Suppression of the amyloidogenic metabolism of APP and the accumulation of Aß by alcadein α in the brain during aging.

Generation and accumulation of amyloid-ß (Aß) protein in the brain are the primary causes of Alzheimer's disease (AD). Alcadeins (Alcs composed of Alcα, Alcß and Alcγ family) are a neuronal membrane protein that is subject to proteolytic processing, as is Aß protein precursor (APP), by APP secretases. Previous observations suggest that Alcs are involved in the pathophysiology of Alzheimer's disease (AD). Here, we generated new mouse AppNL-F (APP-KI) lines with either Alcα- or Alcß-deficient background and analyzed APP processing and Aß accumulation through the aging process. The Alcα-deficient APP-KI (APP-KI/Alcα-KO) mice enhanced brain Aß accumulation along with increased amyloidogenic ß-site cleavage of APP through the aging process whereas Alcß-deficient APP-KI (APP-KI/Alcß-KO) mice neither affected APP metabolism nor Aß accumulation at any age. More colocalization of APP and BACE1 was observed in the endolysosomal pathway in neurons of APP-KI/Alcα-KO mice compared to APP-KI and APP-KI/Alcß-KO mice. These results indicate that Alcα plays an important role in the neuroprotective function by suppressing the amyloidogenic cleavage of APP by BACE1 in the brain, which is distinct from the neuroprotective function of Alcß, in which p3-Alcß peptides derived from Alcß restores the viability in neurons impaired by toxic Aß.

Synthesis and Evaluation of Chloride-Substituted Ramalin Derivatives for Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by the accumulation of amyloid-beta plaques and hyperphosphorylated tau proteins, leading to cognitive decline and neuronal death. However, despite extensive research, there are still no effective treatments for this condition. In this study, a series of chloride-substituted Ramalin derivatives is synthesized to optimize their antioxidant, anti-inflammatory, and their potential to target key pathological features of Alzheimer's disease. The effect of the chloride position on these properties is investigated, specifically examining the potential of these derivatives to inhibit tau aggregation and beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) activity. Our findings demonstrate that several derivatives, particularly RA-3Cl, RA-4Cl, RA-26Cl, RA-34Cl, and RA-35Cl, significantly inhibit tau aggregation with inhibition rates of approximately 50%. For BACE-1 inhibition, Ramalin and RA-4Cl also significantly decrease BACE-1 expression in N2a cells by 40% and 38%, respectively, while RA-23Cl and RA-24Cl showed inhibition rates of 30% and 35% in SH-SY5Y cells. These results suggest that chloride-substituted Ramalin derivatives possess promising multifunctional properties for AD treatment, warranting further investigation and optimization for clinical applications.

Proteomic Analysis Reveals Physiological Activities of Aß Peptide for Alzheimer's Disease.

With the rapid progress in deciphering the pathogenesis of Alzheimer's disease (AD), it has been widely accepted that the accumulation of misfolded amyloid ß (Aß) in the brain could cause the neurodegeneration in AD. Although much evidence demonstrates the neurotoxicity of Aß, the role of Aß in the nervous system are complex. However, more comprehensive studies are needed to understand the physiological effect of Aß40 monomers in depth. To explore the physiological mechanism of Aß, we employed mass spectrometry to investigate the altered proteomic events induced by a lower submicromolar concentration of Aß. Human neuroblastoma SH-SY5Y cells were exposed to five different concentrations of Aß1-40 monomers and collected at four time points. The proteomic analysis revealed the time-course behavior of proteins involved in biological processes, such as RNA splicing, nuclear transport and protein localization. Further biological studies indicated that Aß40 monomers may activate PI3K/AKT signaling to regulate p-Tau, Ezrin and MAP2. These three proteins are associated with dendritic morphogenesis, neuronal polarity, synaptogenesis, axon establishment and axon elongation. Moreover, Aß40 monomers may regulate their physiological forms by inhibiting the expression of BACE1 and APP via activation of the ERK1/2 pathway. A comprehensive exploration of pathological and physiological mechanisms of Aß is beneficial for exploring novel treatment.

