ABSTRACT
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.
Subject(s)
Aging , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides , Amyloid beta-Protein Precursor , Brain , Animals , Mice , Aging/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/genetics , Brain/metabolism , Calcium-Binding Proteins/metabolism , Calcium-Binding Proteins/genetics , Mice, Knockout , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Neurons/metabolismABSTRACT
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.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/genetics , Humans , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Alzheimer Disease/genetics , Animals , Amyloid beta-Peptides/metabolism , Biomarkers/metabolismABSTRACT
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.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , tau Proteins , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Humans , tau Proteins/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Chlorides/chemistry , Antioxidants/pharmacology , Antioxidants/chemical synthesis , Antioxidants/chemistry , Protein Aggregates/drug effects , Cell Line, Tumor , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/chemistryABSTRACT
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.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Proteomics , Humans , Amyloid beta-Peptides/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Proteomics/methods , Cell Line, Tumor , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/genetics , Peptide Fragments/metabolism , Amyloid Precursor Protein Secretases/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/genetics , tau Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Proto-Oncogene Proteins c-akt/metabolism , Amyloid beta-Protein Precursor/metabolism , Proteome/metabolism , Microtubule-Associated Proteins/metabolism , MAP Kinase Signaling SystemABSTRACT
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.
Subject(s)
Astrocytes , Ethanol , Astrocytes/metabolism , Astrocytes/drug effects , Ethanol/metabolism , Ethanol/pharmacology , Animals , Humans , Nervous System Diseases/metabolism , Amyloid beta-Peptides/metabolism , Amyloid/metabolism , Oxidative Stress/drug effects , Amyloid Precursor Protein Secretases/metabolism , Amyloid Precursor Protein Secretases/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Alzheimer Disease/genetics , Cytokines/metabolism , Alcohol Drinking/adverse effects , Alcohol Drinking/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/geneticsABSTRACT
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.
Subject(s)
Aspartic Acid Endopeptidases , Plasmodium falciparum , Protozoan Proteins , Plasmodium falciparum/enzymology , Plasmodium falciparum/metabolism , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/genetics , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/chemistry , Proteolysis , Humans , Subtilisins/metabolism , Catalytic Domain , Protein Domains , Malaria, Falciparum/parasitology , Malaria, Falciparum/metabolism , Erythrocytes/parasitology , Erythrocytes/metabolismABSTRACT
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.
Subject(s)
Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Amyloid Precursor Protein Secretases/chemistry , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/metabolism , Humans , Cluster Analysis , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protease Inhibitors/metabolism , Models, Molecular , Structure-Activity Relationship , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacologyABSTRACT
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.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides , Aspartic Acid Endopeptidases , Brain , Humans , Amyloid Precursor Protein Secretases/cerebrospinal fluid , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/cerebrospinal fluid , Aspartic Acid Endopeptidases/metabolism , Alzheimer Disease/pathology , Alzheimer Disease/cerebrospinal fluid , Male , Aged , Female , Amyloid beta-Peptides/cerebrospinal fluid , Amyloid beta-Peptides/metabolism , Brain/pathology , Brain/metabolism , Positron-Emission Tomography , Aged, 80 and over , Middle Aged , Cognition/physiology , Biomarkers/cerebrospinal fluid , Peptide Fragments/cerebrospinal fluid , Peptide Fragments/metabolismABSTRACT
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.
Subject(s)
Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Fluorescence Resonance Energy Transfer , Fluorescent Dyes , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Humans , Fluorescent Dyes/chemistry , Animals , HEK293 Cells , Single Molecule Imaging/methodsABSTRACT
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.
Subject(s)
Antimalarials , Aspartic Acid Endopeptidases , Drug Resistance , Membrane Transport Proteins , Plasmodium falciparum , Protozoan Proteins , Quinolines , Plasmodium falciparum/genetics , Plasmodium falciparum/drug effects , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Drug Resistance/genetics , Antimalarials/pharmacology , Quinolines/pharmacology , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Membrane Transport Proteins/genetics , Malaria, Falciparum/parasitology , Malaria, Falciparum/drug therapy , Humans , Alleles , Cambodia , Mutation , PiperazinesABSTRACT
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.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Endothelium, Vascular , Humans , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/genetics , Amyloid Precursor Protein Secretases/metabolism , Animals , Endothelium, Vascular/physiopathology , Endothelium, Vascular/metabolism , Alzheimer Disease/physiopathology , Alzheimer Disease/metabolism , Alzheimer Disease/enzymology , Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Cerebrovascular Circulation , Endothelial Cells/metabolism , Endothelial Cells/enzymology , Endothelial Cells/pathology , Brain/metabolism , Brain/physiopathology , Brain/blood supply , Brain/enzymology , Cerebrovascular Disorders/physiopathology , Cerebrovascular Disorders/metabolism , Cerebrovascular Disorders/enzymology , Cerebrovascular Disorders/etiologyABSTRACT
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.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Signal Transduction , Vascular Endothelial Growth Factor Receptor-3 , Zebrafish , Amyloid Precursor Protein Secretases/metabolism , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Animals , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/antagonists & inhibitors , Humans , Mice , Vascular Endothelial Growth Factor Receptor-3/metabolism , Vascular Endothelial Growth Factor Receptor-3/genetics , Zebrafish/metabolism , Zebrafish/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Alzheimer Disease/enzymology , Endothelial Cells/metabolism , Endothelial Cells/enzymology , Endothelial Cells/pathology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Stroke and Alzheimer's disease (AD) are prevalent age-related diseases; however, the relationship between these two diseases remains unclear. In this study, we aimed to investigate the ability of melatonin, a hormone produced by the pineal gland, to alleviate the effects of ischemic stroke leading to AD by observing the pathogenesis of AD hallmarks. We utilized SH-SY5Y cells under the conditions of oxygen-glucose deprivation (OGD) and oxygen-glucose deprivation and reoxygenation (OGD/R) to establish ischemic stroke conditions. We detected that hypoxia-inducible factor-1α (HIF-1α), an indicator of ischemic stroke, was highly upregulated at both the protein and mRNA levels under OGD conditions. Melatonin significantly downregulated both HIF-1α mRNA and protein expression under OGD/R conditions. We detected the upregulation of ß-site APP-cleaving enzyme 1 (BACE1) mRNA and protein expression under both OGD and OGD/R conditions, while 10 µM of melatonin attenuated these effects and inhibited beta amyloid (Aß) production. Furthermore, we demonstrated that OGD/R conditions were able to activate the BACE1 promoter, while melatonin inhibited this effect. The present results indicate that melatonin has a significant impact on preventing the aberrant development of ischemic stroke, which can lead to the development of AD, providing new insight into the prevention of AD and potential stroke treatments.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Melatonin , Neuroblastoma , Melatonin/pharmacology , Humans , Alzheimer Disease/metabolism , Alzheimer Disease/etiology , Alzheimer Disease/pathology , Neuroblastoma/metabolism , Neuroblastoma/pathology , Cell Line, Tumor , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Glucose/metabolism , Amyloid beta-Peptides/metabolism , Oxygen/metabolism , Cell Hypoxia/drug effects , Hypoxia/metabolismABSTRACT
A previous study reported that the ethanolic extract of the edible fern, Diplazium esculentum (Retz.) Sw. (DE), obtained from a non-optimized extraction condition exhibited anti-Alzheimer's disease (AD) properties through the inhibition of a rate-limiting enzyme in amyloid peptide formation, ß-secretase-1 (BACE-1). Nevertheless, a non-optimized or suboptimal extraction may lead to several issues, such as a reduction in extraction efficiency and increased time and plant materials. In this study, extraction of the DE was optimized to obtain appropriate BACE-1 inhibition using a Box-Behnken design (BBD) and response surface methodology (RSM). Data revealed that the optimal extraction condition was 70% (v/v) aqueous ethanol, 50 min extraction time, 30 °C extraction temperature, and 1:30 g/mL solid/liquid ratio, giving BACE-1 inhibition at 56.33%. In addition, the extract also exhibited significant antioxidant activities compared to the non-optimized extraction. Metabolomic phytochemical profiles and targeted phytochemical analyses showed that kaempferol, quercetin, and their derivatives as well as rosmarinic acid were abundant in the extract. The optimized DE extract also acted synergistically with donepezil, an AD drug suppressing BACE-1 activities. Data received from Drosophila-expressing human amyloid precursor proteins (APPs) and BACE-1, representing the amyloid hypothesis, showed that the optimized DE extract penetrated the fly brains, suppressed BACE-1 activities, and improved locomotor functions. The extract quenched the expression of glutathione S transferase D1 (GSTD1), inositol-requiring enzyme (IRE-1), and molecular chaperone-binding immunoglobulin (Bip), while donepezil suppressed these genes and other genes involved in antioxidant and endoplasmic reticulum (ER) stress response, including superoxide dismutase type 1 (SOD1), activating transcription factor 6 (ATF-6), and protein kinase R-like endoplasmic reticulum kinase (PERK). To sum up, the optimized extraction condition reduced extraction time while resulting in higher phytochemicals, antioxidants, and BACE-1 inhibitors.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Antioxidants , Phytochemicals , Plant Extracts , Antioxidants/pharmacology , Antioxidants/chemistry , Antioxidants/isolation & purification , Alzheimer Disease/drug therapy , Plant Extracts/pharmacology , Plant Extracts/chemistry , Phytochemicals/chemistry , Phytochemicals/pharmacology , Phytochemicals/isolation & purification , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Animals , Ferns/chemistry , Humans , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolismABSTRACT
Given continued failure of BACE1 inhibitor programs at symptomatic and prodromal stages of Alzheimer's disease (AD), clinical trials need to target the earlier preclinical stage. However, trial design is complex in this population with negative diagnosis of classical hippocampal amnesia on standard memory tests. Besides recent advances in brain imaging, electroencephalogram, and fluid-based biomarkers, new cognitive markers should be established for earlier diagnosis that can optimize recruitment to BACE1 inhibitor trials in presymptomatic AD. Notably, accelerated long-term forgetting (ALF) is emerging as a sensitive cognitive measure that can discriminate between asymptomatic individuals with high risks for developing AD and healthy controls. ALF is a form of declarative memory impairment characterized by increased forgetting rates over longer delays (days to months) despite normal storage within the standard delays of testing (20-60âmin). Therefore, ALF may represent a harbinger of preclinical dementia and the impairment of systems memory consolidation, during which memory traces temporarily stored in the hippocampus become gradually integrated into cortical networks. This review provides an overview of the utility of ALF in a rational design of next-generation BACE1 inhibitor trials in preclinical AD. I explore potential mechanisms underlying ALF and relevant early-stage biomarkers useful for BACE1 inhibitor evaluation, including synaptic protein alterations, astrocytic dysregulation and neuron hyperactivity in the hippocampal-cortical network. Furthermore, given the physiological role of the isoform BACE2 as an AD-suppressor gene, I also discuss the possible association between the poor selectivity of BACE1 inhibitors and their side effects (e.g., cognitive worsening) in prior clinical trials.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Early Diagnosis , Humans , Alzheimer Disease/drug therapy , Alzheimer Disease/diagnosis , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , AnimalsABSTRACT
Staufen-1 (STAU1) is a double-stranded RNA-binding protein (RBP) involved in a variety of pathological conditions. In this study, we investigated the potential role of STAU1 in Alzheimer's disease (AD), in which two hallmarks are well-established as cerebral ß-amyloid protein (Aß) deposition and Tau-centered neurofibrillary tangles. We found that STAU1 protein level was significantly increased in cells that stably express full-length APP and the brain of APP/PS1 mice, an animal model of AD. STAU1 knockdown, as opposed to overexpression, significantly decreased the protein levels of ß-amyloid converting enzyme 1 (BACE1) and Aß. We further found that STAU1 extended the half-life of the BACE1 mRNA through binding to the 3' untranslated region (3'UTR). Transcriptome analysis revealed that STAU1 enhanced the expression of growth arrest and DNA damage 45 ß (GADD45B) upstream of P38 MAPK signaling, which contributed to STAU1-induced regulation of Tau phosphorylation at Ser396 and Thr181. Together, STAU1 promoted amyloidogenesis by inhibiting BACE1 mRNA decay, and augmented Tau phosphorylation through activating GADD45B in relation to P38 MAPK. Targeting STAU1 that acts on both amyloidogenesis and tauopathy may serve as an optimistic approach for AD treatment.
Subject(s)
Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , RNA-Binding Proteins , tau Proteins , Animals , tau Proteins/metabolism , tau Proteins/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Mice , Phosphorylation , Amyloid Precursor Protein Secretases/metabolism , Amyloid Precursor Protein Secretases/genetics , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/genetics , Humans , Mice, Transgenic , Amyloid beta-Peptides/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Alzheimer Disease/genetics , Cells, Cultured , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/geneticsABSTRACT
In binding free energy calculations, simulations must sample all relevant conformations of the system in order to obtain unbiased results. For instance, different ligands can bind to different metastable states of a protein, and if these protein conformational changes are not sampled in relative binding free energy calculations, the contribution of these states to binding is not accounted for and thus calculated binding free energies are inaccurate. In this work, we investigate the impact of different beta-sectretase 1 (BACE1) protein conformations obtained from x-ray crystallography on the binding of BACE1 inhibitors. We highlight how these conformational changes are not adequately sampled in typical molecular dynamics simulations. Furthermore, we show that insufficient sampling of relevant conformations induces substantial error in relative binding free energy calculations, as judged by a variation in calculated relative binding free energies up to 2 kcal/mol depending on the starting protein conformation. These results emphasize the importance of protein conformational sampling and pose this BACE1 system as a challenge case for further method development in the area of enhanced protein conformational sampling, either in combination with binding calculations or as an endpoint correction.
Subject(s)
Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Thermodynamics , Amyloid Precursor Protein Secretases/chemistry , Amyloid Precursor Protein Secretases/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Humans , Crystallography, X-Ray , LigandsABSTRACT
The common neurodegenerative disorder Alzheimer disease (AD) is characterized by memory dysfunction and cognitive decline in the elderly. Neuropathological features include aggregated ß-amyloid (Aß) accumulation, neuroinflammation, and oxidative stress in the brain. Daphnetin (DAPH), a natural coumarin derivative, has the potential for inhibiting inflammatory and oxidative responses. We explored neuroprotective roles of DAPH treatment in the APP/PS1 transgenic mouse AD model. DAPH ameliorated spatial learning disabilities in Morris water maze tests and reduced Aß deposition, assessed by immunohistochemistry. It also reduced the Aß content in supernatants of neurons from fetal APP/PS1 mice, assessed by cell-based soluble ELISA. Molecular docking and fluorescence resonance energy transfer-based assay results suggested that DAPH could directly inhibit BACE1 activity. Furthermore, in vitro experiments utilizing isolated rat neurons assessing RNA expression profiling, immunofluorescence, TUNEL assay, and Western-blot analysis, suggested the potential of DAPH for regulating BDNF and GM-CSF expression and mitigating Aß1-42-induced cortical injury, synaptic loss, and apoptosis. HO-1 and Nrf2 mRNA and protein expression were also increased in a dose-dependent manner. These results underscore the potential of DAPH as a neuroprotective agent in reversing memory deficits associated with AD and bolster its candidacy as a multitarget natural small-molecule drug for AD patients.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Disease Models, Animal , Heme Oxygenase-1 , Mice, Transgenic , NF-E2-Related Factor 2 , Neurons , Neuroprotective Agents , Umbelliferones , Animals , Alzheimer Disease/pathology , Alzheimer Disease/metabolism , Alzheimer Disease/drug therapy , NF-E2-Related Factor 2/metabolism , Umbelliferones/pharmacology , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Amyloid Precursor Protein Secretases/metabolism , Rats , Aspartic Acid Endopeptidases/metabolism , Mice , Neuroprotective Agents/pharmacology , Heme Oxygenase-1/metabolism , Signal Transduction/drug effects , Amyloid beta-Peptides/metabolism , Rats, Sprague-Dawley , Male , Membrane ProteinsABSTRACT
The endoplasmic reticulum (ER) produces proteins destined to organelles of the endocytic and secretory pathways, the plasma membrane, and the extracellular space. While native proteins are transported to their intra- or extracellular site of activity, folding-defective polypeptides are retro-translocated across the ER membrane into the cytoplasm, poly-ubiquitylated and degraded by 26 S proteasomes in a process called ER-associated degradation (ERAD). Large misfolded polypeptides, such as polymers of alpha1 antitrypsin Z (ATZ) or mutant procollagens, fail to be dislocated across the ER membrane and instead enter ER-to-lysosome-associated degradation (ERLAD) pathways. Here, we show that pharmacological or genetic inhibition of ERAD components, such as the α1,2-mannosidase EDEM1 or the OS9 ERAD lectins triggers the delivery of the canonical ERAD clients Null Hong Kong (NHK) and BACE457Δ to degradative endolysosomes under control of the ER-phagy receptor FAM134B and the LC3 lipidation machinery. Our results reveal that ERAD dysfunction is compensated by the activation of FAM134B-driven ERLAD pathways that ensure efficient lysosomal clearance of orphan ERAD clients.
Subject(s)
Endoplasmic Reticulum-Associated Degradation , Endoplasmic Reticulum , Lysosomes , Membrane Proteins , Lysosomes/metabolism , Humans , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Amyloid Precursor Protein Secretases/metabolism , alpha 1-Antitrypsin/metabolism , alpha 1-Antitrypsin/genetics , Animals , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Protein Folding , Protein Transport , Lectins/metabolism , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/genetics , Mice , HeLa CellsABSTRACT
Alzheimer's disease (AD) is a chronic neurological condition that has become a leading cause of cognitive decline in elder individuals. Hardly any effective medication has been developed to halt the progression of AD due to the disease's complexity. Several theories have been put forward to clarify the mechanisms underlying AD etiology. The identification of amyloid plaques as a hallmark of AD has sparked the development of numerous drugs targeting the players involved in the amyloidogenic pathway, such as the ß-site of amyloid precursor protein cleavage enzyme 1 (BACE1) blockers. Over the last ten years, preclinical and early experimental research has led several pharmaceutical companies to prioritize producing BACE1 inhibitors. Despite all these efforts, earlier discovered inhibitors were discontinued in consideration of another second-generation small molecules and recent BACE1 antagonists failed in the final stages of clinical trials because of the complications associated either with toxicity or effectiveness. In addition to discussing the difficulties associated with development of BACE1 inhibitors, this review aims to provide an overview of BACE1 and offer perspectives on the causes behind the failure of five recent BACE1 inhibitors, that would be beneficial for choosing effective treatment approaches in the future.