Tg2576) to produce a BACE1−/−; APP bigenic strain (Luo et al. 2001, 2003; Ohno et al. 2004, 2007). In BACE1−/−, APP brain extracts, Aβ and C99 fragments are absent (Roberds et al. 2001; Dominguez et al. 2005). Moreover, neuronal cultures prepared from BACE1−/− tissue that were infected with APP-expressing adenovirus show no evidence of Aβ or C99 (Cai et al. 2001). In addition, age-associated cognitive deficits were prevented in BACE1−/−;APP bigenic mice (Luo et al. 2001, 2003; Ohno et al. 2004, 2007; Laird et al. 2005; Nishitomi et al. 2006; McConlogue et al. 2007; Kobayashi et al. 2008). Similarly, lentiviral delivery of BACE1 RNAi attenuated Aβ amyloidosis and rescued memory deficits in APP transgenics (Laird et al. 2005; Singer et al. 2005). The rescue of memory deficits in BACE1−/−;APP mice suggests that BACE1 inhibition has potential to improve cognitive impairment in humans with AD.

To date, the α-secretase-like APP cleavage and low-level cerebral expression of BACE2 argues against a role for BACE2 as the primary β-secretase involved in Aβ generation. Rather, it has been suggested that BACE2 plays a role in Down syndrome pathology (Dominguez et al. 2005) because the gene resides on chromosome 21 (Saunders et al. 1999) and BACE2 is over-expressed in Down syndrome patients (Motonaga et al. 2002; Barbiero et al. 2003). The physiological and pathological role of BACE2 remains unclear. BACE2 is expressed in glial cells and may contribute to Aβ generation within this cell type although the mechanism requires elucidation (Dominguez et al. 2005; Bettegazzi et al. 2011). Glial cells play a role in AD amyloidogenesis, and early evidence for a role for BACE2 in glial amyloidogenic processing in Down syndrome patients suggests further investigation.

BACE1 exhibits all of the putative β-secretase characteristics, and most strikingly, absence of BACE1 in vivo abolishes Aβ formation and subsequent amyloid pathology. Converging evidence from the molecular, biochemical and animal studies described above substantiates BACE1 as the β-secretase.

BACE1

  1. Top of page
  2. Abstract
  3. Identification and validation of BACE1 as the Alzheimer’s β-secretase
  4. BACE1
  5. BACE1 localization
  6. BACE1 knockout mice
  7. BACE1 substrates
  8. Conclusions
  9. Acknowledgements/Conflict of Interests
  10. References

The BACE1 gene is localized to chromosome 11q23.3 (Saunders et al. 1999) and encodes for a ∼70kDa type 1 transmembrane aspartic protease related to the pepsins and retroviral aspartic proteases (Hussain et al. 1999; Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999; Lin et al. 2000). The BACE1 luminal domain contains two aspartic protease active site motifs at amino acids 93–96 and 289–292, with each motif containing the highly conserved sequence defining aspartic proteases, D(T/S)G(T/S) (Fig. 1) (Vassar et al. 1999). BACE1 is synthesized as a 501 amino acid pro-enzyme with a short prodomain in the endoplasmic reticulum (ER) (Hussain et al. 1999; Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999; Capell et al. 2000; Lin et al. 2000). Within the ER, the luminal domain of BACE1 is glycosylated on four Asn residues (Haniu et al. 2000) and transiently acetylated on seven Arg residues (Costantini et al. 2007) (Fig. 1). Once translocated to the Golgi apparatus, complex carbohydrates are attached and the N-terminal prodomain is removed by furin convertases (Hussain et al. 1999; Bennett et al. 2000b; Capell et al. 2000; Benjannet et al. 2001; Creemers et al. 2001). After maturation, BACE1 is transported from the trans-Golgi network to the cell surface where it may be reinternalized into early endosomes (Huse et al. 2000; Walter et al. 2001). The low pH of the late Golgi/trans-Golgi network and early endosomal compartments, coupled with the maturation of BACE1, increases BACE1 enzymatic activity (Vassar et al. 1999).

image

Figure 1.  BACE1 structure and post-translational modifications. Colored rectangles depict BACE1 domains with corresponding amino acid numbers indicated below. Acetylation, glycosylation, S-palmitoylation and phosporylation sites are indicated by Rs, Ns, Cs and S, respectively. The BACE1 catalytic domain is comprised of two aspartic protease active site motifs that are represented by D92TG and D298SG. Three disulfide bonds (S--S) connect amino acids 216–420, 278–443 and 330–380. S, signal peptide; Pro, propetide; TM, transmembrane; C, C-terminal domain.

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BACE1 is phosphorylated on Ser 498, and this phosphorylation together with a C-terminal acidic cluster di-leucine motif (DXXLL) regulates BACE1 recycling between the cell surface and endosomal compartments (Huse et al. 2000; Walter et al. 2001; Pastorino et al. 2002). BACE1 is S-palmitoylated on four Cys residues located at the junction of the transmembrane and cytosolic domains (Fig. 1) (Benjannet et al. 2001; Vetrivel et al. 2009), and this modification facilitates BACE1 partitioning into lipid rafts. Increased targeting of BACE1 to the lipid raft had been suggested to enhance β-secretase processing of APP (Tun et al. 2002; Cordy et al. 2003). However, a recent study has reported that non-raft localized palmitoylation-deficient BACE1 is equally active in APP processing and Aβ secretion as raft-associated palmitoylated BACE1 (Vetrivel et al. 2009). Although BACE1 can process APP in both raft and non-raft environments, a membrane-anchored version of a BACE1 transition-state inhibitor produced by linkage to a sterol moiety appeared more potent as a result of targeting to lipid rafts (Rajendran et al. 2008).

BACE1 localization

  1. Top of page
  2. Abstract
  3. Identification and validation of BACE1 as the Alzheimer’s β-secretase
  4. BACE1
  5. BACE1 localization
  6. BACE1 knockout mice
  7. BACE1 substrates
  8. Conclusions
  9. Acknowledgements/Conflict of Interests
  10. References

BACE1 is localized to the trans-Golgi network and endosomal pathway (Hussain et al. 1999; Vassar et al. 1999; Capell et al. 2000; Huse et al. 2000; Lin et al. 2000), co-localizing with APP in endosomes (Hussain et al. 1999; Kinoshita et al. 2003). As mentioned previously, BACE1 also shuttles between the cell surface and early endosomes (Huse et al. 2000; Walter et al. 2001). Intracellular BACE1 localization is regulated by various adapter proteins. Golgi-localized gamma-ear-containing ADP ribosylation factor-binding (GGA) proteins regulate trafficking of BACE1 between the late Golgi and early endosomes by interacting with the BACE1 C-terminal DXXLL motif via a von Hippel-Lindau domain (He et al. 2002, 2003; von Arnim et al. 2004). Depletion of GGA proteins by RNAi or disruption of phosphorylation of BACE1 on Ser498 increases accumulation of BACE1 in early endosomes, an acidic environment that favors BACE1 cleavage of APP and subsequent Aβ production (He et al. 2005; Wahle et al. 2005; Tesco et al. 2007). Interestingly, GGA3 is a caspase 3 substrate and is degraded during neuronal apoptosis. In the brains of AD patients, in which neuronal apoptosis may occur, the levels of GGA3 are significantly decreased (Tesco et al. 2007). Reduced GGA3 levels increase localization of BACE1 to early endosomes and also stabilize BACE1 by preventing its trafficking to lysosomes where it is degraded. Recently, Kang et al. (Kang et al. 2010) reported that BACE1 is ubiquitinated, and found that GGA3 traffics BACE1 cargo to lysosomes for degredation by binding to ubiquitin. Thus, when GGA3 was over-expressed, BACE1 levels were reduced as was subsequent Aβ formation.

The reticulon/Nogo family members have been identified as negative regulators of BACE1 (He et al. 2004; Murayama et al. 2006). Over-expression of reticulon proteins results in prolonged BACE1 retention in the ER with concomitant decrease in BACE1-mediated APP cleavage (Shi et al. 2009). Sorting nexin 6 is another BACE1-associated protein that influences BACE1 subcellular localization and acts as a negative regulator of BACE1 activity (Okada et al. 2010). Inhibition of sorting nexin 6 increases Aβ as well as retrograde transport of BACE1 to the trans-Golgi network. Sortilin is the most recently identified modulator of BACE1 trafficking (Finan et al. 2011). When over-expressed, sortilin increases BACE1-mediated APP cleavage, while RNAi-mediated knockdown decreases Aβ. Identification and characterization of BACE1 interactors will yield fruitful information regarding novel proteins that control BACE1 trafficking, and hence Aβ production. This has implications for identification of potential AD therapeutic targets.

BACE1 knockout mice

  1. Top of page
  2. Abstract
  3. Identification and validation of BACE1 as the Alzheimer’s β-secretase
  4. BACE1
  5. BACE1 localization
  6. BACE1 knockout mice
  7. BACE1 substrates
  8. Conclusions
  9. Acknowledgements/Conflict of Interests
  10. References

Shortly after the identification of BACE1, several groups undertook efforts to generate BACE1−/− mice. Generation of these mice would address whether BACE1 played a vital role in vivo, as well as provide evidence for potential mechanism-based side effects of anti-BACE1 therapeutics. The following knockout strategies were employed: (i) removal of the ATG start codon via deletion of exon 1 (Cai et al. 2001), (ii) insertion of a β-galactosidase gene downstream of the ATG start codon (Roberds et al. 2001), (iii) removal of the N-terminal active site motif via deletion of exon 2 (Luo et al. 2001), (iv) removal of the C-terminal half of the protease domain (Roberds et al. 2001), and (v) insertion of a neomycin cassette within exon 1 to introduce a premature stop codon (Dominguez et al. 2005). Although β-secretase activity was effectively abolished in the brains of BACE1−/− mice, initial analyses revealed no effect on gross behavioral and neuromuscular function (Roberds et al. 2001), tissue morphology, histology, blood or urine chemistry (Luo et al. 2001; Roberds et al. 2001). Subsequent analyses, however, revealed subtle yet distinct deficits that point to additional physiological roles for BACE1 other than APP cleavage. For example, peripheral sciatic nerves and central optic nerves were hypomyelinated in BACE1−/− mice (Hu et al. 2006; Willem et al. 2006), and when injured, sciatic nerves were slow to remyelinate (Hu et al. 2008). Additionally, BACE1−/− mice revealed an increased frequency of spontaneous and kainate-induced seizures (Kobayashi et al. 2008; Hitt et al. 2010; Hu et al. 2010). It is unclear whether these phenotypes are attributable to the lack of BACE1 in adult or during embryonic or postnatal development. Moreover, Dominguez et al. (Dominguez et al. 2005) noted an increased neonatal lethality among BACE1 null pups that is not attributable to maternal nursing defects.

BACE1 substrates

  1. Top of page
  2. Abstract
  3. Identification and validation of BACE1 as the Alzheimer’s β-secretase
  4. BACE1
  5. BACE1 localization
  6. BACE1 knockout mice
  7. BACE1 substrates
  8. Conclusions
  9. Acknowledgements/Conflict of Interests
  10. References

While the majority of investigative reports focus on BACE1 proteolysis of APP, the recent identification of additional BACE1 substrates hints at lesser-known physiological functions in which BACE1 may be involved. To date, all known BACE1 substrates are transmembrane proteins, many of which function in cell signaling, immune or inflammatory responses, and which suggests a role for BACE1 in this capacity. These include: Golgi-localized membrane-bound a 2,6-sialyltransferase (ST6Gal I) (Kitazume et al. 2001, 2003, 2005), interleukin-1 type II receptor (IL1R2) (Kuhn et al. 2007), P-selectin glycoprotein ligand-1 (Lichtenthaler et al. 2003), APP homologs APLP1 and APLP2 (Eggert et al. 2004; Li and Sudhof 2004; Pastorino et al. 2004; Hemming et al. 2009), low density lipoprotein receptor-related protein (von Arnim et al. 2005; Hemming et al. 2009), and the voltage-gated sodium channel β1-4 subunits (Navβ1-4) (Kim et al. 2005, 2007; Wong et al. 2005). A potential role for BACE1 in modulating sodium currents is also evidenced by cleavage of Navβ1-4 (Kim et al. 2005, 2007; Wong et al. 2005) and alteration of sodium currents in BACE1−/− mice (Dominguez et al. 2005). Moreover, BACE1 has been implicated in the regulation of myelination and myelin sheath thickness via cleavage of neuregulin–1 (Hu et al. 2006; Willem et al. 2006) and neuregulin-3 (Hu et al. 2008). Recently, an unbiased screen for novel BACE1 substrates identified 64 type I transmembrane proteins, three glycophosphatidylinositol-linked and one type II transmembrane protein (Hemming et al. 2009). The majority of the substrates from this screen have yet to be validated; however, several were cleaved by BACE1 in cell culture. These include: ephrin type A receptor (ephrin-A5), Golgi phosphoprotein 4 (GOLIM4), leucine-rich repeats and immunoglobulin-like domains proteins 2 and 3, insulin-like growth factor 2 receptor and semaphorin-4C. Although these studies point to alternative functions for BACE1, further studies are necessary to determine whether these proteins are, in fact, BACE1 substrates in vivo.

Conclusions

  1. Top of page
  2. Abstract
  3. Identification and validation of BACE1 as the Alzheimer’s β-secretase
  4. BACE1
  5. BACE1 localization
  6. BACE1 knockout mice
  7. BACE1 substrates
  8. Conclusions
  9. Acknowledgements/Conflict of Interests
  10. References

BACE1 is the key enzyme initiating Aβ synthesis in vivo, making it a prime drug target for AD treatment. The past decade has shown significant progress in our understanding of BACE1 molecular and cellular properties, and moderate progress identifying and characterizing BACE1 substrates other than APP. The recently identified BACE1 substrates hint at potential roles for BACE1 in immunological and inflammatory responses, modulating sodium currents, and regulating nerve myelination. Further investigations are crucial to define the precise role BACE1 may play in these processes, and the extent to which BACE1 inhibition will influence these essential biological functions. Additional phenotypes resulting from BACE1 deficiency may be revealed in studies of BACE1−/− mice under specific challenges. The collective knowledge acquired from investigations of BACE1 deletion mutants and characterization of BACE1 substrates has downstream implications for the discovery of new AD therapeutic targets and predicting side effects of BACE1 inhibition.

References

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  2. Abstract
  3. Identification and validation of BACE1 as the Alzheimer’s β-secretase
  4. BACE1
  5. BACE1 localization
  6. BACE1 knockout mice
  7. BACE1 substrates
  8. Conclusions
  9. Acknowledgements/Conflict of Interests
  10. References
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