NFkappaB-dependent control of BACE1 promoter transactivation by Abeta42

Beta-amyloid (Abeta) peptides that accumulate in Alzheimer disease are generated from the beta-amyloid precursor protein (betaAPP) by cleavages by beta-secretase BACE1 and by presenilin-dependent gamma-secretase activities. Very few data document a putative cross-talk between these proteases and the regulatory mechanisms underlying such interaction. We show that presenilin deficiency lowers BACE1 maturation and affects both BACE1 activity and promoter transactivation. The specific gamma-secretase inhibitor DFK167 triggers the decrease of BACE1 activity in wild-type but not in presenilin-deficient fibroblasts. This decrease is also elicited by catalytically inactive gamma-secretase. The overexpression of APP intracellular domain (AICD), the gamma/epsilon-secretase-derived C-terminal product of beta-amyloid precursor protein, does not modulate BACE1 activity or promoter transactivation in fibroblasts and does not alter BACE1 expression in AICD transgenic brains of mice. A DFK167-sensitive increase of BACE1 activity is observed in cells overexpressing APPepsilon (the N-terminal product of betaAPP generated by epsilon-secretase cleavage harboring the Abeta domain but lacking the AICD sequence), suggesting that the production of Abeta could account for the modulation of BACE1. Accordingly, we show that HEK293 cells overexpressing wild-type betaAPP exhibit a DFK167-sensitive increase in BACE1 promoter transactivation that is increased by the Abeta-potentiating Swedish mutation. This effect was mimicked by exogenous application of Abeta42 but not Abeta40 or by transient transfection of cDNA encoding Abeta42 sequence. The IkappaB kinase inhibitor BMS345541 prevents Abeta-induced BACE1 promoter transactivation suggesting that NFkappaB could mediate this Abeta-associated phenotype. Accordingly, the overexpression of wild-type or Swedish mutated betaAPP does not modify the transactivation of BACE1 promoter constructs lacking NFkappaB-responsive element. Furthermore, APP/beta-amyloid precursor protein-like protein deficiency does not affect BACE1 activity and expression. Overall, these data suggest that physiological levels of endogenous Abeta are not sufficient per se to modulate BACE1 promoter transactivation but that exacerbated Abeta production linked to wild-type or Swedish mutated betaAPP overexpression modulates BACE1 promoter transactivation and activity via an NFkappaB-dependent pathway.     

Kinetic properties of cathepsin D and BACE 1 indicate the need to search for additional beta-secretase candidate(s)

Many studies suggest that BACE 1 is the genuine beta-secretase; however, this is not undisputed. The wild-type (WT) beta-site of the amyloid precursor protein (APP) present in the worldwide population is cleaved very slowly (kcat/Km: approx. 50 m(-1) s(-1)), while proteases acting on relevant substrates are much more efficient (kcat/Km: 10(4)-10(6) m(-1) s(-1)). Knock-out of BACE 1 in mouse markedly reduces A beta formation. Nevertheless, studies in other systems show that knock-out experiments in rodents and corresponding genetic defects in human may reveal different phenotypes. Considering these issues, we searched for other beta-secretase candidate(s), identified cathepsin D, and evaluated properties of cathepsin D related to BACE 1 that were not examined previously. The kinetic constants (kcat, Km, kcat/Km) for cleaving peptides with beta-sites of the WT or the mutated Swedish families (SW) APP by human BACE 1 and cathepsin D were determined and found to be similar. Western blots reveal that in human brain cathepsin D is approximately 280-fold more abundant than BACE 1. Furthermore, pepstatin A strongly inhibits the cleavage of SW and WT peptides by both brain extracts and cathepsin D, but not by BACE 1. These findings indicate that beta-secretase activity observed in brain extracts is mainly due to cathepsin D. Nevertheless, as both BACE 1 and cathepsin D show poor activity towards the WT beta-site sequence, it is necessary to continue the search for additional beta-secretase candidate(s).     

Apical sorting of beta-secretase limits amyloid beta-peptide production.

