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.     

A comparative molecular dynamics study on BACE1 and BACE2 flap flexibility

Beta-amyloid precursor protein cleavage enzyme1 (BACE1) and beta-amyloid precursor protein cleavage enzyme2 (BACE2), members of aspartyl protease family, are close homologs and have high similarity in their protein crystal structures. However, their enzymatic properties are different, which leads to different clinical outcomes. In this study, we performed sequence analysis and all-atom molecular dynamic (MD) simulations for both enzymes in their ligand-free states in order to compare their dynamical flap behaviors. This is to enhance our understanding of the relationship between sequence, structure and the dynamics of this protein family. Sequence analysis shows that in BACE1 and BACE2, most of the ligand-binding sites are conserved, indicative of their enzymatic property as aspartyl protease members. The other conserved residues are more or less unsystematically localized throughout the structure. Herein, we proposed and applied different combined parameters to define the asymmetric flap motion; the distance, d₁, between the flap tip and the flexible region; the dihedral angle, φ, to account for the twisting motion and the TriCα angle, θ₂ and θ₁. All four combined parameters were found to appropriately define the observed “twisting” motion during the flaps different conformational states. Additional analysis of the parameters indicated that the flaps can exist in an ensemble of conformations, i.e. closed, semi-open and open conformations for both systems. However, the behavior of the flap tips during simulations is different between BACE1 and BACE2. The BACE1 active site cavity is more spacious as compared to that of BACE2. The analysis of 10S loop and 113S loop showed a similar trend to that of flaps, with the BACE1 loops being more flexible and less stable than those of BACE2. We believe that the results, methods and perspectives highlighted in this report would assist researchers in the discovery of BACE inhibitors as potential Alzheimer’s disease therapies.     

Unmasking BACE1 in aging and age-related diseases

The beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) has long been considered a conventional target for Alzheimer’s disease (AD). Unfortunately, AD clinical trials of most BACE1 inhibitors were discontinued due to ineffective cognitive improvement or safety challenges. Recent studies investigating the involvement of BACE1 in metabolic, vascular, and immune functions have indicated a role in aging, diabetes, hypertension, and cancer. These novel BACE1 functions have helped to identify new ‘druggable’ targets for BACE1 against aging comorbidities. In this review, we discuss BACE1 regulation during aging, and then provide recent insights into its enzymatic and nonenzymatic involvement in aging and age-related diseases. Our study not only proposes the perspective of BACE1’s actions in various systems, but also provides new directions for using BACE1 inhibitors and modulators to delay aging and to treat age-related diseases.     

The Alzheimer's disease β-secretase enzyme, BACE1

The pathogenesis of Alzheimer's disease is highly complex. While several pathologies characterize this disease, amyloid plaques, composed of the β-amyloid peptide are hallmark neuropathological lesions in Alzheimer's disease brain. Indeed, a wealth of evidence suggests that β-amyloid is central to the pathophysiology of AD and is likely to play an early role in this intractable neurodegenerative disorder. The BACE1 enzyme is essential for the generation of β-amyloid. BACE1 knockout mice do not produce β-amyloid and are free from Alzheimer's associated pathologies including neuronal loss and certain memory deficits. The fact that BACE1 initiates the formation of β-amyloid, and the observation that BACE1 levels are elevated in this disease provide direct and compelling reasons to develop therapies directed at BACE1 inhibition thus reducing β-amyloid and its associated toxicities. However, new data indicates that complete abolishment of BACE1 may be associated with specific behavioral and physiological alterations. Recently a number of non-APP BACE1 substrates have been identified. It is plausible that failure to process certain BACE1 substrates may underlie some of the reported abnormalities in the BACE1-deficient mice. Here we review BACE1 biology, covering aspects ranging from the initial identification and characterization of this enzyme to recent data detailing the apparent dysregulation of BACE1 in Alzheimer's disease. We pay special attention to the putative function of BACE1 during healthy conditions and discuss in detail the relationship that exists between key risk factors for AD, such as vascular disease (and downstream cellular consequences), and the pathogenic alterations in BACE1 that are observed in the diseased state.     

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.     

Extracellular release of BACE1 holoproteins from human neuronal cells

BACE1 is a membrane-bound aspartyl protease involved in production of the Alzheimer's amyloid beta-protein. The BACE1 ectodomain is partially cleaved to generate soluble BACE1, but the physiological significance of this event is unclear. During our characterization of BACE1 shedding from human neuroblastoma SH-SY5Y cells stably expressing BACE1, we unexpectedly found that detectable amounts of BACE1 holoproteins were released extracellularly along with soluble BACE1. Treatment with the metalloprotease inhibitor, TAPI-1, inhibited BACE1 shedding but increased BACE1 holoprotein release. Soluble and full-length BACE1 were released in parallel, at least partly originating from the plasma membrane. Furthermore, the release of soluble BACE1, but not full-length BACE1, was increased by deletion of the C-terminal dileucine motif, indicating that dysregulated BACE1 sorting affects BACE1 shedding. These findings suggest that the release of BACE1 holoproteins may be a physiologically relevant cellular process.     

Significance of interactions of BACE1–Arg235 with its ligands and design of BACE1 inhibitors with P2 pyridine scaffold

Recently, we reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. Because these inhibitors contained some natural amino acids, we would need to improve their enzymatic stability in vivo and permeability across the blood–brain barrier, so that they become practically useful. Subsequently, non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold, 2,6-pyridenedicarboxylic, chelidamic or chelidonic moiety, at the P₂ position were reported. These inhibitors were designed based on the conformer of docked inhibitor in BACE1. In this study, we discuss the role and significance of interactions between Arg235 of BACE1 and its inhibitor in BACE1 inhibitory mechanism. Moreover, we designed more potent small-sized BACE1 inhibitors with a 2,6-pyridinedicarboxylic scaffold at the P₂ position, that were optimized for the interactions with Arg235 of BACE1.     

Protein expression of BACE1, BACE2 and APP in Down syndrome brains

Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21. The phenotype of DS is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. Several reports have shown that the neuropathology of DS comprises developmental abnormalities and Alzheimer-like lesions such as senile plaques. A key component of senile plaques is amyloid beta-peptide which is generated from the amyloid precursor protein (APP) by sequential action of beta-secretases (BACE1 and BACE2) and gamma-secretase. While BACE1 maps to chromosome 11, APP and BACE2 are located on chromosome 21. To challenge the gene dosage effect and gain insight into the expressional relation between beta-secretases and APP in DS brain, we evaluated protein expression levels of BACE1, BACE2 and APP in fetal and adult DS brain compared to controls. In fetal brain, protein expression levels of BACE2 and APP were comparable between DS and controls. BACE1 was increased, but did not reach statistical significance. In adult brain, BACE1 and BACE2 were comparable between DS and controls, but APP was significantly increased. We conclude that APP overexpression seems to be absent during the development of DS brain up to 18-19 weeks of gestational age. However, its overexpression in adult DS brain could lead to disturbance of normal function of APP contributing to neurodegeneration. Comparable expression of BACE1 and BACE2 speaks against the hypothesis that increased beta-secretase results in (or even underlies) increased production of amyloidogenic A beta fragments. Furthermore, current data indicate that the DS phenotype cannot be fully explained by simple gene dosage effect.     

Carbazole-containing arylcarboxamides as BACE1 inhibitors

β-Secretase (BACE1) is widely recognized as a prime drug target for the treatment of Alzheimer's disease (AD). In this Letter, we report the synthesis and the BACE1 inhibitory activity of novel, variously substituted N-[3-(9H-carbazol-9-yl)-2-hydroxypropyl]-arylcarboxamides. The best results have been obtained with the introduction of a 4-OMe substituent (IC(50)=3.8 μM) or a 3,4-dichloro substituent (IC(50)=2.5 μM) in the amidic aromatic ring. The blood-brain barrier penetration predictions resulted to be promising for this type of compounds. To better understand the structure-activity relationships (SAR) of the new derivatives, a docking study procedure has been applied exploiting different conformational and ionic states of BACE1.     

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.     

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.     

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.     

Calcium enhances the proteolytic activity of BACE1: An in vitro biophysical and biochemical characterization of the BACE1-calcium interaction

BACE1 is a novel type I transmembrane aspartyl protease that has been implicated in the pathogenesis of Alzheimer's disease. Cleavage of the amyloid precursor protein by the β-secretase, BACE1, is the first step in the production of the Aβ peptide and is a prime target for therapeutic intervention. Using circular dichroism, we reveal that the secondary structure of BACE1 in a membrane environment is significantly different from what was determined from the previously resolved crystal structure, and, we provide the first evidence that show differences in stability between the active (pH 4.8) and inactive (pH 7.4) forms of BACE1. In this study we have also examined Ca²⁺ binding to BACE1, the effect of this binding on the secondary and tertiary structural characteristics of BACE1, and the influence of this binding on the specific activity of the purified protein. Circular dichroism and endogenous tryptophan fluorescence measurements demonstrated that the secondary and tertiary structures, respectively, are sensitive to increasing concentrations of Ca²⁺. Isothermal titration calorimetry was then used to characterize the Ca²⁺-BACE1 interaction in more detail. Our results suggest that there is a high affinity of binding (k_d = 2.0 × μM) between Ca²⁺ and BACE1 and that the binding process was exothermic (ΔH = − 3.5 kcal/mol). We also could demonstrate that low concentrations of Ca²⁺ (μM range) significantly increased the proteolytic activity of BACE1. Collectively, these results identify a direct interaction between BACE1 and Ca²⁺ and suggest that under physiological conditions, the function(s) of BACE1 must also be influenced by Ca²⁺.     

Computational analysis of BACE1-ligand complex crystal structures and linear discriminant analysis for identification of BACE1 inhibitors with anti P-glycoprotein binding property

More than 100 years of research on Alzheimer’s disease didn’t yield a potential cure for this dreadful disease. Poor Blood Brain Barrier (BBB) permeability and P-glycoprotein binding of BACE1 inhibitors are the major causes for the failure of these molecules during clinical trials. The design of BACE1 inhibitors with a balance of sufficient affinity to the binding site and little or no interaction with P-glycoproteins is indispensable. Identification and understanding of protein–ligand interactions are essential for ligand optimization process. Structure-based drug design (SBDD) efforts led to a steady accumulation of BACE1-ligand crystal complexes in the PDB. This study focuses on analyses of 153 BACE1-ligand complexes for the direct contacts (hydrogen bonds and weak interactions) observed between protein and ligand and indirect contacts (water-mediated hydrogen bonds), observed in BACE1-ligand complex crystal structures. Intraligand hydrogen bonds were analyzed, with focus on ligand P-glycoprotein efflux. The interactions are dissected specific to subsites in the active site and discussed. The observed protein-ligand and intraligand interactions were used to develop the linear discriminant model for the identification of BACE1 inhibitors with less or no P-glycoprotein binding property. Excellent statistical results and model’s ability to correctly predict a new data-set with an accuracy of 92% is achieved. The results are retrospectively analyzed to give input for the design of potential BACE1 inhibitors.     

BACE1 regulates voltage-gated sodium channels and neuronal activity

BACE1 activity is significantly increased in the brains of Alzheimer's disease patients, potentially contributing to neurodegeneration. The voltage-gated sodium channel (Na(v)1) beta2-subunit (beta2), a type I membrane protein that covalently binds to Na(v)1 alpha-subunits, is a substrate for BACE1 and gamma-secretase. Here, we find that BACE1-gamma-secretase cleavages release the intracellular domain of beta2, which increases mRNA and protein levels of the pore-forming Na(v)1.1 alpha-subunit in neuroblastoma cells. Similarly, endogenous beta2 processing and Na(v)1.1 protein levels are elevated in brains of BACE1-transgenic mice and Alzheimer's disease patients with high BACE1 levels. However, Na(v)1.1 is retained inside the cells and cell surface expression of the Na(v)1 alpha-subunits and sodium current densities are markedly reduced in both neuroblastoma cells and adult hippocampal neurons from BACE1-transgenic mice. BACE1, by cleaving beta2, thus regulates Na(v)1 alpha-subunit levels and controls cell-surface sodium current densities. BACE1 inhibitors may normalize membrane excitability in Alzheimer's disease patients with elevated BACE1 activity.     

Tripeptidic BACE1 inhibitors devised by in-silico conformational structure-based design

Previously reported pentapeptidic BACE1 inhibitors, designed using a substrate-based approach, were used as lead compounds for the further design of non-peptidic BACE1 inhibitors. Although these peptidic and non-peptidic inhibitors, with a hydroxymethylcarbonyl isostere as a substrate transition-state mimic, exhibited potent BACE1 inhibitory activities, their molecular-sizes appeared a little too big (molecular weight of >600daltons) for developing practical anti-Alzheimer's disease drugs. To develop lower weight BACE1 inhibitors, a series of tripeptidic BACE1 inhibitors were devised using a design approach based on the conformation of a virtual inhibitor bound to the BACE1 active site, also called 'in-silico conformational structure-based design'. Although these tripeptidic BACE1 inhibitors contained some natural amino acid residues, they are expected to be useful as lead compounds for developing the next generation BACE1 inhibitors, due to their low molecular size and unique structural features compared with previously reported inhibitors.     

Altered beta-secretase enzyme kinetics and levels of both BACE1 and BACE2 in the Alzheimer's disease brain

beta-Secretase is the rate limiting enzymatic activity in the production of amyloid-beta peptide, the primary component of senile plaque pathology in Alzheimer's disease (AD). This study performed the first comparative analysis of beta-secretase enzyme kinetics in AD and control brain tissue. Results found V(max) values for beta-secretase to be significantly increased, and K(m) values unchanged in AD temporal cortex compared to matched control temporal cortex. The increased V(max) in AD cases, did not correlate with levels of BACE1, and decreased BACE1 and BACE2 levels correlated with the severity of neurofibrillary pathology (I-VI), and synaptic loss in AD. These results indicate that increased V(max) for beta-secretase is a feature of AD pathogenesis and this increase does not correlate directly with levels of BACE1, the principal beta-secretase in brain.     

Mapping of interaction domains mediating binding between BACE1 and RTN/Nogo proteins

BACE1 is a membrane-bound aspartyl protease that specifically cleaves amyloid precursor protein (APP) at the beta-secretase site. Membrane bound reticulon (RTN) family proteins interact with BACE1 and negatively modulate BACE1 activity through preventing access of BACE1 to its cellular APP substrate. Here, we focused our study on RTN3 and further show that a C-terminal QID triplet conserved among mammalian RTN members is required for the binding of RTN to BACE1. Although RTN3 can form homo- or heterodimers in cells, BACE1 mainly binds to the RTN monomer and disruption of the QID triplet does not interfere with the dimerization. Correspondingly, the C-terminal region of BACE1 is required for the binding of BACE1 to RTNs. Furthermore, we show that the negative modulation of BACE1 by RTN3 relies on the binding of RTN3 to BACE1. The knowledge from this study may potentially guide discovery of small molecules that can mimic the effect of RTN3 on the inhibition of BACE1 activity.