Examination of Akt and GSK3ß in BDNF-mediated reductions in BACE1 activity in neuronal cells.

Brain-derived neurotrophic factor (BDNF) content and signaling has been identified as one potential regulator of amyloid precursor protein (APP) processing. Recently published work has demonstrated that BDNF reduces BACE1 activity while also elevating the inhibition of GSK3ß in the prefrontal cortex of male C57BL/6J mice. These results provide evidence that BDNF alters APP processing by reducing BACE1 activity, which may act through GSK3ß inhibition. The purpose of this study was to further explore the role of GSK3ß in BDNF-induced regulation on BACE1 activity. We utilized a cell culture and an in vitro activity assay model to pharmacologically target BDNF and GSK3ß signaling to confirm its involvement in the BDNF response. Treatment of differentiated SH-SY5Y neuronal cells with 75 ng/mL BDNF resulted in elevated pTrkB content, pAkt content, pGSK3ß content, and reduced BACE1 activity. An in vitro BACE1 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF reduced BACE1 activity; however, in the presence of TrkB or Akt inhibition, this effect was abolished. An in vitro ADAM10 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF did not alter ADAM10 activity. However, inhibiting BDNF signaling reduced ADAM10 activity. Collectively these studies suggest that GSK3ß inhibition may be necessary for BDNF-induced reductions in BACE1 activity. These findings will allow for the optimization of future therapeutic strategies by selectively targeting TrkB activation and GSK3ß inhibition.

Oligodendrocytes produce amyloid-ß and contribute to plaque formation alongside neurons in Alzheimer's disease model mice.

Amyloid-ß (Aß) is thought to be neuronally derived in Alzheimer's disease (AD). However, transcripts of amyloid precursor protein (APP) and amyloidogenic enzymes are equally abundant in oligodendrocytes (OLs). By cell-type-specific deletion of Bace1 in a humanized knock-in AD model, APPNLGF, we demonstrate that OLs and neurons contribute to Aß plaque burden. For rapid plaque seeding, excitatory projection neurons must provide a threshold level of Aß. Ultimately, our findings are relevant for AD prevention and therapeutic strategies.

Corilagin improves cognitive impairment in APP/PS1 mice by reducing Aß generation and enhancing synaptic plasticity.

Alzheimer's disease (AD) is closely associated with the neurotoxic effects of amyloid-ß (Aß), leading to synaptic damage, neuronal loss and cognitive dysfunction. Previous in vitro studies have demonstrated the potential of corilagin to counteract Aß-induced oxidative stress, inflammatory injury, and ß-site amyloid precursor protein cleaving enzyme-1 (BACE1) activity in Aß production. However, the in vivo protective effects of corilagin on Alzheimer's disease remain unexplored. The purpose of this study was to investigate the protective effects of corilagin on APP/PS1 mice and the underlying mechanisms. The cognitive function of the mice was assessed by step-through passive avoidance and Morris water maze tests. Nissl staining was used to evaluate neuronal damage in the hippocampus. ELISA and Western blotting analyses were used to determine the associated protein expression. Transmission electron microscopy was utilized to observe the synaptic ultrastructure of hippocampal neurons. Golgi staining was applied to assess dendritic morphology and dendritic spine density in hippocampal pyramidal neurons. Immunohistochemistry and Western blotting were performed to examine the expression of synaptic-associated proteins. The results showed that corilagin improves learning and memory in APP/PS1 mice, reduces hippocampal neuron damage, inhibits BACE1 and reduces Aß generation. It also improves synaptic plasticity and the expression of synaptic-associated proteins. Corilagin effectively reduces Aß generation by inhibiting BACE1, ultimately reducing neuronal loss and enhancing synaptic plasticity to improve synaptic transmission. This study sheds light on the potential therapeutic role of corilagin in Alzheimer's disease.

It's good to know what to BACE the specificity of your inhibitors on.

Production, aggregation, and clearance of the amyloid ß peptide (Aß) are important processes governing the initial pathogenesis of Alzheimer's disease (AD). Inhibition of ß-site amyloid precursor protein (APP) cleaving enzyme (BACE1) (one of two key proteases responsible for Aß production) as an AD-therapeutic approach so far has failed to yield a successful drug. BACE1 and its homologue BACE2 are frequently inhibited by the same inhibitors. Several genetic and cerebral organoid modeling studies suggest that BACE2 has dose-dependent AD-suppressing activity, which makes its unwanted inhibition potentially counterproductive for AD treatment. The in vivo effects of an unwanted cross inhibition of BACE2 have so far been impossible to monitor because of the lack of an easily accessible pharmacodynamic marker specific for BACE2 cleavage. In this issue of the JCI, work led by Stefan F. Lichtenthaler identifies soluble VEGFR3 (sVEGFR3) as a pharmacodynamic plasma marker for BACE2 activity not shared with BACE1.

Edaravone N-benzyl pyridinium derivatives: BACE-1 inhibition, kinetics and in silico binding pose determination.

BACE-1 plays a pivotal role in the production of ß-amyloid (Aß) peptides, implicated in Alzheimer's Disease (AD) pathology. We previously described edaravone N-benzyl pyridinium derivatives (EBPDs) that exhibited multifunctional activity against multiple AD targets. In this study we explored the EBPDs BACE-1 inhibitory activity to potentially enhance the compounds therapeutic profile. The EBPDs exhibited moderate BACE-1 inhibitory activity (IC50 = 44.10 µM - 123.70 µM) and obtained IC50 values between 2.0 and 5.8-fold greater than resveratrol, a known BACE-1 inhibitor (IC50 = 253.20 µM), in this assay. Compound 3 was the most potent inhibitor with an IC50 of 44.10 µM and a Ki of 19.96 µM and a mixed-type mode of inhibition that favored binding in a competitive manner. Molecular docking identified crucial interactions with BACE-1 active site residues, supported by 100 ns MD simulations. The study highlighted the EBPDs therapeutic potential as BACE-1 inhibitors and multifunctional anti-AD therapeutic agents.

The malaria parasite egress protease SUB1 is activated through precise, plasmepsin X-mediated cleavage of the SUB1 prodomain.

BACKGROUND: The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31. METHODS: Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX. RESULTS: We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity. CONCLUSIONS: Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity. GENERAL SIGNIFICANCE: Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.

Network Models of BACE-1 Inhibitors: Exploring Structural and Biochemical Relationships.

This study investigates the clustering patterns of human ß-secretase 1 (BACE-1) inhibitors using complex network methodologies based on various distance functions, including Euclidean, Tanimoto, Hamming, and Levenshtein distances. Molecular descriptor vectors such as molecular mass, Merck Molecular Force Field (MMFF) energy, Crippen partition coefficient (ClogP), Crippen molar refractivity (MR), eccentricity, Kappa indices, Synthetic Accessibility Score, Topological Polar Surface Area (TPSA), and 2D/3D autocorrelation entropies are employed to capture the diverse properties of these inhibitors. The Euclidean distance network demonstrates the most reliable clustering results, with strong agreement metrics and minimal information loss, indicating its robustness in capturing essential structural and physicochemical properties. Tanimoto and Hamming distance networks yield valuable clustering outcomes, albeit with moderate performance, while the Levenshtein distance network shows significant discrepancies. The analysis of eigenvector centrality across different networks identifies key inhibitors acting as hubs, which are likely critical in biochemical pathways. Community detection results highlight distinct clustering patterns, with well-defined communities providing insights into the functional and structural groupings of BACE-1 inhibitors. The study also conducts non-parametric tests, revealing significant differences in molecular descriptors, validating the clustering methodology. Despite its limitations, including reliance on specific descriptors and computational complexity, this study offers a comprehensive framework for understanding molecular interactions and guiding therapeutic interventions. Future research could integrate additional descriptors, advanced machine learning techniques, and dynamic network analysis to enhance clustering accuracy and applicability.