Polarized cells such as neurons and endothelial cells appear to be involved in two invariant pathological features of Alzheimer's disease pathology, namely the formation of senile plaques and cerebral amyloid angiopathy. This implicates polarized sorting mechanisms in the production and accumulation of amyloid beta-peptide (Abeta). We have now studied polarized sorting of beta-secretase (BACE) in Madin-Darby canine kidney (MDCK) cells. The majority of BACE is sorted to the apical surface of MDCK cells where very little beta-amyloid precursor protein (betaAPP) is observed, because betaAPP undergoes basolateral sorting. Consistent with the usage of similar mechanisms for polarized sorting, BACE was also found to be targeted to axons of hippocampal neurons. The remaining basolaterally sorted BACE competes with the highly polarized basolateral alpha-secretase activity. Therefore, substantial amounts of BACE are targeted away from betaAPP to a non-amyloidogenic compartment, a cellular mechanism that limits Abeta generation. In addition, no alpha-secretase activity was observed on the apical side whereas gamma-secretase activity is observed on the basolateral and the apical side. Consistent with this finding, substantial amounts of Abeta can be produced apically upon missorting of betaAPP to the apical surface. These data demonstrate that Abeta production is limited in polarized cells by differential targeting of BACE and its substrate betaAPP. Moreover, our findings suggest that betaAPP may not be a major physiological substrate of BACE.     

BACE1 suppression by RNA interference in primary cortical neurons

Extracellular deposition of amyloid-beta (Abeta) aggregates in the brain represents one of the histopathological hallmarks of Alzheimer's disease (AD). Abeta peptides are generated from proteolysis of the amyloid precursor proteins (APPs) by beta- and gamma-secretases. Beta-secretase (BACE1) is a type I integral membrane glycoprotein that can cleave APP first to generate C-terminal 99- or 89-amino acid membrane-bound fragments containing the N terminus of Abeta peptides (betaCTF). As BACE1 cleavage is an essential step for Abeta generation, it is proposed as a key therapeutic target for treating AD. In this study, we show that small interfering RNA (siRNA) specifically targeted to BACE1 can suppress BACE1 (but not BACE2) protein expression in different cell systems. Furthermore, BACE1 siRNA reduced APP betaCTF and Abeta production in primary cortical neurons derived from both wild-type and transgenic mice harboring the Swedish APP mutant. The subcellular distribution of APP and presenilin-1 did not appear to differ in BACE1 suppressed cells. Importantly, pretreating neurons with BACE1 siRNA reduced the neurotoxicity induced by H2O2 oxidative stress. Our results indicate that BACE1 siRNA specifically impacts on beta-cleavage of APP and may be a potential therapeutic approach for treating AD.     

Alternative pathways for production of beta-amyloid peptides of Alzheimer's disease

This highlight article describes three Alzheimer's disease (AD) studies presented at the 5th General Meeting of the International Proteolysis Society that address enzymatic mechanisms for producing neurotoxic beta-amyloid (Abeta) peptides. One group described the poor kinetics of BACE 1 for cleaving the wild-type (WT) beta-secretase site of APP found in most AD patients. They showed that cathepsin D displays BACE 1-like specificity and cathepsin D is 280-fold more abundant in human brain than BACE 1. Nevertheless, as BACE 1 and cathepsin D show poor activity towards the WT beta-secretase site, they suggested continuing the search for additional beta-secretase(s). The second group reported cathepsin B as an alternative beta-secretase possessing excellent kinetic efficiency and specificity for the WT beta-secretase site. Significantly, inhibitors of cathepsin B improved memory, with reduced amyloid plaques and decreased Abeta(40/42) in brains of AD animal models expressing amyloid precursor protein containing the WT beta-secretase site. The third group addressed isoaspartate and pyroglutamate (pGlu) posttranslational modifications of Abeta. Results showed that cathepsin B, but not BACE 1, efficiently cleaves the WT beta-secretase isoaspartate site. Furthermore, cyclization of N-terminal Glu by glutaminyl cyclase generates highly amyloidogenic pGluAbeta(3-40/42). These presentations suggest cathepsin B and glutaminyl cyclase as potential new AD therapeutic targets.     

New pyrazolyl and thienyl aminohydantoins as potent BACE1 inhibitors: exploring the S2' region

The proteolytic enzyme β-secretase (BACE1) plays a central role in the synthesis of the pathogenic β-amyloid in Alzheimer's disease. SAR studies of the S2' region of the BACE1 ligand binding pocket with pyrazolyl and thienyl P2' side chains are reported. These analogs exhibit low nanomolar potency for BACE1, and demonstrate >50- to 100-fold selectivity for the structurally related aspartyl proteases BACE2 and cathepsin D. Small groups attached at the nitrogen of the P2' pyrazolyl moiety, together with the P3 pyrimidine nucleus projecting into the S3 region of the binding pocket, are critical components to ligand's potency and selectivity. P2' thiophene side chain analogs are highly potent BACE1 inhibitors with excellent selectivity against cathepsin D, but only modest selectivity against BACE2. The cell-based activity of these new analogs tracked well with their increased molecular binding with EC(50) values of 0.07-0.2 μM in the ELISA assay for the most potent analogs.     

