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1.
Article in English | MEDLINE | ID: mdl-30405739

ABSTRACT

Rotifers have been widely used as well-characterized models of aging, since their multiorgan character makes them suitable as in vivo toxicological and lifespan models. Here we report the assessment of four adaptogenic plants and their extracts for the first time in this model. The effects on rotifer viability of extracts and characteristic active markers of Panax ginseng, Withania somnifera, Leuzea carthamoides, and Rhodiola rosea were tested in vivo. The crude extracts were nontoxic to Philodina acuticornis bdelloid rotifers; however, the pure substances of the plants influenced negatively the viability. Ginsenoside Rb1 and secondary metabolites of Withania somnifera exerted deleterious effect on the animals. The aglycone tyrosol and cinnamyl alcohol (from Rhodiola rosea) were more toxic than their glycosides salidroside and rosavin. Although the 20-OH-ecdysone and ajugasterone C (from Leuzea carthamoides) are chemically very similar, the latter was less toxic.

2.
J Neurosci ; 37(30): 7253-7262, 2017 07 26.
Article in English | MEDLINE | ID: mdl-28652412

ABSTRACT

Brain Aß1-42 accumulation is considered an upstream event in pathogenesis of Alzheimer's disease. However, accumulating evidence indicates that other neurochemical changes potentiate the toxicity of this constitutively generated peptide. Here we report that the interaction of Aß1-42 with extracellular Zn2+ is essential for in vivo rapid uptake of Aß1-42 and Zn2+ into dentate granule cells in the normal rat hippocampus. The uptake of both Aß1-42 and Zn2+ was blocked by CaEDTA, an extracellular Zn2+ chelator, and by Cd2+, a metal that displaces Zn2+ for Aß1-42 binding. In vivo perforant pathway LTP was unaffected by perfusion with 1000 nm Aß1-42 in ACSF without Zn2+ However, LTP was attenuated under preperfusion with 5 nm Aß1-42 in ACSF containing 10 nm Zn2+, recapitulating the concentration of extracellular Zn2+, but not with 5 nm Aß1-40 in ACSF containing 10 nm Zn2+ Aß1-40 and Zn2+ were not taken up into dentate granule cells under these conditions, consistent with lower affinity of Aß1-40 for Zn2+ than Aß1-42 Aß1-42-induced attenuation of LTP was rescued by both CaEDTA and CdCl2, and was observed even with 500 pm Aß1-42 Aß1-42 injected into the dentate granule cell layer of rats induced a rapid memory disturbance that was also rescued by coinjection of CdCl2 The present study supports blocking the formation of Zn-Aß1-42 in the extracellular compartment as an effective preventive strategy for Alzheimer's disease.SIGNIFICANCE STATEMENT Short-term memory loss occurs in normal elderly and increases in the predementia stage of Alzheimer's disease (AD). Amyloid-ß1-42 (Aß1-42), a possible causing peptide in AD, is bound to Zn2+ in the extracellular compartment in the hippocampus induced short-term memory loss in the normal rat brain, suggesting that extracellular Zn2+ is essential for Aß1-42-induced short-term memory loss. The evidence is important to find an effective preventive strategy for AD, which is blocking the formation of Zn-Aß1-42 in the extracellular compartment.


Subject(s)
Amyloid beta-Peptides/metabolism , Cognitive Dysfunction/metabolism , Hippocampus/metabolism , Long-Term Potentiation , Memory Disorders/metabolism , Peptide Fragments/metabolism , Zinc/metabolism , Animals , Cognitive Dysfunction/complications , Disease Progression , Extracellular Fluid/metabolism , Male , Memory Disorders/complications , Protein Binding , Rats , Rats, Wistar
3.
Front Neurosci ; 11: 199, 2017.
Article in English | MEDLINE | ID: mdl-28442988

ABSTRACT

Background: Adiponectin and leptin are implicated in the initiation and pathomechanism of Alzheimer's disease (AD). The serum concentrations of these adipokines has been extensively studied in AD, however little is known about their receptors in this disease. Objective: We developed a novel approach to examine whether the receptors of adiponectin (AdipoR1 and -R2) and/or leptin (LepR) can contribute to AD pathomechanism. To achieve this, we investigated the effect of both genetic and environmental factors associated with AD on the expression of these receptors. Method: We used C57BL/6J (WT) and APP(swe)/Presen(e9d)1 (AD) mice. Both strains were exposed to restraint stress (RS) daily for 6h over different time periods. Then, we measured the mRNA expression of AdipoR1, AdipoR2 and LepR and the level of AdipoR1 and AdipoR2 proteins in the hippocampal and prefrontal cortical areas of each mouse. Results: We detected brain region specific transcriptomic changes of adiponectin receptors induced by APP and PS1 transgenes. Both acute and chronic RS caused significant elevations in AdipoR1 mRNA expression in the hippocampus of WT mice. In the prefrontal cortex, the mRNA expression of AdipoR1 followed a biphasic course. In AD mice, RS did not promote any changes in the expression of AdipoR1 mRNA and AdipoR1 protein levels. AdipoR2 mRNA in AD animals, however, showed a significant increase in the prefrontal cortex during RS. Regarding AdipoR1 and AdipoR2 mRNA and protein expression, relevant changes could be measured during stress exposure in both brain areas. Furthermore, stress exposed groups exhibited little change in LepR mRNA expression. Conclusion: Our findings indicate that carrying the transgenes associated with AD induces modification in the expression of both adiponectin receptors. In the case of a normal genetic background, these receptors also appear to be sensitive to environmental factors, while in a genetically determined AD model less response to stress stimuli could be observed. The results suggest that modification of adipokine receptors could also be considered in the therapeutic approach to AD.

4.
Neurochem Int ; 62(1): 58-69, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23153458

ABSTRACT

Disturbances in intraluminal endoplasmic reticulum (ER) Ca(2+) concentration leads to the accumulation of unfolded proteins and perturbation of intracellular Ca(2+) homeostasis, which has a huge impact on mitochondrial functioning under normal and stress conditions and can trigger cell death. Thapsigargin (TG) is widely used to model cellular ER stress as it is a selective and powerful inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+) ATPases. Here we provide a representative proteome-wide picture of ER stress induced by TG in N2a neuroblastoma cells. Our proteomics study revealed numerous significant protein expression changes in TG-treated N2a cell lysates analysed by two-dimensional electrophoresis followed by mass spectrometric protein identification. The proteomic signature supports the evidence of increased bioenergetic activity of mitochondria as several mitochondrial enzymes with roles in ATP-production, tricarboxylic acid cycle and other mitochondrial metabolic processes were upregulated. In addition, the upregulation of the main ER resident proteins confirmed the onset of ER stress during TG treatment. It has become widely accepted that metabolic activity of mitochondria is induced in the early phases in ER stress, which can trigger mitochondrial collapse and subsequent cell death. Further investigations of this cellular stress response in different neuronal model systems like N2a cells could help to elucidate several neurodegenerative disorders in which ER stress is implicated.


Subject(s)
Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Enzyme Inhibitors/pharmacology , Neuroblastoma/pathology , Proteome/genetics , Thapsigargin/pharmacology , Blotting, Western , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Endoplasmic Reticulum Chaperone BiP , Energy Metabolism/drug effects , Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/genetics , Humans , Image Processing, Computer-Assisted , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Chaperones/metabolism , Neurites/drug effects , Spectrum Analysis
5.
Brain Res Bull ; 86(3-4): 217-21, 2011 Oct 10.
Article in English | MEDLINE | ID: mdl-21782906

ABSTRACT

The cell-impermeant oligomer-(e.g. beta-amyloid-, or tubulin-) specific fluorescent dye, bis-ANS (4,4'-bis-1-anilinonaphtalene-8-sulfonate), was successfully used for labeling mechanically damaged but still viable neuron bodies, neurites and neurite cross sections in acute brain slices. Acute hippocampal brain slices of rats were co-stained with bis-ANS and the cell-impermeant, DNA-specific dye propidium iodide (PI) and were then analyzed using fluorescence and confocal microscopes. Both the neuron bodies and the neurites were found to exhibit increased fluorescence intensities, suggesting that using this method they can be detected more easily. In addition, bis-ANS showed good region - but not cell specific co-localization with the neuron-specific fluorescent dye Dil (1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). These two dyes label different neuronal structures: Dil binds specifically to intact cell membranes while bis-ANS can enter cells with compromised cell membranes and then stain the microtubules in the cytoplasm. For a quick (10min) staining of acute brain slices with bis-ANS both HEPES and NaHCO(3) were needed in order to achieve high signal intensity. Labeling with bis-ANS fluorescent dye is an easy method for imaging the neuronal structures on the surface of acute brain slices.


Subject(s)
Anilino Naphthalenesulfonates , Brain/cytology , Fluorescent Dyes , Neurons/ultrastructure , Anilino Naphthalenesulfonates/metabolism , Animals , Carbocyanines , Cell Line, Tumor , Cell Membrane/ultrastructure , Coloring Agents , Cytoplasm/ultrastructure , Fluorescent Dyes/metabolism , Male , Microscopy, Confocal , Microscopy, Fluorescence , Microtubules/ultrastructure , Neurons/physiology , Propidium , Rats , Rats, Wistar , Staining and Labeling , Tubulin/metabolism
6.
J Neurochem ; 117(4): 691-702, 2011 May.
Article in English | MEDLINE | ID: mdl-21388376

ABSTRACT

Alzheimer's disease (AD) is the most prevalent form of neurodegenerative disorders even so the exact pathomechanism is still unclear. Recently, it is widely accepted that amyloid-beta peptide (Aß) toxicity is positively linked to Aß oligomers, which may be responsible for the initiation of AD. For this reason, AD research requires well defined aggregation state and structure of Aß. Precursor peptide 'iso-Aß1-42' makes it possible to use Aß1-42 with well- defined aggregation state for in vitro and in vivo experiments. The aim of this study was to identify protein expression changes from differentiated SH-SY5Y neuroblastoma cells after treatment with oligomeric Aß1-42 prepared in situ from 'iso-Aß1-42'. In our experiment, a cell viability assay revealed a strong and time-dependent toxic effect of oligomeric Aß1-42 which was supported by dramatic morphological changes. Our proteomics study also revealed numerous significant protein expression changes (22 proteins down- and 25 proteins up-regulated) after comparison of the untreated and Aß1-42-treated cell lysates by two-dimensional electrophoresis. From the functional classification of the identified proteins, we found deregulations of proteins involved in metabolic processes, cytoskeleton organisation and protein biosynthesis and a huge number of up-regulated stress proteins displayed oligomeric Aß1-42-induced cell stress.


Subject(s)
Amyloid beta-Peptides/toxicity , Peptide Fragments/toxicity , Proteomics , Amino Acid Sequence , Amyloid beta-Peptides/chemistry , Blotting, Western , Cell Line, Tumor , Cell Survival/drug effects , Electrophoresis, Gel, Two-Dimensional , HSP70 Heat-Shock Proteins/metabolism , Humans , Image Processing, Computer-Assisted , Isomerism , Mass Spectrometry , Molecular Sequence Data , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/classification , Peptide Elongation Factor 2/metabolism , Peptide Fragments/chemistry
7.
Brain Res Bull ; 75(5): 648-54, 2008 Mar 28.
Article in English | MEDLINE | ID: mdl-18355641

ABSTRACT

Alpha-synuclein (alphaSN) plays a major role in numerous neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Intracellular inclusions containing aggregated alphaSN have been reported in Alzheimer's and Parkinson's affected brains. Moreover, a proteolytic fragment of alphaSN, the so-called non-amyloid component of Alzheimer's disease amyloid (NAC) was found to be an integral part of Alzheimer's dementia related plaques. Despite the extensive research on this topic, the exact toxic mechanism of alphaSN remains elusive. We have taken the advantage of an alphaSN overexpressing SH-SY5Y cell line and investigated the effects of classical apoptotic factors (e.g. H(2)O(2), amphotericin B and ruthenium red) and aggregated disease-related peptides on cell viability compared to wild type neuroblastoma cells. It was found that alphaSN overexpressing cells are more sensitive to aggregated peptides treatment than normal expressing counterparts. In contrast, cells containing elevated amount of alphaSN were less vulnerable to classical apoptotic stressors than wild type cells. In addition, alphaSN overexpression is accompanied by altered phenotype, attenuated proliferation kinetics, increased neurite arborisation and decreased cell motility. Based on these results, the alphaSN overexpressing cell lines may represent a good and effective in vitro model for Alzheimer's and Parkinson's disease.


Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/pharmacology , Amyloid/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Neuroblastoma/metabolism , Peptide Fragments/pharmacology , alpha-Synuclein/metabolism , Amphotericin B/pharmacology , Amyloid/chemistry , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Humans , Hydrogen Peroxide/pharmacology , Molecular Weight , Neuroblastoma/physiopathology , Ruthenium Red/pharmacology , Tetrazolium Salts , Thiazoles , Time Factors , alpha-Synuclein/genetics
8.
Brain Res Bull ; 74(1-3): 183-7, 2007 Sep 14.
Article in English | MEDLINE | ID: mdl-17683806

ABSTRACT

The synaptic vesicles in the hippocampal neuronal terminals are abundantly supplied with zinc ions (Zn2+), which can be released into the synaptic cleft. In the glutamatergic systems (e.g. the hippocampus and the amygdala), the vesicular Zn2+ is co-localized with glutamate (Glu). Glu functions as a neurotransmitter, and Zn2+ as a neuromodulator (effecting basic synaptic functions). Electrical stimulation or chemical treatment (e.g. KCl) of hippocampal slices evokes the release of presynaptic vesicular Zn2+ into the synapse, together with Glu. This paper reports on the development of a rapid and simple method with which to assess the vesicular Zn2+ release and the effects of Zn2+-binding chelators in rat acute hippocampal slices. This method uses a 96-well fluorescence plate reader and the well-known zinc-sensitive fluorescence dye, FluoZin-3, which is cell-impermeable. This dye forms a stable complex with Zn2+ (Kd = 15 nM at pH 7.4); the amount of Zn2+ can be measured by fluorometry (lambda ex. 480-485 nm, em. 520-535 nm). Using 96-well plates, we could measure the Zn2+ release with high sensitivity, in at most 10 slices with a 15-s cycle time. This novel method can readily be used for the ex vivo modelling of the stress-evoked neuronal presynaptic Zn2+ release characteristic of neurodegenerative processes (e.g. Alzheimer's disease), or for the testing of Zn2+ chelators including putative drug candidates. This novel fluorescence plate reader method offers a simple, rapid and cost-effective technique for the measurement of vesicular Zn2+ release. It permits the simultaneous measurement of all mechanically undamaged hippocampal slices, regardless of size, thereby reducing the number of rats required experimentally.


Subject(s)
Hippocampus/ultrastructure , Microscopy, Fluorescence/methods , Molecular Probe Techniques , Presynaptic Terminals/metabolism , Synaptic Vesicles/metabolism , Zinc/metabolism , Age Factors , Analysis of Variance , Animals , In Vitro Techniques , Male , Microscopy, Fluorescence/instrumentation , Polycyclic Compounds/pharmacokinetics , Rats , Rats, Wistar , Time Factors
9.
J Biol Chem ; 280(9): 8464-70, 2005 Mar 04.
Article in English | MEDLINE | ID: mdl-15596431

ABSTRACT

Amyloid fibrils are self-associating filamentous structures, the deposition of which is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease and various other disorders. Here we used single molecule manipulation methods to explore the mechanics and structural dynamics of amyloid fibrils. In mechanically manipulated amyloid fibrils, formed from either amyloid beta (Abeta) peptides 1-40 or 25-35, beta-sheets behave as elastic structures that can be "unzipped" from the fibril with constant forces. The unzipping forces were different for Abeta1-40 and Abeta25-35. Unzipping was fully reversible across a wide range of stretch rates provided that coupling, via the beta-sheet, between bound and dissociated states was maintained. The rapid, cooperative zipping together of beta-sheets could be an important mechanism behind the self-assembly of amyloid fibrils. The repetitive force patterns contribute to a mechanical fingerprint that could be utilized in the characterization of different amyloid fibrils.


Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/physiology , Peptide Fragments/chemistry , Adsorption , Humans , Microscopy, Atomic Force , Models, Theoretical , Peptides/chemistry , Protein Structure, Secondary , Spectrophotometry , Time Factors
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