Astrocytes in Amyloid Generation and Alcohol Metabolism: Implications of Alcohol Use in Neurological Disorder(s).

As per the National Survey on Drug Use and Health, 10.5% of Americans aged 12 years and older are suffering from alcohol use disorder, with a wide range of neurological disorders. Alcohol-mediated neurological disorders can be linked to Alzheimer's-like pathology, which has not been well studied. We hypothesize that alcohol exposure can induce astrocytic amyloidosis, which can be corroborated by the neurological disorders observed in alcohol use disorder. In this study, we demonstrated that the exposure of astrocytes to ethanol resulted in an increase in Alzheimer's disease markers-the amyloid precursor protein, Aß1-42, and the ß-site-cleaving enzyme; an oxidative stress marker-4HNE; proinflammatory cytokines-TNF-α, IL1ß, and IL6; lncRNA BACE1-AS; and alcohol-metabolizing enzymes-alcohol dehydrogenase, aldehyde dehydrogenase-2, and cytochrome P450 2E1. A gene-silencing approach confirmed the regulatory role of lncRNA BACE1-AS in amyloid generation, alcohol metabolism, and neuroinflammation. This report is the first to suggest the involvement of lncRNA BACE1-AS in alcohol-induced astrocytic amyloid generation and alcohol metabolism. These findings will aid in developing therapies targeting astrocyte-mediated neurological disorders and cognitive deficits in alcohol users.

Proteolytic cleavage of Golgi glycosyltransferases by SPPL3 and other proteases and its implications for cellular glycosylation.

Glycosylation of proteins and lipids is of fundamental importance in multicellular eukaryotes. The vast diversity of glycan structures observed is generated in the Golgi apparatus by the concerted activity of >100 distinct enzymes, which include glycosyltransferases and other glycan-modifying enzymes. Well-known for decades, the majority of these enzymes is released from the Golgi apparatus and subsequently secreted into the extracellular space following endoproteolytic cleavage, but the underlying molecular mechanisms and the physiological implications have remained unexplored. This review will summarize our current knowledge of Golgi enzyme proteolysis and secretion and will discuss its conceptual implications for the regulation of cellular glycosylation and the organization of the Golgi apparatus. A particular focus will lie on the intramembrane protease SPPL3, which recently emerged as key protease facilitating Golgi enzyme release and has since been shown to affect a multitude of glycosylation-dependent physiological processes.

MGST3 regulates BACE1 protein translation and amyloidogenesis by controlling the RGS4-mediated AKT signaling pathway.

Microsomal glutathione transferase 3 (MGST3) regulates eicosanoid and glutathione metabolism. These processes are associated with oxidative stress and apoptosis, suggesting that MGST3 might play a role in the pathophysiology of Alzheimer's disease. Here, we report that knockdown (KD) of MGST3 in cell lines reduced the protein level of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and the resulting amyloidogenesis. Interestingly, MGST3 KD did not alter intracellular reactive oxygen species level but selectively reduced the expression of apoptosis indicators which could be associated with the receptor of cysteinyl leukotrienes, the downstream metabolites of MGST3 in arachidonic acid pathway. We then showed that the effect of MGST3 on BACE1 was independent of cysteinyl leukotrienes but involved a translational mechanism. Further RNA-seq analysis identified that regulator of G-protein signaling 4 (RGS4) was a target gene of MGST3. Silencing of RGS4 inhibited BACE1 translation and prevented MGST3 KD-mediated reduction of BACE1. The potential mechanism was related to AKT activity, as the protein level of phosphorylated AKT was significantly reduced by silencing of MGST3 and RGS4, and the AKT inhibitor abolished the effect of MGST3/RGS4 on phosphorylated AKT and BACE1. Together, MGST3 regulated amyloidogenesis by controlling BACE1 protein expression, which was mediated by RGS4 and downstream AKT signaling pathway.

The Alzheimer's disease-linked protease BACE2 cleaves VEGFR3 and modulates its signaling.

The ß-secretase ß-site APP cleaving enzyme (BACE1) is a central drug target for Alzheimer's disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, nonhuman primates, and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for safer prevention of Alzheimer's disease.

Heterologous expression of BACE1 and its interaction with Codonopsis pilosula polysaccharides and Lobetyolin.

BACE1, a crucial enzyme in the amyloid-ß deposition theory of Alzheimer's disease (AD), is targeted by Codonopsis pilosula, a traditional tonic believed to impede AD onset. However, the specific active compounds responsible for its effects remain elusive. Our prior network pharmacology research identified C. pilosula polysaccharides (CPPS) and Lobetyolin may serve as potential inhibitors of AD by suppressing amyloidogenesis. Here, we recombinantly expressed BACE1 under varied conditions and assessed its activity using Fluorescence Resonance Energy Transfer technology. Through spectroscopy, molecular docking, and dynamics, we elucidated the interactions of CPPS, Lobetyolin, and BACE1. Optimal BACE1 expression occurred at 22 °C with 0.4 mM IPTG for 6 h, yielding a 72 kDa protein. Enzyme kinetics displayed a maximum rate of 4096 µmol/min and a Michaelis constant of 16 mg/mL for BACE1. Spectroscopic analysis revealed differing binding affinities of the compounds at various temperatures, peaking at 293 K. Lobetyolin exhibited superior binding to BACE1 compared to CPPS, driven by hydrophobic and electrostatic forces. Molecular docking and dynamics highlighted hydrophobic amino acids' role in BACE1 interactions with Lobetyolin and CPPS, with binding energy < -1.2 kcal/mol signifying strong affinities. Notably, Lobetyolin and CPPS showed higher BACE1 affinity than APP, with the Lobetyolin-BACE1 complex being the most stable.

Cerebrovascular Endothelial Dysfunction: Role of BACE1.

Dysfunctional endothelium is increasingly recognized as a mechanistic link between cardiovascular risk factors and dementia, including Alzheimer disease. BACE1 (ß-site amyloid-ß precursor protein-cleaving enzyme 1) is responsible for ß-processing of APP (amyloid-ß precursor protein), the first step in the production of Aß (amyloid-ß) peptides, major culprits in the pathogenesis of Alzheimer disease. Under pathological conditions, excessive activation of BACE1 exerts detrimental effects on endothelial function by Aß-dependent and Aß-independent mechanisms. High local concentration of Aß in the brain blood vessels is responsible for the loss of key vascular protective functions of endothelial cells. More recent studies recognized significant contribution of Aß-independent proteolytic activity of endothelial BACE1 to the pathogenesis of endothelial dysfunction. This review critically evaluates existing evidence supporting the concept that excessive activation of BACE1 expressed in the cerebrovascular endothelium impairs key homeostatic functions of the brain blood vessels. This concept has important therapeutic implications. Indeed, improved understanding of the mechanisms of endothelial dysfunction may help in efforts to develop new approaches to the protection and preservation of healthy cerebrovascular function.

A Plasmodium falciparum genetic cross reveals the contributions of pfcrt and plasmepsin II/III to piperaquine drug resistance.

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC50. We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt. A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage.

Associations of CSF BACE1 with amyloid pathology, neurodegeneration, and cognition in Alzheimer's disease.

