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This study aimed to provide scientific data on the anti-Alzheimer's disease (AD) effects of phenolic compounds from Drynariae Rhizoma (DR) extract using a multi-component approach. Screening of DR extracts, fractions, and the ten phenolic compounds isolated from DR against the key AD-related enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BChE), β-site amyloid precursor protein cleaving enzyme 1 (BACE1), and monoamine oxidase-B (MAO-B) confirmed their significant inhibitory activities. The DR extract was confirmed to have BACE1-inhibitory activity, and the ethyl acetate and butanol fractions were found to inhibit all AD-related enzymes, including BACE1, AChE, BChE, and MAO-B. Among the isolated phenolic compounds, compounds (2) caffeic acid 4-O-β-D-glucopyranoside, (6) kaempferol 3-O-rhamnoside 7-O-glucoside, (7) kaempferol 3-o-b-d-glucopyranoside-7-o-a-L-arabinofuranoside, (8) neoeriocitrin, (9) naringin, and (10) hesperidin significantly suppressed AD-related enzymes. Notably, compounds 2 and 8 reduced soluble Amyloid Precursor Protein β (sAPPβ) and β-secretase expression by over 45% at a concentration of 1.0 μM. In the thioflavin T assay, compounds 6 and 7 decreased Aβ aggregation by approximately 40% and 80%, respectively, and degraded preformed Aβ aggregates. This study provides robust evidence regarding the potential of DR as a natural therapeutic agent for AD, highlighting specific compounds that may contribute to its efficacy. [ABSTRACT FROM AUTHOR]

Background: β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is a key enzyme in the formation of amyloid-β (Aβ) protein. Increasing evidence suggests that BACE1 concentration is a potential biomarker for Alzheimer's disease (AD). Objective: To evaluate the correlations between plasma BACE1 concentration, cognition, and hippocampal volume at different stages of the AD continuum. Methods: Plasma BACE1 concentrations were measured in 32 patients with probable dementia due to AD (ADD), 48 patients with mild cognitive impairment (MCI) due to AD, and 40 cognitively unimpaired (CU) individuals. Memory function was evaluated using the auditory verbal learning test (AVLT), and voxel-based morphometry was used to analyze bilateral hippocampal volumes. Correlation and mediation analyses were performed to investigate the associations between plasma BACE1 concentration, cognition, and hippocampal atrophy. Results: The MCI and ADD groups exhibited elevated BACE1 concentrations compared with the CU group after adjusting for age, sex, and apolipoprotein E (APOE) genotype. Increased BACE1 concentration was found in AD continuum patients who were APOE ɛ4 carriers (p < 0.05). BACE1 concentration was negatively associated with the scores of the subitems of the AVLT and hippocampal volume (p < 0.05, false discovery rate correction) in the MCI group. Moreover, bilateral hippocampal volume mediated the relationship between BACE1 concentration and recognition in the MCI group. Conclusion: BACE1 expression increased in the AD continuum, and bilateral hippocampal volume mediated the effect of BACE1 concentration on memory function in patients with MCI. Research has indicated that the plasma BACE1 concentration might be a biomarker at the early stage of AD. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease amyloid-beta peptides (Aβ) are generated via sequential cleavage of the amyloid precursor protein (APP) by β-secretase (Bace1) and γ-secretase. Though the precise subcellular location(s) of Bace1-mediated APP cleavage remains unresolved, current models suggest APP internalization into Bace1-containing endosomes is a critical step. However, direct evidence for this model is lacking, and previous reports that probed the APP/Bace1 interaction (using co-expressed APP and Bace1 differentially labeled with fluorescent protein tags) did not determine if APP fluorescence originated from full-length APP (fl-APP) molecules that had internalized from the cell surface pool. Methods: We adapted the bungarotoxin-ligand (BTX) system to label surface APP and track internalized fluorescent APP/BTX puncta in rodent primary neurons co-expressing fluorescently-tagged Bace1. Subsequently, we employed imaging and biochemical-based approaches to measure N- and C-terminal APP epitope levels in primary neurons, N2a neuroblastoma, and HeLa cell lines. Results: We hypothesized that surface-labeled APP/BTX puncta would, upon internalization, colocalize with fluorescently-tagged Bace1. Unexpectedly, we observed a dramatic loss of internalized APP in co-transfected neurons and ~ 80–90% loss of surface-resident fl-APP, which we also observed in HeLa and N2a cells. Loss of surface fl-APP could be reversed by a Bace1 inhibitor, suggesting that enhanced Bace1-mediated APP cleavage was responsible for the altered processing and mis-sorting. Importantly, in a C-terminally-tagged APP construct, the majority of C-terminal fluorescence was preserved in HeLa cells despite the loss of N-terminal APP signal. This phenomenon was not only recapitulated in cultured neurons, but also showed a progressive disappearance of the APP N-terminal tag, reflecting continual cleavage of fl-APP by Bace1 away from the cell body. Conclusions: Our results strongly suggested that in APP/Bace1 co-expression approaches, there was significant early and aberrant Bace1-mediated APP cleavage that perturbed fl-APP trafficking from the secretory pathway onwards, resulting in a substantial loss of surface fl-APP, which in turn led to a marked reduction in APP internalization. In C-terminally-tagged APP constructs, a large fraction of the APP fluorescence signal therefore likely arose from fluorescently-tagged β-C-terminal-fragment (β-CTF) or downstream proteolytic derivatives instead of fl-APP. Thus, care is needed in interpreting results where APP is detected only with a C-terminal tag in the presence of Bace1 co-expression, and previous findings may need to be reinterpreted if it is unclear whether fl-APP is present in normal physiological levels. [ABSTRACT FROM AUTHOR]

The β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the main brain β-secretase responsible for the amyloidogenic processing of the amyloid precursor protein (APP). Previous studies have suggested that cerebrospinal fluid (CSF) β-secretase activity may be a candidate diagnostic biomarker for Alzheimer's disease (AD), but biochemical characterization of BACE1 protein in CSF is needed. CSF samples from 19 AD patients and 19 age-matched non-AD controls (n = 19) were classified according to their Aβ42, total tau, and P-tau CSF biomarker levels. We found that β-secretase activity was higher in the CSF of AD subjects than in that of the controls. We found that the majority of the β-secretase activity in the CSF, measured using a peptide substrate homologous to the BACE1 cleavage site, was not inhibited by specific BACE1 inhibitors. We defined enzymatic activity attributable specifically to BACE1 as the activity that was blocked by the specific inhibitors, which is still higher in AD subjects. BACE1 protein levels were characterized by lectin binding, immunoprecipitation, blue native-PAGE, and western blotting using antibodies against specific protein domains. BACE1 was found to be present in human CSF as a mature form of ~ 70 kDa that probably comprised truncated and full-length species, and also as an immature form of ~ 50 kDa that retains the prodomain. CSF-BACE1 was found to assemble into hetero-complexes containing distinct species. Immunoblotting with an antibody against the C-terminus of BACE1 revealed significantly higher levels of the 70-kDa full-length BACE1, while the 50 kDa immature form remained unaltered. When the 70-kDa species was probed with an antibody against the N-terminus of BACE1 (which does not discriminate between truncated and full-length forms), no increase in immunoreactivity was observed, suggesting that truncated forms of BACE1 do not increase in AD. In conclusion, the complexity of BACE1 species in CSF has to be taken into consideration when determining BACE1 activity and protein levels in CSF as biomarkers of AD. [ABSTRACT FROM AUTHOR]

Due to the large size and high flexibility of the catalytic active site of BACE1 enzyme, the development of nonpeptide inhibitors with optimal pharmacological properties is still highly demanding. In this work, we have discovered 2-aminobenzimidazole-containg ether scaffolds having potent and selective inhibitory potentials against BACE1 enzyme. We have synthesized novel 29 compounds and optimization of aryl linker region resulted in highly potent BACE1 inhibitory activities with EC 50 values of 0.05–2.71 μM. The aryloxy-phenyl analogs 20j showed the EC 50 value as low as 0.07 μM in the enzyme assay, whereas, the benzyloxyphenyl dervative 24b was comparatively less effective in the enzyme assay. But interestingly the latter was more effective in the cell assay (EC 50 value 1.2 μM). While comparing synthesized derivatives in the cell assay using PC12-APP SW cell, compound 27f appeared as the most potent BACE1 inhibitor having EC 50 value 0.7 μM. This scaffold also showed high selectivity over BACE2 enzyme and cathepsin D. Furthermore, the research findings were bolstered through the incorporation of molecular docking, molecular dynamics, and DFT studies. We firmly believe that these discoveries will pave the way for the development of a novel class of small-molecule selective BACE1 inhibitors. [Display omitted] • 29 Novel diaryl ether-benzimidazole derivatives exhibit potent BACE1 inhibition. • Some compounds showed the EC 50 value as low as 0.07 μM in the enzyme assay. • Compound 27f most potent BACE1 inhibitor having EC 50 value 0.7 μM in cell assay. • Some derivatives showed SI greater than 15 in BACE2 and cathepsin D inhibitory assays. • The interactions of lead compounds with BACE1 were elucidated by in silico models. [ABSTRACT FROM AUTHOR]

Only palliative therapeutic options exist for the treatment of Alzheimer’s Disease; no new successful drug candidates have been developed in over 15 years. The widely used clinical anticoagulant heparin has been reported to exert beneficial effects through multiple pathophysiological pathways involved in the aetiology of Alzheimer’s Disease, for example, amyloid peptide production and clearance, tau phosphorylation, inflammation and oxidative stress. Despite the therapeutic potential of heparin as a multi-target drug for Alzheimer’s disease, the repurposing of pharmaceutical heparin is proscribed owing to the potent anticoagulant activity of this drug. Here, a heterogenous non-anticoagulant glycosaminoglycan extract, obtained from the shrimp Litopenaeus vannamei, was found to inhibit the key neuronal β-secretase, BACE1, displaying a more favorable therapeutic ratio compared to pharmaceutical heparin when anticoagulant activity is considered.

Amyloid precursor protein (APP) internalization via clathrin-/dynamin-mediated endocytosis (CME) mediated by its YENPTY motif into endosomes containing β-secretase is proposed to be critical for amyloid-beta (Aβ) production. Here, we show that somatodendritic APP internalization in primary rodent neurons is not blocked by inhibiting dynamin or mutating the YENPTY motif, in contrast to non-neuronal cell lines. These phenomena, confirmed in induced human neurons under dynamin inhibition, occur during basal conditions and chemical long-term-depression stimulus, pointing to a clathrin-independent internalization pathway for somatodendritic APP. Mutating the YENPTY motif does not alter APP recycling, degradation, or endolysosomal colocalization. However, both dynamin inhibition and the YENPTY mutant significantly decrease secreted Aβ in neurons, suggesting that internalized somatodendritic APP may not constitute a major source of Aβ. Interestingly, like APP, somatodendritic low-density lipoprotein receptor (LDLR) internalization does not require its CME motif. These results highlight intriguing differences in neuronal internalization pathways and refine our understanding of Aβ production and secretion. [Display omitted] • Somatodendritic APP endocytosis does not require dynamin activity or the YENPTY motif • LDLR endocytosis also does not require its NPVY motif, suggesting APP is not unique • Both dynamin inhibition and the YENPTY mutant reduce Aβ production and secretion • Somatodendritic APP endocytosis can be decoupled from pathways that generate Aβ Aow et al. show that APP internalization in the neuronal somatodendritic compartment is clathrin/dynamin independent, suggesting the presence of specialized endocytic pathways in different neuronal compartments. Internalized somatodendritic APP does not appear to represent a major source of Aβ, indicating that neurons compartmentalize surface APP pools toward downstream processing pathways. [ABSTRACT FROM AUTHOR]

Therapeutic options for Alzheimer’s disease, the most common form of dementia, are currently restricted to palliative treatments. The glycosaminoglycan heparin, widely used as a clinical anticoagulant, has previously been shown to inhibit the Alzheimer’s disease-relevant β-secretase 1 (BACE1). Despite this, the deployment of pharmaceutical heparin for the treatment of Alzheimer’s disease is largely precluded by its potent anticoagulant activity. Furthermore, ongoing concerns regarding the use of mammalian-sourced heparins, primarily due to prion diseases and religious beliefs hinder the deployment of alternative heparin-based therapeutics. A marine-derived, heparan sulphate-containing glycosaminoglycan extract, isolated from the crab Portunus pelagicus, was identified to inhibit human BACE1 with comparable bioactivity to that of mammalian heparin (IC50 = 1.85 μg mL−1 (R2 = 0.94) and 2.43 μg mL−1 (R2 = 0.93), respectively), while possessing highly attenuated anticoagulant activities. The results from several structural techniques suggest that the interactions between BACE1 and the extract from P. pelagicus are complex and distinct from those of heparin.

The β-secretase (BACE1) initiates the generation of toxic amyloid-β peptide (Aβ) from amyloid-β precursor protein (APP), which was widely considered to play a key role in the pathogenesis of Alzheimer's disease (AD). Here, a novel microfluidics-based mobility shift assay (MMSA) was developed, validated, and applied for the screening of BACE1 inhibitors for AD. First, the BACE1 activity assay was established with a new fluorescent peptide substrate (FAM-EVNLDAEF) derived from the Swedish mutant APP, and high-quality ratiometric data were generated in both endpoint and kinetic modes by electrophoretic separation of peptide substrate from the BACE1 cleaved product (FAM-EVNL) before fluorescence quantification. To validate the assay, the inhibition and kinetic parameter values of two known inhibitors (AZD3839 and AZD3293) were evaluated, and the results were in good agreement with those reported by other methods. Finally, the assay was applied to screen for new inhibitors from a 900-compound library in a 384-well format, and one novel hit (IC = 26.5 ± 1.5 μM) was identified. Compared with the common fluorescence-based assays, the primary advantage of the direct MMSA was to discover novel BACE1 inhibitors with lower auto-fluorescence interference, and its superb capability for kinetic study. [ABSTRACT FROM AUTHOR]

Discovering multifunctional agents for the treatment of Alzheimer’s disease (AD) is an attractive therapeutic approach. BACE1 (β-site amyloid precursor protein cleaving enzyme 1) inhibitors may play a pivotal role in treating AD. Therefore, the discovery of novel non-peptide BACE1 inhibitors with desirable blood brain barrier permeability is a favorable approach for treatment. Moreover, the antioxidant potential of a drug could serve as an added value for designing dual-acting therapeutic agents. Here, we report the design, synthesis and biological evaluation of quinazolinone-hydrazone derivatives as new multi-target candidates for the treatment of AD. The compounds were investigated for their in vitro BACE1 inhibitory potential using a FRET-based enzymatic assay and also screened for antioxidant activity using DPPH. Among them, compound 4h bearing a 2,3-dichlorophenyl moiety showed the highest activity with an IC 50 value of 3.7 μM against BACE1. In addition, compound 4i with a 2,4-dihydroxyphenyl scaffold demonstrated moderate BACE1 inhibitory activity (IC 50 = 27.6 μM) with a significant antioxidant effect (IC 50 = 8.4 μM). Furthermore, docking studies revealed strong interaction between compound 4h and the key residues of BACE1 active site. These results demonstrate that quinazolinone-hydrazone derivatives represent a valuable scaffold for the discovery of novel non-peptidic BACE1 inhibitors. [ABSTRACT FROM AUTHOR]

In order to design novel non-peptidic inhibitors of BACE1, many research groups have attempted using computational studies including docking analyses. Since there are too many 3D structures for BACE1 in the protein database, the selection of suitable crystal structures is a key prerequisite for the successful application of molecular docking. We employed a multi-structure docking protocol. In which 615 ligands’ structures were docked into 150 BACE1 structures. The large number of the resultant docking scores were post-processed by different data analysis methods including exploratory data analysis, regression analysis and discriminant analysis. It was found that using one crystal structure for docking did not result in high accuracy for predicting activity of the BACE1 inhibitors. Instead, using of the multi-structural docking scores, post-processed by chemometrics methods arrived to highly accurate predictive models. In this regards, the PDB accession codes of 4B70, 4DVF and 2WEZ could discriminate between active and inactive compounds, with higher accuracy. Clustering of the BACE1 structures based on principal component analysis of the crystallographic structures the revealed that the discriminant structures are in the center of the clusters. Thus, these structures can be selected as predominant crystal structures for docking studies of non-peptidic BACE1 inhibitors. [ABSTRACT FROM AUTHOR]

Summary Endocytosis of amyloid-β precursor protein (APP) is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. Emerging evidence indicates that amyloid-β (Aβ) peptides enter mitochondria and may thereby disrupt mitochondrial function in brains of AD patients and transgenic model mice. However, it remains to be determined whether the β-cleaved C-terminal fragment (C99), another neurotoxic fragment of amyloid precursor protein (APP), may accumulate in mitochondria of neurons affected by AD. Using immunoblotting, digitonin fractionation and immunofluorescence labeling techniques, we found that C99 is targeted to mitochondria, in particular, to the mitoplast (i.e., inner membrane and matrix compartments) in brains of AD transgenic mice (5XFAD model). Furthermore, full-length APP (fl-APP) was also identified in mitochondrial fractions of 5XFAD mice. Remarkably, partial deletion of the β-site APP-cleaving enzyme 1 (BACE1+/−) almost completely abolished mitochondrial targeting of C99 and fl-APP in 5XFAD mice at 6 months of age. However, substantial amounts of C99 and fl-APP accumulation remained in mitochondria of 12-month-old BACE1+/−·5XFAD mouse brains. Consistent with these changes in mitochondrial C99/fl-APP levels, BACE1+/− deletion age-dependently rescued mitochondrial dysfunction in 5XFAD mice, as assessed by cytochrome c release from mitochondria, reduced redox or complex activities and oxidative DNA damage. Moreover, BACE1+/− deletion also improved memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at 6 months but not at 12 months of age. Taken together, our findings suggest that mitochondrial accumulation of C99 and fl-APP may occur through BACE1-dependent mechanisms and contribute to inducing mitochondrial dysfunction and cognitive impairments associated with AD.

c-Jun N-terminal kinases (JNKs), which belong to a mitogen-activated protein kinase (MAPK) family, are involved in the regulation of several physiological functions in mammals and act as mediators of apoptosis, obesity, and memory storage in the brain, including the processes of neuronal de- and regeneration. JNK subfamily is encoded by three separate but related genes: jnk1, jnk2, and jnk3, giving rise to at least ten distinct splice variants of the JNK proteins. JNK3 is thought to be a major contributor to neurodegeneration in mammalian brain. The role of JNK1 in the pathological processes affecting cognitive function, especially in diseases such as Alzheimer's disease (AD), is less clear. In order to evaluate the effects of JNK1 deficiency in an experimental model of familial Alzheimer's disease, double transgenic APPswe/PS1dE9 mice were crossed with the JNK1 heterozygous deficient animals ( jnk1+/−). As expected, a ∼50 % reduction in JNK1 protein levels was observed in the hippocampi of 9-month-old APPswe/PS1dE9/ jnk1+/− mice, compared with the APPswe/PS1dE9 group. JNK1 deficiency resulted in reduced BACE1 expression, suggesting alterations in amyloidogenic pathway. However, no significant inter-group differences in the total number of β-amyloid plaques were observed in the hippocampal region. In addition, protein levels of PPAR gamma coactivator-1α (PGC-1α), a molecule involved in mitochondrial biogenesis and energy homeostasis, were decreased in 9-month-old APPswe/PS1dE9 mice but not in APPswe/PS1dE9/ jnk1+/− animals. Furthermore, JNK1 deficiency did not have an effect on pro-inflammatory marker expression in the hippocampus. Heterozygous deficiency of JNK1 results in the decrease of BACE1 protein levels, which is not accompanied by the reduction in the total number of β-amyloid plaques in the hippocampi of APPswe/PS1dE9 mice. Moreover, PGC-1α expression is restored in APPswe/PS1dE9/ jnk1+/− animals, which indicates a possible role of JNK1 in brain mitochondrial regulation. Nevertheless, our results suggest that partial inhibition of JNK1 is not sufficient to prevent the neuropathological processes in this model. It may be necessary to inhibit both the JNK1 and JNK3 simultaneously, especially as previous studies suggest that JNK3 contributes to AD neuropathology. [ABSTRACT FROM AUTHOR]

Accumulating evidence points to the amyloid-β (Aβ) peptide as the culprit in the pathogenesis of Alzheimer’s disease (AD). β-Site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a protease that is responsible for initiating Aβ production. Although precise mechanisms that trigger Aβ accumulation remain unclear, BACE1 inhibition undoubtedly represents an important intervention that may prevent and/or cure AD. Remarkably, animal model studies with knockouts, virus-delivered small interfering RNAs, immunization and bioavailable small-molecule agents that specifically inhibit BACE1 activity strongly support the idea for the therapeutic BACE1 inhibition. Meanwhile, a growing number of BACE1 substrates besides APP uncover new physiological roles of this protease, raising some concern regarding the safety of BACE1 inhibition. Here, I review recent progress in preclinical studies that have evaluated the efficacies and potential limitations of genetic/pharmacological inhibition of BACE1, with special focus on AD-associated phenotypes including synaptic dysfunction, neuron loss and memory deficits in animal models. [ABSTRACT FROM AUTHOR]

β-Secretase, widely known as β-site APP cleaving enzyme 1 (BACE1), is a membrane-associated protease that cleaves amyloid precursor protein (APP) to generate amyloid β-protein (Aβ). As this cleavage is a pathologically relevant event in Alzheimer’s disease, BACE1 is considered a viable therapeutic target. BACE1 can be regulated at the transcriptional, post-transcriptional, translational, and post-translational levels. Elucidation of the regulatory pathways of BACE1 is critical, not only for understanding the pathological mechanisms of AD but also developing effective therapeutic strategies to inhibit activity of the protease. This mini-review focuses on the post-translational regulation of BACE1, as modulation at this level is closely associated with both physiological and pathological conditions. Current knowledge on the mechanisms underlying such BACE1 regulation and their implications for therapy are discussed. [ABSTRACT FROM AUTHOR]

β-secretase (BACE1) is an aspartyl protease that processes the β-amyloid peptide in the human brain in patients with Alzheimer’s disease. There are two catalytic aspartates (ASP32 and ASP228) in the active domain of BACE1. Although it is believed that the net charge of the Asp dyad is −1, the exact protonation state still remains a matter of debate. We carried out molecular dynamic (MD) simulations for the four protonation states of BACE1 proteins. We applied Glide docking studies to 21 BACE1 inhibitors against the MD extracted conformations. The dynamic results infer that the protein/ligand complex remains stable during the entire simulation course for HD32D228 model. The results show that the hydrogen bonds between the inhibitor and the Asp dyad are maintained in the 10,000th ps snapshot of HD32D228 model. Our results also reveal the significant loop residues in maintaining the active binding conformation in the HD32D228 model. Molecular docking results show that the HD32D228 model provided the best enrichment factor score, suggesting that this model was able to recognize the most active compounds. Our observations provide an evidence for the preference of the anionic state (HD32D228) in BACE1 binding site and are in accord with reported computational data. The protonation state study would provide significant information to assign the correct protonation state for structure-based drug design and docking studies targeting the BACE1 proteins as a tactic to develop potential AD inhibitors. [ABSTRACT FROM AUTHOR]

Background: Accumulating evidence indicates that partial inhibition of β-site APP-cleaving enzyme 1 (BACE1), which initiates amyloid-β (Aβ) production, mitigates Alzheimer's disease (AD)-like pathologies and memory deficits in a battery of transgenic mouse models. However, our previous investigations suggest that therapeutic BACE1 suppression may be beneficial only if targeted on earlier stages of AD and encounter dramatic reductions in efficacy during disease progression. This study was designed to test the possibility that a combination approach, aimed at inhibiting BACE1 and boosting neprilysin (a major Aβ-degrading enzyme) activities, may be able to mechanistically overcome the limited efficacy of anti-Aβ therapy in advanced AD. Results: After crossbreeding between BACE1 heterozygous knockout (BACE1+/-), neprilysin transgenic (NEP) and 5XFAD mice, we analyzed the resultant mice at 12 months of age when 5XFAD controls showed robust amyloid-β (Aβ) accumulation and elevation of BACE1 expression (~2 folds). Although haploinsufficiency lowered BACE1 expression by ~50% in concordance with reduction in gene copy number, profound β-amyloidosis, memory deficits and cholinergic neuron death were no longer rescued in BACE1+/- . 5XFAD mice concomitant with their persistently upregulated BACE1 (i.e., equivalent to wild-type control levels). Notably, neprilysin overexpression not only prevented Aβ accumulation but also suppressed the translation initiation factor eIF2α-associated elevation of BACE1 and lowered levels of the β-secretase-cleaved C-terminal fragment of APP (C99) in NEP . 5XFAD mice. Interestingly, these markers for β-amyloidogenesis in BACE1+/- . NEP . 5XFAD mice were further reduced to the levels reflecting a combination of single BACE1 allele ablation and the abolishment of translational BACE1 upregulation. However, since neprilysin overexpression was striking (~8-fold relative to wild-type controls), memory impairments, cholinergic neuronal loss and β-amyloidosis were similarly prevented in NEP . 5XFAD and BACE1+/- . NEP. 5XFAD mice. Conclusions: Our findings indicate that robust overexpression of neprilysin is sufficient to ameliorate AD-like phenotypes in aged 5XFAD mice. We also found that Aβ-degrading effects of overexpressed neprilysin can block deleterious BACE1-elevating mechanisms that accelerate Aβ production, warranting further study to test whether interventions moderately activating neprilysin may be useful for boosting the limited efficacy of therapeutic BACE1 inhibition in treating AD with established Aβ pathology. [ABSTRACT FROM AUTHOR]

In the present study, we have assessed the biophysical properties of lipid rafts from different brain areas in subjects exhibiting early neuropathological stages of Alzheimer's disease (AD). By means of steady-state fluorescence polarization analyses using two environment-sensitive fluorescent probes, we demonstrate that lipid rafts from cerebellum, and frontal and entorhinal cortices, exhibit different biophysical behaviors depending on the stage of the disease. Thus, while membrane anisotropies were similar in the cerebellum along stages, lipid rafts from frontal and entorhinal cortices at AD stages I/II and AD III were significantly more liquid-ordered than in control subjects, both at the aqueous interface and hydrophobic core of the raft membrane. Thermotropic analyses demonstrated the presence of Arrhenius breakpoints between 28.3-32.0°C, which were not influenced by the disease stage. However, analyses of membrane microviscosity (ηapp) demonstrate that frontal and entorhinal lipid rafts are notably more viscous and liquid-ordered all across the membrane from early stages of the disease. These physicochemical alterations in lipid rafts do not correlate with changes in cholesterol or sphingomyelin levels, but to reduced unsaturation index and increased saturate/polyunsaturated ratios in phospholipid acyl chains. Moreover, we demonstrate that β-secretase/AβPP (amyloid-β protein precursor) interaction and lipid raft microviscosity are strongly, and positively, correlated in AD frontal and entorhinal cortices. These observations strengthens the hypothesis that physical properties of these microdomains modulate the convergence of amyloidogenic machinery toward lipid rafts, and also points to a critical role of polyunsaturated fatty acids in amyloidogenic processing of AβPP. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) and vascular dementia are responsible for up to 90% of dementia cases. According to the World Health Organization (WHO), a staggering number of 35.6 million people are currently diagnosed with dementia. Blocking disease progression or preventing AD altogether is desirable for both social and economic reasons and recently focus has shifted to a new and promising drug: the b-secretase inhibitor. Much of AD research has investigated the amyloid cascade hypothesis, which postulates that AD is caused by changes in amyloid beta (Aβ) stability and aggregation. Blocking Aβ production by inhibiting the first protease required for its generation, b-secretase/BACE1, may be the next step in blocking AD progression. In April 2012, promising phase I data on inhibitor MK-8931 was presented. This drug reduced Aβ cerebral spinal fluids (CSF) levels up to 92% and was well tolerated by patients. In March 2013 data was added from a one week trial in 32 mild to moderate AD patients, showing CSF Aβ levels decreased up to 84%. However, b-site APP cleaving enzyme 1 (BACE1) inhibitors require further research. First, greatly reducing Aβ levels through BACE1 inhibition may have harmful side effects. Second, BACE1 inhibitors have yet to pass clinical trial phase II/III and no data on possible side effects on AD patients are available. And third, there remains doubt about the clinical efficacy of BACE1 inhibitors. In moderate AD patients, Aβ plaques have already been formed. BACE1 inhibitors prevent production of new Aβ plaques, but hypothetically do not influence already existing Aβ peptides. Therefore, BACE1 inhibitors are potentially better at preventing AD instead of having therapeutic use. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) and vascular dementia are responsible for up to 90% of dementia cases. According to the World Health Organization (WHO), a staggering number of 35.6 million people are currently diagnosed with dementia. Blocking disease progression or preventing AD altogether is desirable for both social and economic reasons and recently focus has shifted to a new and promising drug: the β-secretase inhibitor. Much of AD research has investigated the amyloid cascade hypothesis, which postulates that AD is caused by changes in amyloid beta (Aβ) stability and aggregation. Blocking Aβ production by inhibiting the first protease required for its generation, β-secretase/BACE1, may be the next step in blocking AD progression. In April 2012, promising phase I data on inhibitor MK-8931 was presented. This drug reduced Aβ CSF levels up to 92% and was well tolerated by patients. In March 2013 data was added from a one week trial in 32 mild to moderate AD patients, showing CSF Aβ levels decreased up to 84%. However, BACE1 inhibitors require further research. First, greatly reducing Aβ levels through BACE1 inhibition may have harmful side effects. Second, BACE1 inhibitors have yet to pass clinical trial phase II/III and no data on possible side effects on AD patients are available. And third, there remains doubt about the clinical efficacy of BACE1 inhibitors. In moderate AD patients, Aβ plaques have already been formed. BACE1 inhibitors prevent production of new Aβ plaques, but hypothetically do not influence already existing Aβ peptides. Therefore, BACE1 inhibitors are potentially better at preventing AD instead of having therapeutic use. [ABSTRACT FROM AUTHOR]

Abstract: We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited potent activities in enzymatic and cellular assays (KMI-429 in particular inhibited Aβ production in vivo), these inhibitors contained some natural amino acids that seemed to be required to improve enzymatic stability in vivo and permeability across the blood–brain barrier, so as to be practical drug. Recently, we synthesized non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold at the P2 position. Herein we report the SAR study of BACE1 inhibitors possessing this heterocyclic scaffold, a chelidonic or 2,6-pyridinedicarboxylic moiety. [Copyright &y& Elsevier]

β-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the β-secretase that initiates Aβ production in Alzheimer's disease (AD). BACE1 levels are increased in AD, which could contribute to pathogenesis, yet the mechanism of BACE1 elevation is unclear. Furthermore, the normal function of BACE1 is poorly understood. We localized BACE1 in the brain at both the light and electron microscopic levels to gain insight into normal and pathophysiologic roles of BACE1 in health and AD, respectively. Our findings provide the first ultrastructural evidence that BACE1 localizes to vesicles (likely endosomes) in normal hippocampal mossy fiber terminals of both non-transgenic and APP transgenic (5XFAD) mouse brains. In some instances, BACE1-positive vesicles were located near active zones, implying a function for BACE1 at the synapse. In addition, BACE1 accumulated in swollen dystrophic autophagosome-poor presynaptic terminals surrounding amyloid plaques in 5XFAD cortex and hippocampus. Importantly, accumulations of BACE1 and APP co-localized in presynaptic dystrophies, implying increased BACE1 processing of APP in peri-plaque regions. In primary cortical neuron cultures, treatment with the lysosomal protease inhibitor leupeptin caused BACE1 levels to increase; however, exposure of neurons to the autophagy inducer trehalose did not reduce BACE1 levels. This suggests that BACE1 is degraded by lysosomes but not by autophagy. Our results imply that BACE1 elevation in AD could be linked to decreased lysosomal degradation of BACE1 within dystrophic presynaptic terminals. Elevated BACE1 and APP levels in plaque-associated presynaptic dystrophies could increase local peri-plaque Aβ generation and accelerate amyloid plaque growth in AD. [ABSTRACT FROM AUTHOR]

β-Secretase ( BACE1) cleavage of the amyloid precursor protein ( APP) represents the initial step in the formation of the Alzheimer's disease associated amyloidogenic Aβ peptide. Substantive evidence indicates that APP processing by BACE1 is dependent on intracellular sorting of this enzyme. Nonetheless, knowledge of the intracellular trafficking pathway of internalised BACE1 remains in doubt. Here we show that cell surface BACE1 is rapidly internalised by the AP2/clathrin dependent pathway in transfected cells and traffics to early endosomes and Rab11-positive, juxtanuclear recycling endosomes, with very little transported to the TGN as has been previously suggested. Moreover, BACE1 is predominantly localised to the early and recycling endosome compartments in different cell types, including neuronal cells. In contrast, the majority of internalised wild-type APP traffics to late endosomes/lysosomes. To explore the relevance of the itinerary of BACE1 on APP processing, we generated a BACE1 chimera containing the cytoplasmic tail of TGN38 ( BACE/ TGN38), which cycles between the cell surface and TGN in an AP2-dependent manner. Wild-type BACE1 is less efficient in Aβ production than the BACE/ TGN38 chimera, highlighting the relevance of the itinerary of BACE1 on APP processing. Overall the data suggests that internalised BACE1 and APP diverge at early endosomes and that Aβ biogenesis is regulated in part by the recycling itinerary of BACE1. [ABSTRACT FROM AUTHOR]

Abstract: The β-site APP cleaving enzyme (BACE1) is required for the production of β-amyloid peptides, which give rise to β-amyloid (Aβ) deposits in the brains of Alzheimer’s disease (AD) patients. In brains, BACE1 is primarily expressed by neurons, however BACE1 expression has also been observed in reactive astrocytes in close proximity to β-amyloid plaques in the brains of aged Tg2576 AD model mice. To date, the direct effects of Aβ on BACE1 gene expression in astrocytes is unknown. We found that Aβ42 or Aβ25–35 treatment induced BACE1 expression in primary astrocytes as well as human astrocytoma cell line. Aβ neurotoxicity has been associated with the disruption of intracellular calcium homeostasis both in neurons and in glial cells. Here, we demonstrated that NFAT4, a transcription factor tightly regulated by the calcium/calmodulin-dependent phosphatase, calcineurin, was activated in astrocytes applied with calcium ionophore or Aβ. Aβ-activated NFAT4 proteins were associated with astrocytic BACE1 gene expression via direct interaction with the BACE1 promoter region. [Copyright &y& Elsevier]

Abstract: Recently, we reported substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell assays. We also designed and synthesized non-peptidic and small-sized inhibitors possessing a heterocyclic scaffold at the P2 position. By studying the structure–activity relationship of these inhibitors, we found that the σ–π interaction of an inhibitor with the BACE1-Arg235 side chain played a key role in the inhibition mechanism. Hence, we optimized the inhibitors with a focus on their P2 regions. In this Letter, a series of novel BACE1 inhibitors possessing a 5-nitroisophthalic scaffold at the P2 position are described along with the results of the related structure–activity relationship study. These small-sized inhibitors are expected improved membrane permeability and bioavailability. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are small non-coding RNA regulators of protein synthesis that function as “fine-tuning” tools of gene expression in development and tissue homeostasis. Their profiles are significantly altered in neurodegenerative diseases such as Alzheimer''s disease (AD) that is characterized by both amyloid-β (Aβ) and tau deposition in brain. A key challenge remains in determining how changes in miRNA profiles translate into biological function in a physiological and pathological context. The key lies in identifying specific target genes for deregulated miRNAs and understanding which pathogenic factors trigger their deregulation. Here we review the literature about the intricate network of miRNAs surrounding the regulation of the amyloid precursor protein (APP) from which Aβ is derived by proteolytic cleavage. Normal brain function is highly sensitive to any changes in APP metabolism and miRNAs function at several steps to ensure that the correct APP end product is produced and in the right form and abundance. Disruptions in this miRNA regulatory network may therefore alter Aβ production, which in turn can affect miRNA expression. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Mitochondrial dysfunction is an early feature of Alzheimer''s disease (AD) and may play an important role in the pathogenesis of disease. Emerging evidence indicates that amyloid-β (Aβ) peptides enter mitochondria and may thereby disrupt mitochondrial function in brains of AD patients and transgenic model mice. However, it remains to be determined whether the β-cleaved C-terminal fragment (C99), another neurotoxic fragment of amyloid precursor protein (APP), may accumulate in mitochondria of neurons affected by AD. Using immunoblotting, digitonin fractionation and immunofluorescence labeling techniques, we found that C99 is targeted to mitochondria, in particular, to the mitoplast (i.e., inner membrane and matrix compartments) in brains of AD transgenic mice (5XFAD model). Furthermore, full-length APP (fl-APP) was also identified in mitochondrial fractions of 5XFAD mice. Remarkably, partial deletion of the β-site APP-cleaving enzyme 1 (BACE1+/−) almost completely abolished mitochondrial targeting of C99 and fl-APP in 5XFAD mice at 6months of age. However, substantial amounts of C99 and fl-APP accumulation remained in mitochondria of 12-month-old BACE1+/−·5XFAD mouse brains. Consistent with these changes in mitochondrial C99/fl-APP levels, BACE1+/− deletion age-dependently rescued mitochondrial dysfunction in 5XFAD mice, as assessed by cytochrome c release from mitochondria, reduced redox or complex activities and oxidative DNA damage. Moreover, BACE1+/− deletion also improved memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at 6months but not at 12months of age. Taken together, our findings suggest that mitochondrial accumulation of C99 and fl-APP may occur through BACE1-dependent mechanisms and contribute to inducing mitochondrial dysfunction and cognitive impairments associated with AD. [Copyright &y& Elsevier]

Abstract: A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (β-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer’s disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer’s disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution. [Copyright &y& Elsevier]

The enzyme β-secretase (BACE1) is essentially involved in the production of cerebral amyloidogenic pathology in Alzheimer's disease (AD). The measurement of BACE1 activity in cerebrospinal fluid (CSF) has been reported, which may render CSF measurement of BACE1 a potential biomarker candidate of AD. In order to investigate whether BACE1 protein activity is correlated with regional brain atrophy in AD, we investigated the association between CSF levels of BACE1 and MRI-assessed hippocampus volume in patients with AD (n = 30). An increase in CSF-BACE1 activity was associated with decreased left and right hippocampus volume corrected for global head volume in the AD patients. Boot-strapped regression analysis showed that increased CSF levels of BACE1 activity were associated with increased CSF concentration of total tau but not amyloid-β1-42 in AD. White matter hyperintensities did not influence the results. BACE1 activity and protein levels were significantly increased in AD compared to 19 elderly healthy controls. Thus, the CSF biomarker candidate of BACE1 activity was associated with hippocampus atrophy in AD in a robust manner and may reflect neurotoxic amyloid-β-related processes. [ABSTRACT FROM AUTHOR]

The diagnosis of Alzheimer's disease (AD) relies principally on clinical criteria for probable and possible AD as defined by the NINCDS-ADRDRA. The field is desperately lacking of biological markers to assist with AD diagnosis and verification of treatment efficacy. According to the Consensus Report of the Working Group on Molecular and Biochemical Markers of Alzheimer's Disease, in order to qualify as a biomarker the sample in question must adhere to certain basic requirements, including the ability to: reflect AD pathology and differentiate it from other dementia with an 80% sensitivity; be reliable and reproducible; be easy to perform and analyze; remain relatively inexpensive. Beta secretases are crucial enzymes in the pathogenesis of AD. Given its primary role in brain amyloidogenesis and its ubiquitous expression, one may consider measuring peripheral BACE1 levels and activity as biomarkers of AD, like performed in the brain and cerebrospinal fluid. However, very little is known about the periphery and whether peripheral BACE1 is involved in AD pathogenesis or mirrors AD progression. Moreover, no investigation has focused on the possibility of monitoring peripheral BACE1 to assess the efficiency of BACE1 inhibitors during the course of clinical trials. Part of the problem may be attributed to the lack of sensitive molecular tools which are absolutely necessary to use BACE1 as a biomarker. In this review we evaluate the progress and feasibility of developing BACE1 as a biomarker for AD in different tissues. [ABSTRACT FROM AUTHOR]

β-secretase, a key enzyme involved in amyloid-β generation, is an attractive candidate for Alzheimer's disease therapy. Transition-state inhibitors of β-secretase are designed to achieve specificity. However, these inhibitors bind only to the active conformation of the enzyme and as the active β-secretase is sequestered in subcellular compartments, new strategies have to be implemented. We propose that membrane-anchoring of β-secretase inhibitors would render them endocytosis-competent thereby enabling the inhibitors to reach these compartments that harbor active β-secretase. By choosing cholesterol as a membrane anchor, we also enrich the inhibitor in lipid rafts where much of the β-secretase is present. In addition, membrane-anchoring of soluble inhibitors reduces the dimensionality of the inhibitor and consequently increases the inhibitor concentration at the target membrane plane. Such inhibitors have great potential in terms of substrate selectivity and reduced side effects. Not only for β-secretase, this strategy could be applied for many membrane targets that are localized either at the plasma membrane or in the endocytic compartments. [ABSTRACT FROM AUTHOR]

The diagnosis of Alzheimer's disease (AD) relies principally on clinical criteria for probable and possible AD as defined by the NINCDS-ADRDRA. The field is desperately lacking of biological markers to assist with AD diagnosis and verification of treatment efficacy. According to the Consensus Report of the Working Group on Molecular and Biochemical Markers of Alzheimer's Disease, in order to qualify as a biomarker the sample in question must adhere to certain basic requirements, including the ability to: reflect AD pathology and differentiate it from other dementia with an 80% sensitivity; be reliable and reproducible; be easy to perform and analyze; remain relatively inexpensive. Beta secretases are crucial enzymes in the pathogenesis of AD. Given its primary role in brain amyloidogenesis and its ubiquitous expression, one may consider measuring peripheral BACE1 levels and activity as biomarkers of AD, like performed in the brain and cerebrospinal fluid. However, very little is known about the periphery and whether peripheral BACE1 is involved in AD pathogenesis or mirrors AD progression. Moreover, no investigation has focused on the possibility of monitoring peripheral BACE1 to assess the efficiency of BACE1 inhibitors during the course of clinical trials. Part of the problem may be attributed to the lack of sensitive molecular tools which are absolutely necessary to use BACE1 as a biomarker. In this review we evaluate the progress and feasibility of developing BACE1 as a biomarker for AD in different tissues. [ABSTRACT FROM AUTHOR]

β-secretase, a key enzyme involved in amyloid-β generation, is an attractive candidate for Alzheimer's disease therapy. Transition-state inhibitors of β-secretase are designed to achieve specificity. However, these inhibitors bind only to the active conformation of the enzyme and as the active β-secretase is sequestered in subcellular compartments, new strategies have to be implemented. We propose that membrane-anchoring of β-secretase inhibitors would render them endocytosis-competent thereby enabling the inhibitors to reach these compartments that harbor active β-secretase. By choosing cholesterol as a membrane anchor, we also enrich the inhibitor in lipid rafts where much of the β-secretase is present. In addition, membrane-anchoring of soluble inhibitors reduces the dimensionality of the inhibitor and consequently increases the inhibitor concentration at the target membrane plane. Such inhibitors have great potential in terms of substrate selectivity and reduced side effects. Not only for β-secretase, this strategy could be applied for many membrane targets that are localized either at the plasma membrane or in the endocytic compartments. [ABSTRACT FROM AUTHOR]

Abstract: BACE1 and presenilin (PS)/γ-secretase are primary proteolytic enzymes responsible for the generation of pathogenic amyloid β-peptides (Aβ) in Alzheimer''s disease. We and others have found that β-subunits of the voltage-gated sodium channel (Navβs) also undergo sequential proteolytic cleavages mediated by BACE1 and PS/γ-secretase. In a follow-up study, we reported that elevated BACE1 activity regulates total and surface expression of voltage-gated sodium channels (Nav1 channels) and thereby modulates sodium currents in neuronal cells and mouse brains. In this review, we focus on the molecular mechanism of how BACE1 and PS/γ-secretase regulate Nav1 channels in neuronal cells. We will also discuss potential physiological and pathological roles of BACE1- and PS/γ-secretase-mediated processing of Navβs in relation to Nav1 channel function. [Copyright &y& Elsevier]

Abstract: A novel class of pyridinyl aminohydantoins was designed and prepared as highly potent BACE1 inhibitors. Compound (S)-4g showed excellent potency with IC50 of 20nM for BACE1. X-ray crystallography indicated that the interaction between pyridine nitrogen and the tryptophan Trp76 was a key feature in the S2′ region of the enzyme that contributed to increased potency. [Copyright &y& Elsevier]

J. Neurochem. (2009) 112, 1045–1053. β-Secretase is the rate limiting enzymatic activity in the production of the amyloid-β peptide (Aβ) and is thought to be involved in Alzheimer’s disease (AD) pathogenesis. Although BACE1 (β-site APP Cleaving Enzyme 1, EC 3.4.23.46) has received significant attention, the related BACE2 (EC 3.4.23.45) has not. Though BACE2 is also expressed in the brain, its potential role in AD has not been resolved. In this study, we compared the activities of both BACE1 and BACE2, which were isolated from the same samples of frontal cortex from both AD-affected individuals and age-matched controls. BACE1 activity showed a significant positive correlation with the amount of extractable Aβ, and BACE1 protein and activity were significantly increased in AD cases. Unexpectedly, there were substantial total amounts of BACE2 protein and enzymatic activity in the human brain. BACE2 activity did not change significantly in the AD brain, and was not related to Aβ concentration. These data indicate that BACE1 likely accounts for most of the Aβ produced in the human brain, and that BACE2 activity is not a likely contributor. However, as both forms of BACE compete for the same substrate pool, even small changes in BACE2 activity could have consequences for human disease. [ABSTRACT FROM AUTHOR]

Although transgenic mouse models of Alzheimer’s disease (AD) recapitulate amyloid-β (Aβ)-related pathologies and cognitive impairments, previous studies have mainly evaluated their hippocampus-dependent memory dysfunctions using behavioral tasks such as the water maze and fear conditioning. However, multiple memory systems become impaired in AD as the disease progresses and it is important to test whether other forms of memory are affected in AD models. This study was designed to use conditioned taste aversion (CTA) and contextual fear conditioning paradigms to compare the phenotypes of hippocampus-independent and -dependent memory functions, respectively, in 5XFAD amyloid precursor protein/presenilin-1 transgenic mice that harbor five familial AD mutations. Although both types of memory were significantly impaired in 5XFAD mice, the onset of CTA memory deficits (∼9 months of age) was delayed compared with that of contextual memory deficits (∼6 months of age). Furthermore, 5XFAD mice that were genetically engineered to have reduced levels of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) (BACE1+/−·5XFAD) exhibited improved CTA memory, which was equivalent to the performance of wild-type controls. Importantly, elevated levels of cerebral β-secretase-cleaved C-terminal fragment (C99) and Aβ peptides in 5XFAD mice were significantly reduced in BACE1+/−·5XFAD mice. Furthermore, Aβ deposition in the insular cortex and basolateral amygdala, two brain regions that are critically involved in CTA performance, was also reduced in BACE1+/−·5XFAD compared with 5XFAD mice. Our findings indicate that the CTA paradigm is useful for evaluating a hippocampus-independent form of memory defect in AD model mice, which is sensitive to rescue by partial reductions of the β-secretase BACE1 and consequently of cerebral Aβ. [ABSTRACT FROM AUTHOR]

Abstract: Cyclization of recently reported linear phosphino dipeptide isostere inhibitors of BACE1 via side chain olefin metathesis yielded macrocyclic BACE1 inhibitors. The most potent compound II-P1 (IC50 of 47nM) and the corresponding linear analog I were tested for serum stability. The approach led to three times prolonged half life serum stability of 44min for the macrocyclic inhibitor II-P1 compared to the linear compound I. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Recently, we reported substrate-based β-secretase (BACE1) inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. These inhibitors showed potent BACE1 inhibitory activities (∼1.2nM IC50). In order to improve in vivo enzymatic stability and permeability across the blood–brain barrier, these penta-peptidic inhibitors would need to be further optimized. On the other hand, non-peptidic inhibitors possessing isophthalic residue at the P2 position were reported from other research groups. We selected isophthalic-type aromatic residues at the P2 position and an HMC isostere at the P1 position as lead compounds. On the basis of the design approach focused on the conformer of docked inhibitor in BACE1, we found novel non-peptidic and small-sized BACE1 inhibitors possessing a 2,6-pyridinedicarboxylic, chelidamic or chelidonic residue at the P2 position. [Copyright &y& Elsevier]

Abstract: Recently, we reported potent BACE1 inhibitors KMI-429, -684, and -574 possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell assays, especially, KMI-429 was confirmed to significantly inhibit Aβ production in vivo. However, acidic moieties at the P4 and positions of KMI-compounds were thought to be unfavorable for membrane permeability across the blood–brain barrier. Herein, we replaced acidic moieties at the P4 position with other hydrogen bond acceptor groups, and these inhibitors exhibited improved BACE1 inhibitory activities in cultured cells. In this study, we replaced the acidic moieties at the position with non-acidic and low molecular sized moieties. [Copyright &y& Elsevier]

Abstract: β-Site APP cleavage enzyme 1 (BACE1) is the β-secretase responsible for generating amyloid-β (Aβ) peptides in Alzheimer''s disease (AD). Previous studies suggest that activation of protein kinase C (PKC) modulates the β-secretase-mediated cleavage of APP and reduces the production of Aβ. The mechanism of PKC-mediated modulation of β-secretase activity, however, remains elusive. We report here that activation of PKC modulated β-secretase activity through either suppressing the accumulation or promoting the translocation of BACE1 protein in a cell type-dependent manner. We found that activation of PKC suppressed the accumulation of BACE1 protein in fibroblasts through an enhancement of intracellular protease activities. In neurons, activation of PKC did not alter the expression level of BACE1, but led to more BACE1 translocated to the cell surface, resulting in a decreased cleavage of APP at the β1 site. Together, Our findings provide novel mechanisms of PKC-mediated modulation of β-secretase activity, suggesting that alteration of the intracellular trafficking of BACE1 may serve as a useful therapeutic strategy to lower the production of Aβ in AD. [Copyright &y& Elsevier]

Abstract: The design and synthesis of a novel series of potent BACE1 hydroxyethylamine inhibitors. These inhibitors feature hydrogen bonding substituents at the C-5 position of the isophthalamide ring with improved selectivity over cathepsin D. [Copyright &y& Elsevier]

Abstract: BACE1 is a membrane-bound aspartyl protease involved in production of the Alzheimer’s amyloid β-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. [Copyright &y& Elsevier]

Data that have accumulated for well over a decade have implicated the β-amyloid (Aβ) peptide as a central player in the pathogenesis of Alzheimer’s disease (AD). Amyloid plaques, composed primarily of Aβ progressively form in the brains of AD patients, and mutations in three genes (amyloid precursor protein [APP] and presenilin 1 and 2 [PS1 and PS2]) cause early-onset familial AD (FAD) by directly increasing production of the toxic, plaque-promoting Aβ42 peptide. Given the strong association between Aβ and AD, it is likely that therapeutic strategies to lower the levels of Aβ in the brain should prove beneficial for the treatment of AD. One such strategy could involve inhibiting the enzymes that generate Aβ. Aβ is a product of catabolism of the large type-I membrane protein APP. Two proteases, called β- and γ-secretase, endoproteolyze APP to liberate the Aβ peptide. Recently, the molecules responsible for these proteolytic activities have been identified. Several lines of evidence suggest that the PS1 and PS2 proteins are γ-secretase, and the identity of β-secretase has been shown to be the novel transmembrane aspartic protease, β-site APP-cleaving enzyme 1 (BACE1; also called Asp2 and memapsin 2). BACE2, a protease homologous to BACE1, was also identified, and together the two enzymes define a new family of transmembrane aspartic proteases. BACE1 exhibits all the functional properties of β-secretase, and as the key enzyme that initiates the formation of Aβ, BACE1 is an attractive drug target for AD. This review discusses the identification and initial characterization of BACE1 and BACE2, and summarizes recent studies of BACE1 knockout mice that have validated BACE1 as the authentic β-secretase in vivo. [ABSTRACT FROM AUTHOR]