The aberrantly expressed microRNAs (miRNAs) including miR-29c-3p have been reported in the brains of Alzheimer's disease (AD) patients in recent researches. Nevertheless, the functional role and underlying molecular mechanism of miR-29c-3p in AD pathogenesis are still not well elucidated. The purpose of this study was to examine whether miR-29c-3p regulated beta-Ameyloid (Abeta)-induced neurotoxicity by targeting beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1). The expressions of miR 29c 3p and BACE1 mRNA and protein levels in Abeta-treated PC12 cellular AD model were examined by qRT-PCR and western blot analyses. Luciferase reporter assay verified the potential target of miR 29c 3p. Cell viability, apoptosis, and caspase-3 activity in PC12 cells were detected by the MTT assay, flow cytometry, and caspase-3 activity assay, respectively. Our results indicated that miR-29c-3p downregulation and BACE1 upregulation existed in the cellular AD model of PC12 cells. Moreover, miR-29c-3p directly inhibited BACE1 expression. miR-29c-3p overexpression and BACE1 knockdown strengthened Abeta-induced cell apoptosis, and caspase-3 activity in PC12 cells, which was partially eliminated by over-expression of BACE1. Conversely, BACE1 knockdown reversed the miR-29c-3p inhibition- mediated inhibitory effect on Abeta-induced cell toxicity, apoptosis, and caspase-3 activity in PC12 cells. Considering, miR-29c-3p attenuated Abeta-induced neurotoxicity through targeting BACE1 in an cellular AD model of PC12, providing a potential therapeutic target for AD treatment.
- MeSH
- Alzheimerova nemoc * metabolismus MeSH
- amyloidní beta-protein toxicita genetika metabolismus MeSH
- amyloidový prekurzorový protein beta genetika metabolismus MeSH
- aspartátové endopeptidasy genetika metabolismus MeSH
- kaspasa 3 metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- mikro RNA * genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- sekretasy genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In this issue, Ballin et al. report on their analysis of the substrate repertoire of SPPL2a and b intramembrane proteases. Based on the previous studies of their closest homologues, SPPL2c, SPPL3 and SPP, the authors hypothesized that SPPL2a/b proteases may cleave a subset of SNARE proteins. Indeed, four R-SNARE proteins, VAMP1, 2, 3 and 4, were cleaved by SPPL2a/b, both in overexpression assays and at endogenous levels. These findings have been validated by analysis of SPPL2a/b double knock-out mice tissues, which implicates these proteases in the regulation of SNARE protein turnover in vivo. The study of Ballin et al. also provides material for future studies of factors determining substrate specificity of SPPLs, as they cleave different subsets of the tail-anchored SNARE proteins. Comment on: https://doi.org/10.1111/febs.16610.
Although neurocognitive deficit is the best-recognized indicator of Alzheimer's disease (AD), psychotic and other noncognitive symptoms are the prime cause of institutionalization. BACE1 is the rate-limiting enzyme in the production of Aβ of AD, and one of the promising therapeutic targets in countering cognitive decline and amyloid pathology. Changes in BACE1 activity have also emerged to cause significant noncognitive neuropsychiatric symptoms and impairments of circadian rhythms, as evident from clinical trials and reports in transgenic models. In this study, we consider key characteristics of BACE1 with its contribution to neurocognitive deficit and other psychiatric symptoms of AD. We argue that a growing list of noncognitive mental impairments related to pharmacological modulation of BACE1 might present a major obstacle in clinical translation of emerging therapeutic leads targeting this protease. The adverse effects of BACE1 inhibition on mental health call for a revision of treatment strategies that assume indiscriminate inhibition of this key protease, and stress the need for further mechanistic and translational studies.
- MeSH
- Alzheimerova nemoc * MeSH
- amyloidní beta-protein MeSH
- aspartátové endopeptidasy MeSH
- lidé MeSH
- sekretasy * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
ADAM10 and ADAM17 are proteases that affect multiple signalling pathways by releasing molecules from the cell surface. As their substrate specificities partially overlaps, we investigated their concurrent role in liver regeneration and fibrosis, using three liver-specific deficient mouse lines: ADAM10- and ADAM17-deficient lines, and a line deficient for both proteases. In the model of partial hepatectomy, double deficient mice exhibited decreased AKT phosphorylation, decreased release of EGFR activating factors and lower shedding of HGF receptor c-Met. Thus, simultaneous ablation of ADAM10 and ADAM17 resulted in inhibited EGFR signalling, while HGF/c-Met signalling pathway was enhanced. In contrast, antagonistic effects of ADAM10 and ADAM17 were observed in the model of chronic CCl4 intoxication. While ADAM10-deficient mice develop more severe fibrosis manifested by high ALT, AST, ALP and higher collagen deposition, combined deficiency of ADAM10 and ADAM17 surprisingly results in comparable degree of liver damage as in control littermates. Therefore, ADAM17 deficiency is not protective in fibrosis development per se, but can ameliorate the damaging effect of ADAM10 deficiency on liver fibrosis development. Furthermore, we show that while ablation of ADAM17 resulted in decreased shedding of TNF RI, ADAM10 deficiency leads to increased levels of soluble TNF RI in serum. In conclusion, hepatocyte-derived ADAM10 and ADAM17 are important regulators of growth receptor signalling and TNF RI release, and pathological roles of these proteases are dependent on the cellular context.
- MeSH
- fibróza metabolismus MeSH
- játra * metabolismus patologie MeSH
- kultivované buňky MeSH
- membránové proteiny fyziologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nemoci jater * metabolismus patologie MeSH
- primární buněčná kultura MeSH
- protein ADAM10 fyziologie MeSH
- protein ADAM17 fyziologie MeSH
- regenerace jater * MeSH
- sekretasy fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
β-secretase (BACE1) has been regarded as a prime target for the development of amyloid beta (Aβ) lowering drugs in the therapy of Alzheimer ́s disease (AD). Although the enzyme was discovered in 1991 and helped to formulate the Aβ hypothesis as one of the very important features of AD etiopathogenesis, progress in AD treatment utilizing BACE1 inhibitors has remained limited. Moreover, in the last years, major pharmaceutical companies have discontinued clinical trials of five BACE1 inhibitors that had been strongly perceived as prospective. In our review, the Aβ hypothesis, the enzyme, its functions, and selected substrates are described. BACE1 inhibitors are classified into four generations. Those that underwent clinical trials displayed adverse effects, including weight loss, skin rashes, worsening of neuropsychiatric symptoms, etc. Some inhibitors could not establish a statistically significant risk-benefit ratio, or even scored worse than placebo. We still believe that drugs targeting BACE1 may still hide some potential, but a different approach to BACE1 inhibition or a shift of focus to modulation of its trafficking and/or post-translational modification should now be followed.
Alzheimer's disease is the most common cause of dementia, currently afflicting almost 40 million patients worldwide. According to the amyloid cascade hypothesis, the pathogenesis of the disease could be slowed down or even stopped by the inhibition of beta-secretase, making this aspartic acid protease a potentially important drug target site. Capillary electrophoresis is a promising technique for screening putative enzyme inhibitors due to highly effective separations, minuscule sample and other chemicals consumption, compatibility with a variety of detection techniques, and high throughput via automation. This chapter presents a method based on capillary electrophoresis coupled to mass spectrometry detection for kinetic and inhibition assays of the beta-secretase reaction with a decapeptide derived from an amyloid precursor protein.
- MeSH
- Alzheimerova nemoc farmakoterapie metabolismus MeSH
- amyloidový prekurzorový protein beta metabolismus MeSH
- aspartátové endopeptidasy metabolismus MeSH
- elektroforéza kapilární metody MeSH
- hmotnostní spektrometrie metody MeSH
- inhibitory enzymů farmakologie MeSH
- inhibitory proteas farmakologie MeSH
- kinetika MeSH
- lidé MeSH
- plošný screening metody MeSH
- sekretasy antagonisté a inhibitory MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of β-secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized β-secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor. The applicability of the capillary electrophoresis integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods.
- MeSH
- Alzheimerova nemoc farmakoterapie metabolismus MeSH
- bioreaktory * MeSH
- elektroforéza kapilární MeSH
- enzymy imobilizované antagonisté a inhibitory chemie metabolismus MeSH
- HEK293 buňky MeSH
- inhibitory enzymů chemie farmakologie MeSH
- kinetika MeSH
- lidé MeSH
- peptidy chemie farmakologie MeSH
- sekretasy antagonisté a inhibitory chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of β-secretase (BACE1), a key enzyme of the progression of Alzheimer's disease. Compatibility of the enzyme assay with MALDI TOF MS was achieved, providing both a complex protocol including a desalting step designed for rigorous kinetic studies and a simple mix-and-measure protocol designed for high-throughput inhibitor screening. In comparison with fluorescent or colorimetric assays, MALDI TOF MS represents a sensitive, fast, and label-free technique with minimal sample preparation. In contrast to other MS-based methodological approaches typically used in drug discovery processes, such as a direct injection MS or MS-coupled liquid chromatography or capillary electrophoresis, MALDI TOF MS enables direct analysis and is a highly suitable approach for high-throughput screening. The method's applicability is strongly supported by the high correlation of the acquired kinetic and inhibition parameters with data from the literature as well as from our previous research. Graphical abstract ᅟ.
- MeSH
- Alzheimerova nemoc enzymologie MeSH
- aminokyseliny antagonisté a inhibitory MeSH
- HEK293 buňky MeSH
- heterocyklické sloučeniny bicyklické farmakologie MeSH
- kinetika MeSH
- kyseliny pikolinové farmakologie MeSH
- lidé MeSH
- preklinické hodnocení léčiv metody MeSH
- pyrimidinony farmakologie MeSH
- sekretasy antagonisté a inhibitory metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
γ-Secretase is an integral membrane protein complex and is involved in the cleavage of the amyloid precursor protein APP to produce amyloid-β peptides. Amyloid-β peptides are considered causative agents for Alzheimer's disease and drugs targeted at γ-secretase are investigated as therapeutic treatments. We synthesized new carprofen derivatives, which showed γ-secretase modulating activity and determined their precise position, orientation, and dynamics in lipid membranes by combining neutron diffraction, solid-state NMR spectroscopy, and molecular dynamics simulations. Our data indicate that the carprofen derivatives are inserted into the membrane interface, where the exact position and orientation depends on the lipid phase. This knowledge will help to understand the docking of carprofen derivatives to γ-secretase and in the design of new potent drugs. The approach presented here promises to serve as a general guideline how drug/target interactions in membranes can be analyzed in a comprehensive manner.
- MeSH
- Alzheimerova nemoc metabolismus MeSH
- karbazoly metabolismus farmakologie MeSH
- lidé MeSH
- lipidové dvojvrstvy * MeSH
- magnetická rezonanční spektroskopie metody MeSH
- sekretasy účinky léků metabolismus MeSH
- simulace molekulární dynamiky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Alzheimer's disease (AD) is a progressive neurodegenerative disorder and currently there is no efficient treatment. The classic drug-design strategy based on the "one-molecule-one-target" paradigm was found to be ineffective in the case of multifactorial diseases like AD. A novel multi-target-directed ligand strategy based on the assumption that a single compound consisting of two or more distinct pharmacophores is able to hit multiple targets has been proposed as promising. Herein, we investigated 7-methoxytacrine - memantine heterodimers developed with respect to the multi-target-directed ligand theory. The spectroscopic, microscopic and cell culture methods were used for systematic investigation of the interference of the heterodimers with β-secretase (BACE1) activity, Aβ peptide amyloid fibrillization (amyloid theory) and interaction with M1 subtype of muscarinic (mAChRs), nicotinic (nAChRs) acetylcholine receptors (cholinergic theory) and N-methyl-d-aspartate receptors (NMDA) (glutamatergic theory). The drug-like properties of selected compounds have been evaluated from the point of view of blood-brain barrier penetration and cell proliferation. We have confirmed the multipotent effect of novel series of compounds. They inhibited effectively Aβ peptide amyloid fibrillization and affected the BACE1 activity. Moreover, they have AChE inhibitory potency but they could not potentiate cholinergic transmission via direct interaction with cholinergic receptors. All compounds were reported to act as an antagonist of both M1 muscarinic and muscle-type nicotinic receptors. We have found that 7-methoxytacrine - memantine heterodimers are able to hit multiple targets associated with Alzheimer's disease and thus, have a potential clinical impact for slowing or blocking the neurodegenerative process related to this disease.
- MeSH
- Alzheimerova nemoc farmakoterapie metabolismus MeSH
- amantadin analogy a deriváty farmakologie MeSH
- amyloidní beta-protein antagonisté a inhibitory metabolismus MeSH
- CHO buňky MeSH
- cholinesterasy metabolismus MeSH
- cílená molekulární terapie MeSH
- Cricetulus MeSH
- dimerizace MeSH
- HEK293 buňky MeSH
- inhibitory enzymů chemie farmakologie MeSH
- lidé MeSH
- receptor muskarinový M1 antagonisté a inhibitory metabolismus MeSH
- receptory cholinergní metabolismus MeSH
- receptory N-methyl-D-aspartátu metabolismus MeSH
- sekretasy antagonisté a inhibitory metabolismus MeSH
- takrin analogy a deriváty chemie farmakologie MeSH
- Xenopus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