Β-site amyloid precursor protein (APP) cleaving enzyme (BACE1) is a crucial protease in the production of amyloid-ß (Aß) in Alzheimer's disease (AD) patients. However, the side effects observed in clinical trials of BACE1 inhibitors, including reduction in brain volume and cognitive worsening, suggest that the exact role of BACE1 in AD pathology is not fully understood. To further investigate this, we examined cerebrospinal fluid (CSF) levels of BACE1 and its cleaved product sAPPß that reflects BACE1 activity in the China Aging and Neurodegenerative Disorder Initiative cohort. We found significant correlations between CSF BACE1 or sAPPß levels and CSF Aß40, Aß42, and Aß42/Aß40 ratio, but not with amyloid deposition detected by 18F-Florbetapir PET. Additionally, CSF BACE1 and sAPPß levels were positively associated with cortical thickness in multiple brain regions, and higher levels of sAPPß were linked to increased cortical glucose metabolism in frontal and supramarginal areas. Interestingly, individuals with higher baseline levels of CSF BACE1 exhibited slower rates of brain volume reduction and cognitive worsening over time. This suggests that increased levels and activity of BACE1 may not be the determining factor for amyloid deposition, but instead, may be associated with increased neuronal activity and potentially providing protection against neurodegeneration in AD.

Inhibition of BACE1 affected both its Aß producing and degrading activities and increased Aß42 and Aß40 levels at high-level BACE1 expression.

The beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the predominant ß-secretase, cleaving the amyloid precursor protein (APP) via the amyloidogenic pathway. In addition, BACE1 as an amyloid degrading enzyme (ADE), cleaves Aß to produce the C-terminally truncated non-toxic Aß fragment Aß34 which is an indicator of amyloid clearance. Here, we analyzed the effects of BACE1 inhibitors on its opposing enzymatic functions, i.e., amyloidogenic (Aß producing) and amyloidolytic (Aß degrading) activities, using cell culture models with varying BACE1/APP ratios. Under high-level BACE1 expression, low-dose inhibition unexpectedly yielded a two-fold increase in Aß42 and Aß40 levels. The concomitant decrease in Aß34 and secreted APPß levels suggested that the elevated Aß42 and Aß40 levels were due to the attenuated Aß degrading activity of BACE1. Notably, the amyloidolytic activity of BACE1 was impeded at lower BACE1 inhibitor concentrations compared to its amyloidogenic activity, thereby suggesting that the Aß degrading activity of BACE1 was more sensitive to inhibition than its Aß producing activity. Under endogenous BACE1 and APP levels, "low-dose" BACE1 inhibition affected both the Aß producing and degrading activities of BACE1, i.e., significantly increased Aß42/Aß40 ratio and decreased Aß34 levels, respectively. Further, we incubated recombinant BACE1 with synthetic Aß peptides and found that BACE1 has a higher affinity for Aß substrates over APP. In summary, our results suggest that stimulating BACE1's ADE activity and halting Aß production without decreasing Aß clearance could still be a promising therapeutic approach with new, yet to be developed, BACE1 modulators.

Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules.

The prevailing but not undisputed amyloid cascade hypothesis places the ß-site of APP cleaving enzyme 1 (BACE1) center stage in Alzheimer's Disease pathogenesis. Here, we investigated functional properties of BACE1 with novel tag- and antibody-free labeling tools, which are conjugates of the BACE1-inhibitor IV (also referred to as C3) linked to different impermeable Alexa Fluor dyes. We show that these fluorescent small molecules bind specifically to BACE1, with a 1:1 labeling stoichiometry at their orthosteric site. This is a crucial property especially for single-molecule and super-resolution microscopy approaches, allowing characterization of the dyes' labeling capabilities in overexpressing cell systems and in native neuronal tissue. With multiple colors at hand, we evaluated BACE1-multimerization by Förster resonance energy transfer (FRET) acceptor-photobleaching and single-particle imaging of native BACE1. In summary, our novel fluorescent inhibitors, termed Alexa-C3, offer unprecedented insights into protein-protein interactions and diffusion behavior of BACE1 down to the single molecule level.