A non-amyloidogenic function of BACE-2 in the secretory pathway

beta-Site amyloid precursor protein cleavage enzyme (BACE)-1 and BACE-2 are members of a novel family of membrane-bound aspartyl proteases. While BACE-1 is known to cleave beta-amyloid precursor protein (betaAPP) at the beta-secretase site and to be required for the generation of amyloid beta-peptide (Abeta), the role of its homologue BACE-2 in amyloidogenesis is less clear. We now demonstrate that BACE-1 and BACE-2 have distinct specificities in cleavage of betaAPP in cultured cells. Radiosequencing of the membrane-bound C-terminal cleavage product revealed that BACE-2 cleaves betaAPP in the middle of the Abeta domain between phenylalanines 19 and 20, resulting in increased secretion of APPs-alpha- and p3-like products and reduced production of Abeta species. This cleavage can occur in the Golgi and later secretory compartments. We also demonstrate that BACE-1-mediated cleavage of betaAPP at Asp1 of the Abeta domain can occur as early as in the endoplasmic reticulum, while cleavage at Glu11 occurs in later compartments. These data indicate that the distinct specificities of BACE-1 and BACE-2 in their cleavage of betaAPP differentially affect the generation of Abeta.     

Beta-secretase cleavage at amino acid residue 34 in the amyloid beta peptide is dependent upon gamma-secretase activity

The amyloid beta peptides (Abeta) are the major components of the senile plaques characteristic of Alzheimer's disease. Abeta peptides are generated from the cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Abeta peptides. Gamma-secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Abeta peptides. The production of Abeta peptides is, therefore, dependent on the activities of both BACE and gamma-secretase. The cleavage of APP by BACE is believed to be a prerequisite for gamma-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Abeta-34 site) in the Abeta region is dependent on gamma-secretase activity. In vitro, the Abeta-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Abeta-34 site by BACE1 or BACE2 occurred only when Abeta 1- 40 peptide, a gamma-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Abeta 1-34 species. More importantly, the cellular production of Abeta 1-34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known gamma-secretase inhibitors in a concentration-dependent manner. These gamma-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that gamma-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Abeta-34 site. Therefore, BACE and gamma-secretase activity can be mutually dependent.     

Phenotypic and biochemical analyses of BACE1- and BACE2-deficient mice

Beta-secretase (BACE1) is the rate-limiting protease for the generation of the amyloid beta-peptide (Abeta) in Alzheimer disease. Mice in which the bace1 gene is inactivated are reported to be healthy. However, the presence of a homologous gene encoding BACE2 raises the possibility of compensatory mechanisms. Therefore, we have generated bace1, bace2, and double knockout mice. We report here that BACE1 mice display a complex phenotype. A variable but significant number of BACE1 offspring died in the first weeks after birth. The surviving mice remained smaller than their littermate controls and presented a hyperactive behavior. Electrophysiologically, subtle alterations in the steady-state inactivation of voltage-gated sodium channels in BACE1-deficient neurons were observed. In contrast, bace2 knockout mice displayed an overall healthy phenotype. However, a combined deficiency of BACE2 and BACE1 enhanced the bace1-/- lethality phenotype. At the biochemical level, we have confirmed that BACE1 deficiency results in an almost complete block of Abeta generation in neurons, but not in glia. As glia are 10 times more abundant in brain compared with neurons, our data indicate that BACE2 could indeed contribute to Abeta generation in the brains of Alzheimer disease and, in particular, Down syndrome patients. In conclusion, our data challenge the general idea of BACE1 as a safe drug target and call for some caution when claiming that no major side effects should be expected from blocking BACE1 activity.     

Biochemical and kinetic characterization of BACE1: investigation into the putative species-specificity for beta- and beta'-cleavage sites by human and murine BACE1

Beta-amyloid peptides (Abeta) are produced by a sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The lack of Abeta production in beta-APP cleaving enzyme (BACE1)(-/-) mice suggests that BACE1 is the principal beta-secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild-type and BACE1(-/-) mice supports cleavage of APP at the canonical beta-secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as beta', resulting in Abeta peptides starting at Glu11. The apparent inability of human BACE1 to make this beta'-cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species-specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the beta'-cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the beta- and beta'-cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the beta- and beta'-sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated beta'-cleavage site. It does not appear to be a significant species specificity to this cleavage.     

Receptor tyrosine kinases positively regulate BACE activity and Amyloid-β production through enhancing BACE internalization

Amyloid-β （Aβ） peptide, the primary constituent of senile plaques in Alzheimer＇s disease （AD）, is generated by β-secretase- and y-secretase-mediated sequential proteolysis of the amyloid precursor protein （APP）. The aspartic protease, β -site APP cleavage enzyme （BACE）, has been identified as the main β-secretase in brain but the regulation of its activity is largely unclear. Here, we demonstrate that both BACE activity and subsequent Aβ production are enhanced after stimulation of receptor tyrosine kinases （RTKs）, such as the receptors for epidermal growth factor （EGF） and nerve growth factor （NGF）, in cultured cells as well as in mouse hippocampus. Furthermore, stimulation of RTKs also induces BACE internalization into endosomes and Golgi apparatus. This enhancement of BACE activity and A β production upon RTK activation could be specifically inhibited by Src family kinase inhibitors and by depletion of endogenous c-Src with RNAi, and could be mimicked by over-expressed c-Src. Moreover, blockage of BACE internalization by a dominant negative form of Rab5 also abolished the enhancement of BACE activity and Aβ production, indicating the requirement of BACE internalization for the enhanced activity. Taken together, our study presents evidence that BACE activity and Aβ production are under the regulation of RTKs and this is achieved via RTK-stimulated BACE internalization, and suggests that an aberration of such regulation might contribute to pathogenic Aβ production.     

Post-translational processing of beta-secretase (beta-amyloid-converting enzyme) and its ectodomain shedding. The pro- and transmembrane/cytosolic domains affect its cellular activity and amyloid-beta production

Processing of the beta-amyloid precursor protein (betaAPP) by beta- and gamma-secretases generates the amyloidogenic peptide Abeta, a major factor in the etiology of Alzheimer's disease. Following the recent identification of the beta-secretase beta-amyloid-converting enzyme (BACE), we herein investigate its zymogen processing, molecular properties, and cellular trafficking. Our data show that among the proprotein convertase family members, furin is the major converting enzyme of pro-BACE into BACE within the trans-Golgi network of HK293 cells. While we demonstrate that the 24-amino acid prosegment is required for the efficient exit of pro-BACE from the endoplasmic reticulum, it may not play a strong inhibitory role since we observe that pro-BACE can produce significant quantities of the Swedish mutant betaAPP(sw) beta-secretase product C99. BACE is palmitoylated at three Cys residues within its transmembrane/cytosolic tail and is sulfated at mature N-glycosylated moieties. Data with three different antibodies show that a small fraction of membrane-bound BACE is shed into the medium and that the extent of ectodomain shedding is palmitoylation-dependent. Overexpression of full-length BACE causes a significant increase in the production of C99 and a decrease in the alpha-secretase product APPsalpha. Although there is little increase in the generation of Abeta by full-length BACE, overexpression of either a soluble form of BACE (equivalent to the shed form) or one lacking the prosegment leads to enhanced Abeta levels. These findings suggest that the shedding of BACE may play a role in the amyloidogenic processing of betaAPP.     

Di-substituted pyridinyl aminohydantoins as potent and highly selective human β-secretase (BACE1) inhibitors

The identification of highly selective small molecule di-substituted pyridinyl aminohydantoins as β-secretase inhibitors is reported. The more potent and selective analogs demonstrate low nanomolar potency for the BACE1 enzyme as measured in a FRET assay, and exhibit comparable activity in a cell-based (ELISA) assay. In addition, these pyridine-aminohydantoins are highly selectivity (>500×) against the other structurally related aspartyl proteases BACE2, cathepsin D, pepsin and renin.Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on the previously reported aminohydantoin 3a. We have taken advantage of the amino acid difference between the BACE1 and BACE2 at the S2′ pocket (BACE1 Pro₇₀ changed to BACE2 Lys₈₆) to build ligands with >500-fold selectivity against BACE2. The addition of large substituents on the targeted ligand at the vicinity of this aberration has generated a steric conflict between the ligand and these two proteins, thus impacting the ligand’s affinity and selectivity. These ligands have also shown an exceptional selectivity against cathepsin D (>5000-fold) as well as the other aspartyl proteases mentioned. One of the more potent compounds (S)-39 displayed an IC₅₀ value for BACE1 of 10 nM, and exhibited cellular activity with an EC₅₀ value of 130 nM in the ELISA assay.     

The caspase-derived C-terminal fragment of betaAPP induces caspase-independent toxicity and triggers selective increase of Abeta42 in mammalian cells

During its physiopathological maturation, the beta-amyloid precursor protein undergoes several distinct proteolytic events by activities called secretases. In Alzheimer's disease, the main histological hallmark called senile plaque is clearly linked to the overproduction of the amyloid peptides Abeta40 and Abeta42, two highly aggregable betaAPP-derived fragments generated by combined cleavages by beta- and gamma-secretases. Recently, an alternative hydrolytic pathway was described, involving another category of proteolytic activities called caspases, responsible for the production of a 31 amino acids betaAPP C-terminal fragment called C31. C31 was reported to lower the viability of N2a cells but the exact mechanisms mediating C31-toxicity remained to be established. Here we show that the transient transfection of pSV2 vector encoding C31 lowers by about 80% TSM1 neuronal cells viability. Arguing against a C31-stimulated apoptotic response, we demonstrate by combined enzymatic and immunological approaches that C31 expression did not modulate basal or staurosporine-induced caspase 3-like activity and pro-caspase-3 activation. Furthermore, C31 did not modify Bax and p53 expressions, poly-(ADP-ribose)-polymerase cleavage and cytochrome c translocation into the cytosol. However, we established that C31 overexpression triggers selective increase of Abeta42 but not Abeta40 production by HEK293 cells expressing wild-type betaAPP751. Altogether, our data demonstrate that C31 induces a caspase-independent toxicity in TSM1 neurons and potentiates the pathogenic betaAPP maturation pathway by increasing selectively Abeta42 species in wild type-betaAPP-expressing human cells.     

BACE1 interacts with nicastrin

Beta-amyloid peptide (Abeta) is generated through the proteolytic cleavage of beta-amyloid precursor protein (APP) by beta- and gamma-secretases. The beta-secretase, BACE1, initiates Abeta formation followed by gamma-cleavage within the APP transmembrane domain. Although BACE1 localizes in the transGolgi network (TGN), its physiological substrates and modulators are not known. In addition, the relationship to other secretase(s) also remains unidentified. Here, we demonstrate that BACE1 binds to nicastrin, a component of gamma-secretase complexes, in vitro, and that nicastrin activates beta-secretase activity in COS-7 cells.     

Substrate and inhibitor profile of BACE (beta-secretase) and comparison with other mammalian aspartic proteases.

The full-length and ectodomain forms of beta-site APP cleavage enzyme (BACE) have been cloned, expressed in Sf9 cells, and purified to homogeneity. This aspartic protease cleaves the amyloid precursor protein at the beta-secretase site, a critical step in the Alzheimer's disease pathogenesis. Comparison of BACE to other aspartic proteases such as cathepsin D and E, napsin A, pepsin, and renin revealed little similarity with respect to the substrate preference and inhibitor profile. On the other hand, these parameters are all very similar for the homologous enzyme BACE2. Based on a collection of decameric substrates, it was found that BACE has a loose substrate specificity and that the substrate recognition site in BACE extends over several amino acids. In common with the aspartic proteases mentioned above, BACE prefers a leucine residue at position P1. Unlike cathepsin D etc., BACE accepts polar or acidic residues at positions P2'0 and P1 but prefers bulky hydrophobic residues at position P3. BACE displays poor kinetic constants toward its known substrates (wild-type substrate, SEVKM/DAEFR, K(m) = 7 microm, K(cat) = 0.002 s(-1); Swedish mutant, SEVNL/DAEFR, K(m) = 9 microm, K(cat) = 0.02 s(-1)). A new substrate (VVEVDA/AVTP, K(m) = 1 microm, K(cat) = 0.004) was identified by serendipity.     

The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology

Amyloid plaques, composed of the amyloid beta-protein (Abeta), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. Abeta fulfills a central role in AD pathogenesis, and reduction of Abeta levels should prove beneficial for AD treatment. Abeta generation is initiated by proteolysis of amyloid precursor protein (APP) by the beta-secretase enzyme BACE1. Bace1 knockout (Bace1(-/-)) mice have validated BACE1 as the authentic beta-secretase in vivo. BACE1 is essential for Abeta generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1(-/-) mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking.