Cíl: Cílem práce bylo porovnat metody ELISA pro stanovení hladin tripletu proteinů – celkového (t-tau) a fosforylovaného tau (p-tau) proteinů a β-amyloidu (1– 42) (Aβ42) v mozkomíšním moku (MMM) od dvou výrobců Fujirebio a EUROIMMUN a zároveň vytvořit orientační vodítka pro normální koncentrace u české populace od kognitivně zdravých starších osob. Soubor a metodika: Koncentrace likvorového tripletu proteinů od 38 kognitivně zdravých osob starších 55 let (19 mužů, průměrný věk 67 ± 8 let) byly změřeny jak soupravami ELISA INNOTEST, tak soupravami EUROIMMUN AG. Výsledky: Výsledky srovnání metod pro t-tau a p-tau proteiny a Aβ42 pomocí rozdílového grafu dle Blanda a Altmana ukazují, že hodnoty rozdílů mezi metodami ležely v rozsahu limitů shody (průměr ± 1,96 SD) až na ojedinělé výjimky odlehlých hodnot. Hodnoty korelačních koeficientů svědčí pro významnou shodu pro soupravy na stanovení t-tau proteinu. Na základě našich výsledků a zkušeností navrhujeme rozdělit koncentrace likvorového tripletu na tři skupiny: 1. snížené; 2. nerozhodné a hraniční; 3. zvýšené koncentrace v ng/ l na základě našeho konsenzu takto: t-tau protein < 280– 400 >, p181-tau protein < 50– 60 > a Aβ42 < 430– 480 >. Závěr: Soupravy ELISA obou výrobců jsou vhodné ke stanovení tripletu proteinů v MMM. Pro jejich referenční rozmezí lze orientačně využít naše výsledky, založené na reálném měření vzorků pečlivě vybraných a vyšetřených zdravých starších osob z ČR, nebo normy na základě našeho společného názoru.

Aim: The aim of the study was to compare ELISA methods for determination of triplet protein levels – total (t-tau) and phosphorylated tau (p-tau) proteins and β-amyloid (1– 42) (Aβ42) in cerebrospinal fl uid (CSF) from manufacturers Fujirebio and EUROIMMUN; and to provide guidance for normal concentrations from cognitively healthy elderly people. Patients and methods: The group consisted of 38 cognitively healthy persons over 55 years of age (19 men) whose average age was 67 ± 8 years. In each CSF sample, we examined the protein triplet by both ELISA INNOTEST and EUROIMMUN AG kits. Results: A comparison of ELISA methods for determination of t-tau and p-tau proteins and Aβ42 us ing the Bland and Altman difference graph shows that the differences between methods, with the exception of isolated outliers, were within the range of compliance (mean ± 1.96 SD). Cor relation coeffi cient values suggest significant agreement for t-tau protein as say kits. Based on our results and experience, we propose to clas sify CSF concentrations into three groups: 1. decreased; 2. questionable and borderline; 3. increased concentrations in ng/ L based on our consensus as follows: t-tau protein < 280– 400 >, p181-tau protein < 50– 60 > and Aβ42 < 430– 480 >. Conclusion: ELISA kits from two producers are suitable for determination of the triplet proteins in CSF. Our results based on real measurements of samples of carefully selected and examined healthy elderly people from the Czech Republic or the norms based on our consensus recom mendation can be used as reference range of the triplet.

• MeSH
• Alzheimer Disease * diagnosis   MeSH
• Amyloid beta-Peptides * analysis   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Clinical Studies as Topic MeSH
• Middle Aged MeSH
• Humans MeSH
• Cerebrospinal Fluid MeSH
• tau Proteins analysis   MeSH
• Cerebrospinal Fluid Proteins * analysis   MeSH
• Aged MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH

It is suggested that intracellular tau protein (τ), when released extracellularly upon neuron degeneration, could evoke direct toxic effects on the cholinergic neurotransmitter system through muscarinic receptors and thus contribute to the pathogenesis of Alzheimer's disease. In this study, we evaluated the in vitro effects of six naturally occurring monomeric τ isoforms on rat hippocampal synaptosomal choline transporters CHT1 (large transmembrane proteins associated with high-affinity choline transport and vulnerable to actions of amyloid β peptides (Aβ) applied in vitro or in vivo). Some τ isoforms at nM concentrations inhibited choline transport in a dose- and time-dependent saturable manner (352 = 441 > 410 = 383 > 381 = 412) and effects were associated with changes in the Michaelis constant rather than in maximal velocity. Moreover, the actions of τ 352/441 were not influenced by previous depolarisation of synaptosomes or by previous depletion of membrane cholesterol. Specific binding of [3H]hemicholinium-3 was not significantly altered by τ 352/441 at higher nM concentrations. Results of in vitro tests on CHT1 transporters from cholesterol-depleted synaptosomes supported interactions between Aβ 1-40 and τ 352. In addition, we developed surface plasmon resonance biosensors to monitor complexes of Aβ 1-42 and τ 352 using a sandwich detection format. It seems, therefore, that protein τ, similar to Aβ peptides, can contribute to the pathogenesis of Alzheimer's disease through its actions on CHT1 transporters. However, the interaction mechanisms are quite different (τ probably exerts its effects through direct interactions of microtubule binding repeats with extracellular portions of the CHT1 protein without influencing the choline recognition site, Aβ rather through lipid rafts in the surrounding membranes). An N-terminal insert of τ is not necessary but the N-terminal projection domain plays a role. The developed biosensor will be used to detect Aβ-τ complexes in cerebrospinal fluid in order to evaluate them as prospective biomarkers of Alzheimer's disease.

• MeSH
• Amyloid beta-Peptides metabolism   MeSH
• Hippocampus metabolism   MeSH
• Rats MeSH
• Rats, Wistar MeSH
• Surface Plasmon Resonance MeSH
• Cation Transport Proteins metabolism   MeSH
• tau Proteins metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Amyloid β peptides appear to play a role in physiological processes; however, they are also involved in the pathogenesis of Alzheimer disease. Their actions under normal conditions are probably mediated by soluble monomeric L-isoforms at low concentrations, perhaps via highly specific interactions. On the contrary, toxic effects of aggregated natural L-isoforms/synthetic D-isoforms on membranes are very similar, but synthetic reverse/random L: -isoforms without pronounced aggregation properties are not toxic. Our previous work reported interactions of non-aggregated/aggregated L-isoforms of amyloid β peptides 1-40/1-42 with racemic 24-hydroxycholesterol. In this study, stereospecificity in the interactions of natural 24(S)hydroxycholesterol (cerebrosterol) or synthetic 24(R)hydroxycholesterol with soluble fragment 1-40 was evaluated by means of an in vitro test based on increased vulnerability of the hemicholinium-3 sensitive high-affinity choline uptake system in rat hippocampal cholesterol-depleted synaptosomes to the actions of amyloid β; computational simulations were also performed. Our results suggest that: (1) 24(S)hydroxycholesterol interacts with L-peptide 1-40 but not with the reverse L-peptide 40-1, (2) 24(R)hydroxycholesterol does not interact with L-peptide 1-40 or reverse 40-1, and (3) both enantiomers can probably interact with D-peptide 1-40. Therefore, the binding of 24(S)hydroxycholesterol is not fully stereospecific and the interaction could not reflect a physiological mechanism. Data from the computational simulation indicate that the hydrophobic core of the amyloid β molecule interacts with the hydrophobic part of 24(S)hydroxycholesterol, but no hydrogen bonds with high stability were found. Using this procedure, globular amyloid β could retain 24(S)hydroxycholesterol and thus contribute to its pathological accumulation in the brains of patients with Alzheimer disease.

• MeSH
• Amyloid beta-Peptides metabolism   MeSH
• Hippocampus metabolism   MeSH
• Hydroxycholesterols metabolism   MeSH
• Rats MeSH
• Peptide Fragments metabolism   MeSH
• Rats, Wistar MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Mounting evidence suggests that the neuronal cell membrane is the main site of oligomer-mediated neuronal toxicity of amyloid-β peptides in Alzheimer's disease. To gain a detailed understanding of the mutual interference of amyloid-β oligomers and the neuronal membrane, we carried out microseconds of all-atom molecular dynamics (MD) simulations on the dimerization of amyloid-β (Aβ)42 in the aqueous phase and in the presence of a lipid bilayer mimicking the in vivo composition of neuronal membranes. The dimerization in solution is characterized by a random coil to β-sheet transition that seems on pathway to amyloid aggregation, while the interactions with the neuronal membrane decrease the order of the Aβ42 dimer by attenuating its propensity to form a β-sheet structure. The main lipid interaction partners of Aβ42 are the surface-exposed sugar groups of the gangliosides GM1. As the neurotoxic activity of amyloid oligomers increases with oligomer order, these results suggest that GM1 is neuroprotective against Aβ-mediated toxicity.

• MeSH
• Amyloid chemistry   MeSH
• Amyloid beta-Peptides chemistry   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• G(M1) Ganglioside metabolism   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Lipid Bilayers metabolism   MeSH
• Protein Multimerization * MeSH
• Neurons metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The interaction of amyloid β-peptide (Aβ) with the iron-storage protein ferritin was studied in vitro. We have shown that Aβ during fibril formation process is able to reduce Fe(III) from the ferritin core (ferrihydrite) to Fe(II). The Aβ-mediated Fe(III) reduction yielded a two-times-higher concentration of free Fe(II) than the spontaneous formation of Fe(II) by the ferritin itself. We suggest that Aβ can also act as a ferritin-specific metallochaperone-like molecule capturing Fe(III) from the ferritin ferrihydrite core. Our observation may partially explain the formation of Fe(II)-containing minerals in human brains suffering by neurodegenerative diseases.

• MeSH
• Amyloid chemistry   MeSH
• Amyloid beta-Peptides chemistry   metabolism   MeSH
• Ferritins chemistry   metabolism   MeSH
• Humans MeSH
• Oxidation-Reduction MeSH
• Iron metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In this perspective we summarize current knowledge of the effect of monosialoganglioside GM1 on the membrane-mediated aggregation of the β-amyloid (Aβ) peptide. GM1 has been suggested to be actively involved in the development of Alzheimer's disease due to its ability to seed the aggregation of Aβ. However, GM1 is known to be neuroprotective against Aβ-induced toxicity. Here we suggest that the two scenarios are not mutually exclusive but rather complementary, and might depend on the organization of GM1 in membranes. Improving our understanding of the molecular details behind the role of gangliosides in neurodegenerative amyloidoses might help in developing disease-modifying treatments.

• MeSH
• Amyloid beta-Peptides chemistry   metabolism   MeSH
• G(M1) Ganglioside chemistry   metabolism   MeSH
• Humans MeSH
• Brain metabolism   MeSH
• Protein Aggregation, Pathological metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

The accumulation of amyloid-β (Aβ) peptide is thought to be a major causative mechanism of Alzheimer's disease. Aβ accumulation could be caused by dysregulated processing of amyloid precursor protein, yielding excessive amounts of Aβ, and/or by inefficient proteolytic degradation of the peptide itself. Several proteases have been described as Aβ degradation enzymes, most notably metalloendopeptidases, aspartic endopeptidases, and some exopeptidases. Recently a report suggested that another metallopeptidase, glutamate carboxypeptidase II (GCPII), can also cleave Aβ. GCPII is a zinc exopeptidase that cleaves glutamate from N-acetyl-L-aspartyl-L-glutamate in the central nervous system and from pteroylpoly-γ-glutamate in the jejunum. GCPII has been proposed as a promising therapeutic target for disorders caused by glutamate neurotoxicity. However, an Aβ-degrading activity of GCPII would compromise potential pharmaceutical use of GCPII inhibitors, because the enzyme inhibition might lead to increased Aβ levels and consequently to Alzheimer's disease. Therefore, we analyzed the reported Aβ-degrading activity of GCPII using highly purified recombinant enzyme and synthetic Aβ. We did not detect any Aβ degradation activity of GCPII or its homologue even under prolonged incubation at a high enzyme to substrate ratio. These results are in good agreement with the current detailed structural understanding of the substrate specificity and enzyme-ligand interactions of GCPII.

• MeSH
• Amyloid beta-Peptides chemistry   metabolism   MeSH
• Antigens, Surface genetics   metabolism   MeSH
• Biocatalysis drug effects   MeSH
• Dipeptides metabolism   MeSH
• Glutamate Carboxypeptidase II antagonists & inhibitors   genetics   metabolism   MeSH
• Mass Spectrometry MeSH
• Hydrolysis MeSH
• Catalytic Domain MeSH
• Humans MeSH
• Molecular Structure MeSH
• Neprilysin genetics   metabolism   MeSH
• Organophosphorus Compounds pharmacology   MeSH
• Peptide Fragments chemistry   metabolism   MeSH
• Proteolysis MeSH
• Recombinant Proteins metabolism   MeSH
• Substrate Specificity MeSH
• Tritium MeSH
• Chromatography, High Pressure Liquid MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Alzheimer's Disease (AD) is a neurodegenerative disorder with an increasing impact on society. Because currently available therapy has only a short-term effect, a huge number of novel compounds are developed every year exploiting knowledge of the various aspects of AD pathophysiology. To better address the pathological complexity of AD, one of the most extensively pursued strategies by medicinal chemists is based on Multi-target-directed Ligands (MTDLs). Donepezil is one of the currently approved drugs for AD therapy acting as an acetylcholinesterase inhibitor. In this review, we have made an extensive literature survey focusing on donepezil-derived MTDL hybrids primarily targeting on different levels cholinesterases and amyloid beta (Aβ) peptide. The targeting includes direct interaction of the compounds with Aβ, AChE-induced Aβ aggregation, inhibition of BACE-1 enzyme, and modulation of biometal balance thus impeding Aβ assembly.

• MeSH
• Acetylcholinesterase metabolism   MeSH
• Alzheimer Disease drug therapy   metabolism   MeSH
• Amyloid beta-Peptides metabolism   MeSH
• Cholinesterase Inhibitors pharmacology   therapeutic use   MeSH
• Donepezil analogs & derivatives   MeSH
• Humans MeSH
• Protein Aggregation, Pathological drug therapy   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

OBJECTIVE: In a multicenter cohort of probable dementia with Lewy bodies (DLB), we tested the hypothesis that β-amyloid and tau biomarker positivity increases with age, which is modified by APOE genotype and sex, and that there are isolated and synergistic associations with the clinical phenotype. METHODS: We included 417 patients with DLB (age 45-93 years, 31% women). Positivity on β-amyloid (A+) and tau (T+) biomarkers was determined by CSF β-amyloid1-42 and phosphorylated tau in the European cohort and by Pittsburgh compound B and AV-1451 PET in the Mayo Clinic cohort. Patients were stratified into 4 groups: A-T-, A+T-, A-T+, and A+T+. RESULTS: A-T- was the largest group (39%), followed by A+T- (32%), A+T+ (15%), and A-T+ (13%). The percentage of A-T- decreased with age, and A+ and T+ increased with age in both women and men. A+ increased more in APOE ε4 carriers with age than in noncarriers. A+ was the main predictor of lower cognitive performance when considered together with T+. T+ was associated with a lower frequency of parkinsonism and probable REM sleep behavior disorder. There were no significant interactions between A+ and T+ in relation to the clinical phenotype. CONCLUSIONS: Alzheimer disease pathologic changes are common in DLB and are associated with the clinical phenotype. β-Amyloid is associated with cognitive impairment, and tau pathology is associated with lower frequency of clinical features of DLB. These findings have important implications for diagnosis, prognosis, and disease monitoring, as well as for clinical trials targeting disease-specific proteins in DLB. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that in patients with probable DLB, β-amyloid is associated with lower cognitive performance and tau pathology is associated with lower frequency of clinical features of DLB.

• MeSH
• Amyloid beta-Peptides cerebrospinal fluid   metabolism   MeSH
• Apolipoprotein E4 genetics   MeSH
• Biomarkers metabolism   MeSH
• Lewy Body Disease classification   complications   metabolism   physiopathology   MeSH
• Phenotype MeSH
• Cognitive Dysfunction etiology   physiopathology   MeSH
• Cohort Studies MeSH
• Middle Aged MeSH
• Humans MeSH
• Peptide Fragments cerebrospinal fluid   MeSH
• Positron-Emission Tomography MeSH
• tau Proteins cerebrospinal fluid   metabolism   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Age Factors MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Multifunctional mitochondrial enzyme 17β-hydroxysteroid dehydrogenase type 10 plays a role in the development of Alzheimer's disease. However, changes in its expression in the brain or cerebrospinal fluid are not fully specific for this type of dementia. Our previous study revealed that complexes of the enzyme and amyloid β in cerebrospinal fluid could serve as a more specific biomarker of Alzheimer's disease than either the enzyme or amyloid β individually when compared to autoimmune multiple sclerosis. In this study, enzyme-linked immunosorbent assay and the surface plasmon resonance biosensor method were used to analyse cerebrospinal fluid of patients with various neuroinflammatory diseases. Significant differences in the levels of the total enzyme, complexes, amyloid β 1-42 and total τ/phospho-τ were found in Alzheimer's disease patients while differences in complexes, total amyloid β and amyloid β 1- 42 were observed in patients with neuroinflammatory diseases (except for multiple sclerosis) when compared to non-neuroinflammatory controls. The interactions of the enzyme with amyloid β appeared to depend strongly on neuroinflammation-sensitive amyloid β. Our data demonstrated that oligomerisation/aggregation of intracellular amyloid β peptides was important in Alzheimer's disease while extracellular amyloid β could play a role in neuroinflammatory diseases. Phospho-τ is currently the best biomarker of Alzheimer's disease.

• MeSH
• 17-Hydroxysteroid Dehydrogenases MeSH
• Alzheimer Disease complications   MeSH
• Amyloid beta-Peptides MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Humans MeSH
• Central Nervous System Diseases complications   MeSH
• Vascular Diseases complications   MeSH
• Peripheral Nervous System Diseases complications   MeSH
• Statistics, Nonparametric MeSH
• Peptide Fragments MeSH
• Surface Plasmon Resonance MeSH
• Aged MeSH
• Inflammation complications   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH