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Find video protocols related to scientific articles indexed in Pubmed.
Abeta(1-42) enhances neuronal excitability in the CA1 via NR2B subunit-containing NMDA receptors.
Neural Plast.
PUBLISHED: 06-13-2014
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Neuronal hyperexcitability is a phenomenon associated with early Alzheimer's disease. The underlying mechanism is considered to involve excessive activation of glutamate receptors; however, the exact molecular pathway remains to be determined. Extracellular recording from the CA1 of hippocampal slices is a long-standing standard for a range of studies both in basic research and in neuropharmacology. Evoked field potentials (fEPSPs) are regarded as the input, while spiking rate is regarded as the output of the neuronal network; however, the relationship between these two phenomena is not fully clear. We investigated the relationship between spontaneous spiking and evoked fEPSPs using mouse hippocampal slices. Blocking AMPA receptors (AMPARs) with CNQX abolished fEPSPs, but left firing rate unchanged. NMDA receptor (NMDAR) blockade with MK801 decreased neuronal spiking dose dependently without altering fEPSPs. Activating NMDARs by small concentration of NMDA induced a trend of increased firing. These results suggest that fEPSPs are mediated by synaptic activation of AMPARs, while spontaneous firing is regulated by the activation of extrasynaptic NMDARs. Synaptotoxic Abeta(1-42) increased firing activity without modifying evoked fEPSPs. This hyperexcitation was prevented by ifenprodil, an antagonist of the NR2B NMDARs. Overall, these results suggest that Abeta(1-42) induced neuronal overactivity is not dependent on AMPARs but requires NR2B.
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Simultaneous changes of spatial memory and spine density after intrahippocampal administration of fibrillar a?1-42 to the rat brain.
Biomed Res Int
PUBLISHED: 03-16-2014
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Several animal models of Alzheimer's disease have been used in laboratory experiments. Intrahippocampal injection of fibrillar amyloid-beta (fA?) peptide represents one of the most frequently used models, mimicking A? deposits in the brain. In our experiment synthetic fA?1-42 peptide was administered to rat hippocampus. The effect of the A? peptide on spatial memory and dendritic spine density was studied. The fA?1-42-treated rats showed decreased spatial learning ability measured in Morris water maze (MWM). Simultaneously, fA?1-42 caused a significant reduction of the dendritic spine density in the rat hippocampus CA1 region. The decrease of learning ability and the loss of spine density were in good correlation. Our results prove that both methods (MWM and dendritic spine density measurement) are suitable for studying A?-triggered neurodegeneration processes.
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Special lipid-based diets alleviate cognitive deficits in the APPswe/PS1dE9 transgenic mouse model of Alzheimer's disease independent of brain amyloid deposition.
J. Nutr. Biochem.
PUBLISHED: 01-22-2014
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Dietary fish oil, providing n3 polyunsaturated fatty acids like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), associates with reduced dementia risk in epidemiological studies and reduced amyloid accumulation in Alzheimer mouse models. We now studied whether additional nutrients can improve the efficacy of fish oil in alleviating cognitive deficits and amyloid pathology in APPswe/PS1dE9 transgenic and wild-type mice. We compared four isocaloric (5% fat) diets. The fish oil diet differed from the control diet only by substituted fish oil. Besides fish oil, the plant sterol diet was supplemented with phytosterols, while the Fortasyn diet contained as supplements precursors and cofactors for membrane synthesis, viz. uridine-monophosphate; DHA and EPA; choline; folate; vitamins B6, B12, C and E; phospholipids and selenium. Mice began the special diets at 5 months and were sacrificed at 14 months after behavioral testing. Transgenic mice, fed with control chow, showed poor spatial learning, hyperactivity in exploring a novel cage and reduced preference to explore novel odors. All fish-oil-containing diets increased exploration of a novel odor over a familiar one. Only the Fortasyn diet alleviated the spatial learning deficit. None of the diets influenced hyperactivity in a new environment. Fish-oil-containing diets strongly inhibited ?- and ?-secretase activity, and the plant sterol diet additionally reduced amyloid-? 1-42 levels. These data indicate that beneficial effects of fish oil on cognition in Alzheimer model mice can be enhanced by adding other specific nutrients, but this effect is not necessarily mediated via reduction of amyloid accumulation.
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Highly immunoreactive IgG antibodies directed against a set of twenty human proteins in the sera of patients with amyotrophic lateral sclerosis identified by protein array.
PLoS ONE
PUBLISHED: 01-01-2014
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Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disorder, is characterized by the progressive and selective loss of upper and lower motor neurons. Diagnosis of this disorder is based on clinical assessment, and the average survival time is less than 3 years. Injections of IgG from ALS patients into mice are known to specifically mark motor neurons. Moreover, IgG has been found in upper and lower motor neurons in ALS patients. These results led us to perform a case-control study using human protein microarrays to identify the antibody profiles of serum samples from 20 ALS patients and 20 healthy controls. We demonstrated high levels of 20 IgG antibodies that distinguished the patients from the controls. These findings suggest that a panel of antibodies may serve as a potential diagnostic biomarker for ALS.
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Epitaxial assembly dynamics of mutant amyloid ?25-35_N27C fibrils explored with time-resolved scanning force microscopy.
Biophys. Chem.
PUBLISHED: 07-21-2013
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Amyloid ?25-35 (A?25-35) is a toxic fragment of Alzheimers beta peptide. We have previously shown that A?25-35 fibrils form a trigonally oriented network on mica by epitaxial growth mechanisms. Chemical reactivity can be furnished to the fibril by introducing a cysteine residue (A?25-35_N27C) while maintaining oriented assembly properties. Previously we have shown that fibril binding to mica is strongly influenced by KCl concentration. In the present work we explored the kinetics of epitaxial assembly of the mutant fibrils at different peptide and KCl concentrations by using in situ time-resolved AFM. We measured the length of A?25-35_N27C fibrils as a function of time. Increasing free peptide concentration enhanced fibril growth rate, and the critical peptide concentration of fibril assembly was 3.92?M. Increasing KCl concentration decreased the number of fibrils bound to the mica surface, and above 20mM KCl fibril formation was completely abolished even at high peptide concentrations. By modulating peptide and KCl concentrations in the optimal ranges established here the complexity of the A?25-35_N27C network can be finely tuned.
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Tenascin-C deficiency ameliorates Alzheimers disease-related pathology in mice.
Neurobiol. Aging
PUBLISHED: 04-07-2013
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Alzheimers disease (AD) is a neurodegenerative disease characterized by deposits of amyloid ? peptide (A?) and microglia-driven inflammatory activation. Tenascin-C (tnc) is an extracellular matrix protein that is upregulated in inflammation and induces further inflammatory responses. We hypothesized that tnc contributes to the inflammatory pathology in AD. Using real-time polymerase chain reaction, we observed that tnc gene transcription was upregulated in cultured microglia after A? challenge and in the brain of an AD mouse model that overexpresses mutated amyloid precursor protein (APP) in neural cells. By cross-breeding APP-transgenic mice and tenascin-C-deficient mice, we demonstrated using real-time polymerase chain reaction, Western blot analysis, enzyme-linked immunosorbent assay, and immunohistochemistry that tnc deficiency reduces pro- but enhances anti-inflammatory activation in the mutated APP-transgenic mouse brain, associated with a reduced cerebral A? load and higher levels of the postsynaptic density protein 95. Thus, our study indicates that functional inhibition of tnc exerts beneficial effects on AD pathogenesis, suggesting a potential for tnc as a new therapeutic target in AD.
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Overexpression of Hsp27 ameliorates symptoms of Alzheimers disease in APP/PS1 mice.
Cell Stress Chaperones
PUBLISHED: 04-04-2013
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Hsp27 belongs to the small heat shock protein family, which are ATP-independent chaperones. The most important function of Hsp27 is based on its ability to bind non-native proteins and inhibit the aggregation of incorrectly folded proteins maintaining them in a refolding-competent state. Additionally, it has anti-apoptotic and antioxidant activities. To study the effect of Hsp27 on memory and synaptic functions, amyloid-? (A?) accumulation, and neurodegeneration, we generated transgenic mice overexpressing human Hsp27 protein and crossed with APPswe/PS1dE9 mouse strain, a mouse model of Alzheimers disease (AD). Using different behavioral tests, we found that spatial learning was impaired in AD model mice and was rescued by Hsp27 overexpression. Electrophysiological recordings have revealed that excitability of neurons was significantly increased, and long-term potentiation (LTP) was impaired in AD model mice, whereas they were normalized in Hsp27 overexpressing AD model mice. Using anti-amyloid antibody, we counted significantly less amyloid plaques in the brain of APPswe/PS1dE9/Hsp27 animals compared to AD model mice. These results suggest that overexpression of Hsp27 protein might ameliorate certain symptoms of AD.
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Penetratin and derivatives acting as antibacterial agents.
Chem Biol Drug Des
PUBLISHED: 03-13-2013
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The synthesis, in vitro evaluation and conformational study of penetratin and structurally related derivatives acting as antibacterial agents are reported. Among the compounds evaluated here, two methionine sulphoxide derivatives (RQIKIWFQNRRM[O]KWKK-NH2 and RQIKIFFQNRRM[O]KFKK-NH2 ) exhibited the strongest antibacterial effect in this series. In order to better understand the antimicrobial activity obtained for these peptides, we performed an exhaustive conformational analysis using different approaches. Molecular dynamics simulations were performed using two different media (water and trifluoroethanol/water). The results of these theoretical calculations were corroborated using experimental CD measurements. The electronic study for these peptides was carried out using molecular electrostatic potentials obtained from RHF/6-31G(d) calculations. In addition, the non-apeptide RQIRRWWQR-NH2 showed strong inhibitory action against the Gram-negative and Gram-positive bacteria tested in this study.
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TLR2 is a primary receptor for Alzheimers amyloid ? peptide to trigger neuroinflammatory activation.
J. Immunol.
PUBLISHED: 12-23-2011
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Microglia activated by extracellularly deposited amyloid ? peptide (A?) act as a two-edged sword in Alzheimers disease pathogenesis: on the one hand, they damage neurons by releasing neurotoxic proinflammatory mediators (M1 activation); on the other hand, they protect neurons by triggering anti-inflammatory/neurotrophic M2 activation and by clearing A? via phagocytosis. TLRs are associated with A?-induced microglial inflammatory activation and A? internalization, but the mechanisms remain unclear. In this study, we used real-time surface plasmon resonance spectroscopy and conventional biochemical pull-down assays to demonstrate a direct interaction between TLR2 and the aggregated 42-aa form of human A? (A?42). TLR2 deficiency reduced A?42-triggered inflammatory activation but enhanced A? phagocytosis in cultured microglia and macrophages. By expressing TLR2 in HEK293 cells that do not endogenously express TLR2, we observed that TLR2 expression enabled HEK293 cells to respond to A?42. Through site-directed mutagenesis of tlr2 gene, we identified the amino acids EKKA (741-744) as a critical cytoplasmic domain for transduction of inflammatory signals. By coexpressing TLR1 or TLR6 in TLR2-transgenic HEK293 cells or silencing tlrs genes in RAW264.7 macrophages, we observed that TLR2-mediated A?42-triggered inflammatory activation was enhanced by TLR1 and suppressed by TLR6. Using bone marrow chimeric Alzheimers amyloid precursor transgenic mice, we observed that TLR2 deficiency in microglia shifts M1- to M2-inflammatory activation in vivo, which was associated with improved neuronal function. Our study demonstrated that TLR2 is a primary receptor for A? to trigger neuroinflammatory activation and suggested that inhibition of TLR2 in microglia could be beneficial in Alzheimers disease pathogenesis.
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Interactions of pathological hallmark proteins: tubulin polymerization promoting protein/p25, beta-amyloid, and alpha-synuclein.
J. Biol. Chem.
PUBLISHED: 08-08-2011
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The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with ?-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of ?-synuclein with ?-amyloid (A?) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble A? oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric A? with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the A?(42) tightly bound to TPPP/p25 (K(d) = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of A?(42), ?-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with A? was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with A? can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of A? and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.
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Tianeptine potentiates AMPA receptors by activating CaMKII and PKA via the p38, p42/44 MAPK and JNK pathways.
Neurochem. Int.
PUBLISHED: 07-25-2011
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Impairments of cellular plasticity appear to underlie the pathophysiology of major depression. Recently, elevated levels of phosphorylated AMPA receptor were implicated in the antidepressant effect of various drugs. Here, we investigated the effects of an antidepressant, Tianeptine, on synaptic function and GluA1 phosphorylation using murine hippocampal slices and in vivo single-unit recordings. Tianeptine, but not imipramine, increased AMPA receptor-mediated neuronal responses both in vitro and in vivo, in a staurosporine-sensitive manner. Paired-pulse ratio was unaltered by Tianeptine, suggesting a postsynaptic site of action. Tianeptine, 10 ?M, enhanced the GluA1-dependent initial phase of LTP, whereas 100 ?M impaired the latter phases, indicating a critical role of GluA1 subunit phosphorylation in the excitation. Tianeptine rapidly increased the phosphorylation level of Ser(831)-GluA1 and Ser(845)-GluA1. Using H-89 and KN-93, we show that the activation of both PKA and CaMKII is critical in the effect of Tianeptine on AMPA responses. Moreover, the phosphorylation states of Ser(217/221)-MEK and Thr(183)/Tyr(185)-p42MAPK were increased by Tianeptine and specific kinase blockers of the MAPK pathways (PD 98095, SB 203580 and SP600125) prevented the effects of Tianeptine. Overall these data suggest that Tianeptine potentiates several signaling cascades associated with synaptic plasticity and provide further evidence that a major mechanism of action for Tianeptine is to act as an enhancer of glutamate neurotransmission via AMPA receptors.
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Functional changes in transcriptomes of the prefrontal cortex and hippocampus in a mouse model of anxiety.
Pharmacol Rep
PUBLISHED: 05-24-2011
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Anxiety is a multi-etiology disorder influenced by both genetic background and environment. To study the impact of a genetic predisposition, we developed a novel mouse model of anxiety using a combination of crossbreeding and behavioral selection. Comparison of the transcriptomes from the prefrontal cortex and hippocampus of anxious and control mice revealed that the numbers of significantly up- and down-regulated genes were modest, comprising approximately 2% of the tested genes. Functional analysis of the significantly altered gene sets showed that functional groups such as nervous system development, behavior, glial cell differentiation and synaptic transmission were significantly enriched among the up-regulated genes, whereas functional groups such as potassium ion transport, Wnt signaling and neuropeptidergic signaling were significantly enriched among the down-regulated genes. Many of the identified genes and functional groups have been previously linked to the molecular biology of anxiety, while several others, such as transthyretin, vasoactive intestinal polypeptide and various potassium ion channels, are novel or not as well described in this context. Supporting the gene expression data, we also found increased excitability in the hippocampi of anxious mice, which can be a phenotypic result of decreased potassium channel density. Our transcriptome screen showed that the initiation and/or effect of anxiety involve multiple pathways and cellular processes. The identified novel genes and pathways could be involved in the molecular pathogenesis of anxiety and provide potential targets for further drug development.
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A novel application of the fluorescent dye bis-ANS for labeling neurons in acute brain slices.
Brain Res. Bull.
PUBLISHED: 04-27-2011
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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.
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Effect of the beta-sheet-breaker peptide LPFFD on oriented network of amyloid ?25-35 fibrils.
J. Mol. Recognit.
PUBLISHED: 04-20-2011
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Amyloid fibrils are self-associating filamentous structures deposited in extracellular tissue in various neurodegenerative and protein misfolding disorders. It has been shown that beta-sheet-breaker (BSB) peptides may interfere with amyloid fibril assembly. Although BSB peptides are prospective therapeutic agents in amyloidosis, there is ambiguity about the mechanisms and generality of their action. In the present work we analyzed the effect of the BSB peptide LPFFD on the growth kinetics, morphologic, and mechanical properties of amyloid ?25-35 (A?25-35) fibrils assembled in an oriented array on mica surface. A?25-35 is thought to represent the biologically active, toxic fragment of the full-length A? peptide. Growth kinetics and morphologic features were analyzed using in situ atomic force microscopy in the presence of various concentrations of LPFFD. We found that the addition of LPFFD only slightly altered the assembly kinetics of A?25-35 fibrils. Already formed fibrils did not disassemble in the presence of high concentrations of LPFFD. The mechanical stability of the fibrils was explored with force spectroscopy methods. The nanomechanical behavior of A?25-35 fibrils is characterized by the appearance of force staircases which correspond to the force-driven unzipping and dissociation of several protofilaments. In the presence of LPFFD single-plateau force traces dominated. The effects of LPFFD on A?25-35 fibril assembly and stability suggest that inter-protofilament interactions were slightly weakened. Complete disassembly of fibrils, however, was not observed. Thus, under the conditions explored here, LPFFD may not be considered as a BSB peptide with generalized beta-sheet breaking properties.
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Proteomic study of the toxic effect of oligomeric A?1-42 in situ prepared from iso-A?1-42.
J. Neurochem.
PUBLISHED: 03-28-2011
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Alzheimers 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 (A?) toxicity is positively linked to A? oligomers, which may be responsible for the initiation of AD. For this reason, AD research requires well defined aggregation state and structure of A?. Precursor peptide iso-A?1-42 makes it possible to use A?1-42 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 A?1-42 prepared in situ from iso-A?1-42. In our experiment, a cell viability assay revealed a strong and time-dependent toxic effect of oligomeric A?1-42 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 A?1-42-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 A?1-42-induced cell stress.
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Protein array based interactome analysis of amyloid-? indicates an inhibition of protein translation.
J. Proteome Res.
PUBLISHED: 02-22-2011
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Oligomeric amyloid-? is currently of interest in amyloid-? mediated toxicity and the pathogenesis of Alzheimers disease. Mapping the amyloid-? interaction partners could help to discover novel pathways in disease pathogenesis. To discover the amyloid-? interaction partners, we applied a protein array with more than 8100 unique recombinantly expressed human proteins. We identified 324 proteins as potential interactors of oligomeric amyloid-?. The Gene Ontology functional analysis of these proteins showed that oligomeric amyloid-? bound to multiple proteins with diverse functions both from extra and intracellular localizations. This undiscriminating binding phenotype indicates that multiple protein interactions mediate the toxicity of the oligomeric amyloid-?. The most highly impacted cellular system was the protein translation machinery. Oligomeric amyloid-? could bind to altogether 24 proteins involved in translation initiation and elongation. The binding of amyloid-? to purified rat hippocampal ribosomes validated the protein array results. More importantly, in vitro translation assays showed that the oligomeric amyloid-? had a concentration dependent inhibitory activity on translation. Our results indicate that the inhibited protein synthesis is one of the pathways that can be involved in the amyloid-beta induced neurotoxicity.
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Docosahexaenoic acid reduces amyloid beta production via multiple pleiotropic mechanisms.
J. Biol. Chem.
PUBLISHED: 02-15-2011
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Alzheimer disease is characterized by accumulation of the ?-amyloid peptide (A?) generated by ?- and ?-secretase processing of the amyloid precursor protein (APP). The intake of the polyunsaturated fatty acid docosahexaenoic acid (DHA) has been associated with decreased amyloid deposition and a reduced risk in Alzheimer disease in several epidemiological trials; however, the exact underlying molecular mechanism remains to be elucidated. Here, we systematically investigate the effect of DHA on amyloidogenic and nonamyloidogenic APP processing and the potential cross-links to cholesterol metabolism in vivo and in vitro. DHA reduces amyloidogenic processing by decreasing ?- and ?-secretase activity, whereas the expression and protein levels of BACE1 and presenilin1 remain unchanged. In addition, DHA increases protein stability of ?-secretase resulting in increased nonamyloidogenic processing. Besides the known effect of DHA to decrease cholesterol de novo synthesis, we found cholesterol distribution in plasma membrane to be altered. In the presence of DHA, cholesterol shifts from raft to non-raft domains, and this is accompanied by a shift in ?-secretase activity and presenilin1 protein levels. Taken together, DHA directs amyloidogenic processing of APP toward nonamyloidogenic processing, effectively reducing A? release. DHA has a typical pleiotropic effect; DHA-mediated A? reduction is not the consequence of a single major mechanism but is the result of combined multiple effects.
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Systems biology of Alzheimers disease: how diverse molecular changes result in memory impairment in AD.
Neurochem. Int.
PUBLISHED: 01-19-2011
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Alzheimers disease (AD) is a protein misfolding-based rapid cognitive impairment in the aging brain. Because of its very widespread molecular background, AD has been approached using genomic and proteomic methods and has accumulated a large body of data during the last 15 years. In this review, we summarize the systems biology data on AD and pay particular attention to the proteomic changes in AD. Applying a systems biology model of the synapse, we attempt to integrate protein changes and provide an explanation of why seemingly diverse molecular changes result in memory impairment. We also summarize the present state of cerebrospinal fluid (CSF) and blood biomarker studies for the diagnosis of AD as well as the results of proteomic studies in tissue cultures and animal models. Finally, we give a systems biology model of AD explaining how AD can develop in an individual manner in each particular subject but always results in a rapidly developing dementia and memory impairment.
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Exploring and characterizing the folding processes of Lys- and Arg-containing Ala-based peptides: a molecular dynamics study.
Comput Biol Chem
PUBLISHED: 01-11-2011
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In this study, molecular dynamics simulations were carried out on Lys- and Arg-containing Ala-based peptides (i.e. Ace-(AAAAK)(n)A-NH(2) and Ace-(AAAAR)(n)A-NH(2), where n=1-4), in order to explore and characterize their folding processes. For the oligopeptides, the evolution of ?-helical structure with regard to the whole conformation, as well as to each residue was investigated, and the helix-forming propensities were characterized. On the basis of the helicity curves, representing the alteration of average helicity as a function of time, the typical time values describing the folding processes and subprocesses were identified. In the case of each peptide, the evolution and role of helix-stabilizing, non-local and side-chain-to-backbone H-bonds were examined. The appearing i?i+4 H-bonds pointed out the role of these interactions in the stabilization of ?-helical conformations, while the occurring i?i+3 H-bonds indicated the presence of ?-turn or 3(10)-helical structures. Studying the formation and role of non-local and side-chain-to-backbone H-bonds led to the observation that these types of interactions produced an effect on the evolution of helical conformations, as well as on the folding processes.
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Characterisation of the variation of mouse brain proteome by two-dimensional electrophoresis.
J Proteomics
PUBLISHED: 01-04-2011
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Neuroproteomics is aimed to study the molecular organisation of the nervous system at the protein level. Two-dimensional electrophoresis is the most frequently used technique in quantitative proteomics. The aim of this study was to assess the experimental and biological variations on this proteomic platform using mouse brain tissue. Mice are the most generally used lab animals for modelling human disease or investigating the effect of a drug-candidate or a treatment. Experimental design plays a crucial role in quantitative proteomics, hence understanding and minimizing the variables is essential. Our results indicate that the technical variance dominantly contributes to the total variance in mouse brain and the genetic background has a negligible effect on the total variation. The results also characterise the anticipated variation using mouse brain for proteomic study hence they should be useful for future experimental design in other proteomics laboratories.
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Myeloid differentiation factor 88-deficient bone marrow cells improve Alzheimers disease-related symptoms and pathology.
Brain
PUBLISHED: 11-28-2010
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Alzheimers disease is characterized by extracellular deposits of amyloid ? peptide in the brain. Increasing evidence suggests that amyloid ? peptide injures neurons both directly and indirectly by triggering neurotoxic innate immune responses. Myeloid differentiation factor 88 is the key signalling molecule downstream to most innate immune receptors crucial in inflammatory activation. For this reason, we investigated the effects of myeloid differentiation factor 88-deficient bone marrow cells on Alzheimers disease-related symptoms and pathology by establishing bone marrow chimeric amyloid ? peptide precursor transgenic mice, in which bone marrow cells differentiate into microglia and are recruited to amyloid ? peptide deposits. We observed that myeloid differentiation factor 88-deficient bone marrow reconstruction reduced both inflammatory activation and amyloid ? peptide burden in the brain. In addition, synaptophysin, a marker of neuronal integrity, was preserved and the expression of neuronal plasticity-related genes, ARC and NMDA-R1, was increased. Thus, myeloid differentiation factor 88-deficient microglia significantly improved the cognitive function of amyloid ? peptide precursor protein transgenic mice. Myeloid differentiation factor 88-deficiency enhanced amyloid ? peptide phagocytosis by microglia/macrophages and blunted toxic inflammatory activation. Both the expression of amyloid ? peptide precursor protein and amyloid ? peptide degrading enzymes and also the efflux of amyloid ? peptide from brain parenchyma were unaffected by myeloid differentiation factor 88-deficient microglia. By contrast, the activity of ?-secretase was increased. ?-Secretase is expressed primarily in neurons, with relatively little expression in astrocytes and microglia. Therefore, microglial replenishment with myeloid differentiation factor 88-deficient bone marrow cells might improve cognitive functions in Alzheimers disease mouse models by enhancing amyloid ? peptide phagocytosis and reducing inflammatory activation. These results could offer a new therapeutic option that might delay the progression of Alzheimers disease.
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Effects of estrogen on beta-amyloid-induced cholinergic cell death in the nucleus basalis magnocellularis.
Neuroendocrinology
PUBLISHED: 08-18-2010
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Alzheimer disease is characterized by accumulation of ?-amyloid (A?) and cognitive dysfunctions linked to early loss of cholinergic neurons. As estrogen-based hormone replacement therapy has beneficial effects on cognition of demented patients, and it may prevent memory impairments, we investigated the effect of estrogen-pretreatment on A?-induced cholinergic neurodegeneration in the nucleus basalis magnocellularis (NBM). We tested which A? species induces the more pronounced cholinotoxic effect in vivo. We injected different A? assemblies in the NBM of mice, and measured cholinergic cell and cortical fiber loss. Spherical A? oligomers had the most toxic effect. Pretreatment of ovariectomized mice with estrogen before A? injection decreased cholinergic neuron loss and partly prevented fiber degeneration. By using proteomics, we searched for proteins involved in estrogen-mediated protection and in A? toxicity 24 h following injection. The change in expression of, e.g., DJ-1, NADH ubiquinone oxidoreductase, ATP synthase, phosphatidylethanolamine-binding protein 1, protein phosphatase 2A and dimethylarginine dimethylaminohydrolase 1 support our hypothesis that A? induces mitochondrial dysfunction, decreases MAPK signaling, and increases NOS activation in NBM. On the other hand, altered expression of, e.g., MAP kinase kinase 1 and 2, protein phosphatase 1 and 2A by A? might increase MAPK suppression and NOS signaling in the cortical target area. Estrogen pretreatment reversed most of the changes in the proteome in both areas. Our experiments suggest that regulation of the MAPK pathway, mitochondrial pH and NO production may all contribute to A? toxicity, and their regulation can be prevented partly by estrogen pretreatment.
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Penetratin analogues acting as antifungal agents.
Eur J Med Chem
PUBLISHED: 07-27-2010
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The synthesis, in vitro evaluation, and conformational study of penetratin analogues acting as antifungal agents are reported. Different peptides structurally related with penetratin were evaluated. Analogues of penetratin rich in Arg, Lys and Trp amino acids were tested. In addition, HFRWRQIKIWFQNRRM[O]KWKK-NH(2), a synthetic 20 amino acid peptide was also evaluated. These penetratin analogues displayed antifungal activity against human pathogenic strains including Candida albicans and Cryptococcus neoformans. In contrast, Tat peptide, a well-known cell penetrating peptide, did not show a significant antifungal activity against fungus tested here. We also performed a conformational study by means experimental and theoretical approaches (CD spectroscopic measurements and MD simulations). The electronic structure analysis was carried out from Molecular Electrostatic Potentials (MEP) obtained by using RHF/6-31G ab initio calculations. Our experimental and theoretical results permitted us to identify a topographical template which may provide a guide for the design of new peptides with antifungal effects.
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New antifungal peptides. Synthesis, bioassays and initial structure prediction by CD spectroscopy.
Bioorg. Med. Chem. Lett.
PUBLISHED: 04-30-2010
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The synthesis, in vitro evaluation and conformational study of KKWKMRRNQFWIKIQR-NH(2), HFRWRQIKIWFQNRRMKWKK-NH(2) and RQPKIWFPNRRKPWKK-NH(2) acting as antifungal agents are reported. These peptides displayed a moderate but significant antifungal effect against both pathogenic fungi Candida albicans and Cryptococcus neoformans. The conformational analysis of these peptides was carried out using both theoretical and experimental methods.
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Different electrophysiological actions of 24- and 72-hour aggregated amyloid-beta oligomers on hippocampal field population spike in both anesthetized and awake rats.
Brain Res.
PUBLISHED: 03-31-2010
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Diffusible oligomeric assemblies of the amyloid beta-protein (Abeta) could be the primary factor in the pathogenic pathway leading to Alzheimers disease (AD). Converging lines of evidence support the notion that AD begins with subtle alterations in synaptic efficacy, prior to the occurrence of extensive neuronal degeneration. Recently, however, a shared or overlapping pathogenesis for AD and epileptic seizures occurred as aberrant neuronal hyperexcitability, as well as nonconvulsive seizure activity were found in several different APP transgenic mouse lines. This generated a renewed attention to the well-known comorbidity of AD and epilepsy and interest in how Abeta oligomers influence neuronal excitability. In this study therefore, we investigated the effect of various in vitro-aged Abeta(1-42) oligomer solutions on the perforant pathway-evoked field potentials in the ventral hippocampal dentate gyrus in vivo. Firstly, Abeta oligomer solutions (1 microl, 200 microM) which had been aggregated in vitro for 0, 24 or 72h were injected into the hippocampus of urethane-anesthetized rats, in parallel with in vitro physico-chemical characterization of Abeta oligomerization (atomic force microscopy, thioflavin-T fluorescence). We found a marked increase of hippocampal population spike (pSpike) after injection of the 24-h Abeta oligomer solution and a decrease of the pSpike amplitude after injection of the 72-h Abeta oligomer. Since urethane anesthesia affects the properties of hippocampal evoked potentials, we repeated the injection of these two Abeta oligomer solutions in awake, freely moving animals. Evoked responses to perforant pathway stimulation revealed a 70% increase of pSpike amplitude 50 min after the 24-h Abeta oligomer injection and a 55% decrease after the 72-h Abeta oligomer injection. Field potentials, that reflect synaptic potentials, were not affected by the Abeta injection. These results demonstrate that oligomeric Abeta aggregates elicit opposite electrophysiological effects on neuronal excitability which depend on their degree of oligomerization.
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[The effects of duloxetine on beta-actin stress response in rat brain].
Neuropsychopharmacol Hung
PUBLISHED: 03-23-2010
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Depression is a frequent prodromal symptom of Alzheimers disease (AD). Stress factors play an important role in the etiopathology of both diseases, since increased corticosteroid levels caused by chronic stress indirectly induce neuronal damage. The aim of our experiments was to evaluate the changes induced by stress in the transcription of amyloid precursor protein (APP), mitogen activated protein kinase-1 (MAPK-1) and beta-actin, of which the latest plays a leading role in synaptic plasticity. Additionally we intended to examine how duloxetine - a serotonin-norepinephrin reuptake inhibitor type antidepressant - would modify the stress-induced changes. Wistar rats were exposed to immobilization stress for five hours daily through 21 days, while part of the animals received 45 mg/bwkg of duloxetine. At the end of the third week total RNA was purified from the cortex and hippocampus. The amount of beta-actin, APP and MAPK-1 mRNA was determined by real time PCR method. On protein level, semiquantitative measurement was performed by Western blot. The expression of beta-actin mRNA in the animals exposed to stress was four times as intense as in the control group. The increase in the beta-actin mRNA levels was repressed by the duloxetine treatment. In the case of APP and MAPK-1 no changes were detected. According to the Western blot results, the antidepressant treatment slightly, the drug along with the stress treatment strongly decreased the amount of the beta-actin protein. Our findings indicate that antidepressant treatment with duloxetine could play a protective role against the chronic stress-induced changes in the nervous system, such as disorders of synaptic plasticity, and the consequent cognitive dysfunctions in case of both affective disorders and AD.
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New small-size peptides possessing antifungal activity.
Bioorg. Med. Chem.
PUBLISHED: 09-03-2009
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The synthesis, in vitro evaluation, and conformational study of a new series of small-size peptides acting as antifungal agents are reported. In a first step of our study we performed a conformational analysis using Molecular Mechanics calculations. The electronic study was carried out using Molecular electrostatic potentials (MEPs) obtained from RHF/6-31G calculations. On the basis of the theoretical predictions three small-size peptides, RQWKKWWQWRR-NH(2), RQIRRWWQWRR-NH(2), and RQIRRWWQW-NH(2) were synthesized and tested. These peptides displayed a significant antifungal activity against human pathogenic strains including Candida albicans and Cryptococcus neoformans. Our experimental and theoretical results allow the identification of a topographical template which can serve as a guide for the design of new compounds with antifungal properties for potential therapeutic applications against these pathogenic fungi.
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In silico study of full-length amyloid beta 1-42 tri- and penta-oligomers in solution.
J Phys Chem B
PUBLISHED: 08-04-2009
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Amyloid oligomers are considered to play causal roles in the pathogenesis of amyloid-related degenerative diseases including Alzheimers disease. Using MD simulation techniques, we explored the contributions of the different structural elements of trimeric and pentameric full-length Abeta1-42 aggregates in solution to their stability and conformational dynamics. We found that our models are stable at a temperature of 310 K, and converge toward an interdigitated side-chain packing for intermolecular contacts within the two beta-sheet regions of the aggregates: beta1 (residues 18-26) and beta2 (residues 31-42). MD simulations reveal that the beta-strand twist is a characteristic element of Abeta-aggregates, permitting a compact, interdigitated packing of side chains from neighboring beta-sheets. The beta2 portion formed a tightly organized beta-helix, whereas the beta1 portion did not show such a firm structural organization, although it maintained its beta-sheet conformation. Our simulations indicate that the hydrophobic core comprising the beta2 portion of the aggregate is a crucial stabilizing element in the Abeta aggregation process. On the basis of these structure-stability findings, the beta2 portion emerges as an optimal target for further antiamyloid drug design.
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Controlled in situ preparation of A beta(1-42) oligomers from the isopeptide "iso-A beta(1-42)", physicochemical and biological characterization.
Peptides
PUBLISHED: 07-23-2009
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Beta-amyloid (A beta) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimers disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic A beta peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of A beta peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-A beta(1-42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to A beta(1-42) by O-->N acyl migration in a short period of time. We prepared the same precursor peptide using Boc-chemistry and studied the transformation to A beta(1-42) by acyl migration. The peptide conformation and aggregation processes were studied by several methods (circular dichroism, atomic force and transmission electron microscopy, dynamic light scattering). The biological activity of the synthetic A beta(1-42) was measured by ex vivo (long-term potentiation studies in rat hippocampal slices) and in vivo experiments (spatial learning of rats). It was proven that O-->N acyl migration of the precursor isopeptide results in a water soluble oligomeric mixture of neurotoxic A beta(1-42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.
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Intranasal delivery of human beta-amyloid peptide in rats: effective brain targeting.
Cell. Mol. Neurobiol.
PUBLISHED: 06-30-2009
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(1) Intranasal administration is a non-invasive and effective way for the delivery of drugs to brain that circumvents the blood-brain barrier. The aims of the study were to test a nasal delivery system for human beta-amyloid (A beta) peptides, to measure the delivery of the peptides to brain regions, and to test their biological activity in rats. (2) A beta(1-42), in the form of a mixture of oligomers, protofibrils, and fibrils was dissolved in a nasal formulation containing hydrophobic, hydrophylic, and mucoadhesive components. The peptide solution was administered intranasally to rats as a single dose or in repeated doses. (3) Nasally injected A beta labeled with the blue fluorescent dye amino-methyl coumarinyl acetic acid (AMCA) could be detected by fluorescent microscopy in the olfactory bulb and frontal cortex. The concentration of the peptide was quantified by fluorescent spectroscopy, and a significant amount of AMCA-A beta peptide could be detected in the olfactory bulb. Unlabeled A beta also reached the olfactory bulb and frontal cortex of rats as evidenced by intense immunostaining. (4) In behavioral experiments, nasal A beta treatment did not affect anxiety levels (open-field test) and short-term memory (Y-maze test), but significantly impaired long-term spatial memory in the Morris water maze. The treatments did not result in A beta immunization. (5) The tested intranasal delivery system could successfully target a bioactive peptide into the central nervous system and provides a basis for developing a non-invasive and cost effective, new model to study amyloid-induced dysfunctions in the brain.
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Aspartic acid scaffold in bradykinin B1 antagonists.
J. Pept. Sci.
PUBLISHED: 04-21-2009
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Several novel bradykinin B1 receptor (B1R) antagonists were synthesized utilizing a new aspartic acid scaffold. This core is derived from the highly potent dihydroquinoxalinone scaffold published recently by researchers at Merck (Ha et al. Biochem. Biophys. Res. Commun. 2005, 331, 159-166). Despite the considerably limited chemical space of B1 antagonists, the synthesized compounds still showed significant biological activity. None of the four most potent compounds showed significant activity on the bradykinin B2 receptor (B2R), consequently they can be considered as valuable starting points for designing more potent and selective B1 antagonists. Furthermore, the synthesis of these aspartic acid derivatives is much simpler than that of the original Merck compounds suggesting efficient parallel synthesis approaches during their optimization. Docking known and novel B1 antagonists into the refined B1R homology model including the second extracellular loop (EC2) underlined the importance of this loop in ligand binding. Comparative binding mode analysis revealed that our novel compounds bind similar to the dihydroquinoxalinone template. Our results indicate that the rigid core of the dihydroquinoxalinone containing B1 antagonists is not crucial for maintaining B1 activity.
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Penetratin and derivatives acting as antifungal agents.
Eur J Med Chem
PUBLISHED: 04-02-2009
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The synthesis, in vitro evaluation, and conformational study of RQIKIWFQNRRMKWKK-NH(2) (penetratin) and related derivatives acting as antifungal agents are reported. Penetratin and some of its derivatives displayed antifungal activity against the human opportunistic pathogenic standardized ATCC strains Candida albicans and Cryptococcus neoformans as well as clinical isolates of C. neoformans. Among the compounds tested, penetratin along with the nonapeptide RKWRRKWKK-NH(2) and the tetrapeptide RQKK-NH(2) exhibited significant antifungal activities against the Cryptococcus species. A comprehensive conformational analysis on the peptides reported here using three different approaches, molecular mechanics, simulated annealing and molecular dynamics simulations, was carried out. The experimental and theoretical results allow us to identify a topographical template which may provide a guide for the design of new compounds with antifungal characteristics against C. neoformans.
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Amyloid beta-induced neuronal hyperexcitability triggers progressive epilepsy.
J. Neurosci.
PUBLISHED: 03-20-2009
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Alzheimers disease is associated with an increased risk of unprovoked seizures. However, the underlying mechanisms of seizure induction remain elusive. Here, we performed video-EEG recordings in mice carrying mutant human APPswe and PS1dE9 genes (APdE9 mice) and their wild-type littermates to determine the prevalence of unprovoked seizures. In two recording episodes at the onset of amyloid beta (Abeta) pathogenesis (3 and 4.5 months of age), at least one unprovoked seizure was detected in 65% of APdE9 mice, of which 46% had multiple seizures and 38% had a generalized seizure. None of the wild-type mice had seizures. In a subset of APdE9 mice, seizure phenotype was associated with a loss of calbindin-D28k immunoreactivity in dentate granular cells and ectopic expression of neuropeptide Y in mossy fibers. In APdE9 mice, persistently decreased resting membrane potential in neocortical layer 2/3 pyramidal cells and dentate granule cells underpinned increased network excitability as identified by patch-clamp electrophysiology. At stimulus strengths evoking single-component EPSPs in wild-type littermates, APdE9 mice exhibited decreased action potential threshold and burst firing of pyramidal cells. Bath application (1 h) of Abeta1-42 or Abeta25-35 (proto-)fibrils but not oligomers induced significant membrane depolarization of pyramidal cells and increased the activity of excitatory cell populations as measured by extracellular field recordings in the juvenile rodent brain, confirming the pathogenic significance of bath-applied Abeta (proto-)fibrils. Overall, these data identify fibrillar Abeta as a pathogenic entity powerfully altering neuronal membrane properties such that hyperexcitability of pyramidal cells culminates in epileptiform activity.
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Altered functional protein networks in the prefrontal cortex and amygdala of victims of suicide.
PLoS ONE
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Probing molecular brain mechanisms related to increased suicide risk is an important issue in biological psychiatry research. Gene expression studies on post mortem brains indicate extensive changes prior to a successful suicide attempt; however, proteomic studies are scarce. Thus, we performed a DIGE proteomic analysis of post mortem tissue samples from the prefrontal cortex and amygdala of suicide victims to identify protein changes and biomarker candidates of suicide. Among our matched spots we found 46 and 16 significant differences in the prefrontal cortex and amygdala, respectively; by using the industry standard t test and 1.3 fold change as cut off for significance. Because of the risk of false discoveries (FDR) in these data, we also made FDR adjustment by calculating the q-values for all the t tests performed and by using 0.06 and 0.4 as alpha thresholds we reduced the number of significant spots to 27 and 9 respectively. From these we identified 59 proteins in the cortex and 11 proteins in the amygdala. These proteins are related to biological functions and structures such as metabolism, the redox system, the cytoskeleton, synaptic function, and proteolysis. Thirteen of these proteins (CBR1, DPYSL2, EFHD2, FKBP4, GFAP, GLUL, HSPA8, NEFL, NEFM, PGAM1, PRDX6, SELENBP1 and VIM,) have already been suggested to be biomarkers of psychiatric disorders at protein or genome level. We also pointed out 9 proteins that changed in both the amygdala and the cortex, and from these, GFAP, INA, NEFL, NEFM and TUBA1 are interacting cytoskeletal proteins that have a functional connection to glutamate, GABA, and serotonin receptors. Moreover, ACTB, CTSD and GFAP displayed opposite changes in the two examined brain structures that might be a suitable characteristic for brain imaging studies. The opposite changes of ACTB, CTSD and GFAP in the two brain structures were validated by western blot analysis.
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Intraneuronal ?-amyloid and its interactions with proteins and subcellular organelles.
Electrophoresis
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Amyloidogenic aggregation and misfolding of proteins are linked to neurodegeneration. The mechanism of neurodegeneration in Alzheimers disease, which gives rise to severe neuronal death and memory loss, is not yet fully understood. The amyloid hypothesis remains the most accepted theory for the pathomechanism of the disease. It was suggested that ?-amyloid accumulation may play a key role in initiating the neurodegenerative processes. The recent intracellular ?-amyloid (iA?) hypothesis emphasizes the primary role of iA? to initiate the disease by interaction with cytoplasmic proteins and cell organelles, thereby triggering apoptosis. Sophisticated methods (proteomics, protein microarray, and super resolution microscopy) have been used for studying iA? interactions with proteins and membraneous structures. The present review summarizes the studies on the origin of iA? and the base of its neurotoxicity: interactions with cytosolic proteins and several cell organelles such as endoplasmic reticulum, endosomes, lysosomes, ribosomes, mitochondria, and the microtubular system.
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Proteome-wide study of endoplasmic reticulum stress induced by thapsigargin in N2a neuroblastoma cells.
Neurochem. Int.
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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.
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Conformational dynamics of titin PEVK explored with FRET spectroscopy.
Biophys. J.
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The proline-, glutamate-, valine-, and lysine-rich (PEVK) domain of the giant muscle protein titin is thought to be an intrinsically unstructured random-coil segment. Various observations suggest, however, that the domain may not be completely devoid of internal interactions and structural features. To test the validity of random polymer models for PEVK, we determined the mean end-to-end distances of an 11- and a 21-residue synthetic PEVK peptide, calculated from the efficiency of the fluorescence resonance energy transfer (FRET) between an N-terminal intrinsic tryptophan donor and a synthetically added C-terminal IAEDANS acceptor obtained in steady-state and time-resolved experiments. We find that the contour-length scaling of mean end-to-end distance deviates from predictions of a purely statistical polymer chain. Furthermore, the addition of guanidine hydrochloride decreased, whereas the addition of salt increased the FRET efficiency, pointing at the disruption of structure-stabilizing interactions. Increasing temperature between 10 and 50°C increased the normalized FRET efficiency in both peptides but with different trajectories, indicating that their elasticity and conformational stability are different. Simulations suggest that whereas the short PEVK peptide displays an overall random structure, the long PEVK peptide retains residual, loose helical configurations. Transitions in the local structure and dynamics of the PEVK domain may play a role in the modulation of passive muscle mechanics.
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Increased tau phosphorylation and impaired presynaptic function in hypertriglyceridemic ApoB-100 transgenic mice.
PLoS ONE
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ApoB-100 is the major protein component of cholesterol- and triglyceride-rich LDL and VLDL lipoproteins in the serum. Previously, we generated and partially described transgenic mice overexpressing the human ApoB-100 protein. Here, we further characterize this transgenic strain in order to reveal a possible link between hypeprlipidemia and neurodegeneration.
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Synthesis and cytotoxic activity of 4-N-carboxybutyl-5-fluorocytosyl-Arg-Gln-Trp-Arg-Arg-Trp-Trp-Gln-Arg-NH?.
Bioorg. Med. Chem. Lett.
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The chemical synthesis of 4-N-carboxybutyl-5-fluorocytosine (II) in solution phase starting from 5-fluorocytosine and the solid phase synthesis of Arg-Gln-Trp-Arg-Arg-Trp-Trp-Gln-Arg-NH(2) attached to the 4-N-carboxybutyl-5-fluorocytosine residue at the N-terminus of the peptide (III) via peptide bond formation is reported. The target compound exhibited a significant cytotoxic activity against a culture of HepG2 cells. In addition our results demonstrated that this new compound affect cell viability, produce mitochondrial dysfunction as well as interfere with intracellular calcium homeostasis control; leading to cell malfunction and death.
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GluA1 phosphorylation alters evoked firing pattern in vivo.
Neural Plast.
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AMPA and NMDA receptors convey fast synaptic transmission in the CNS. Their relative contribution to synaptic output and phosphorylation state regulate synaptic plasticity. The AMPA receptor subunit GluA1 is central in synaptic plasticity. Phosphorylation of GluA1 regulates channel properties and trafficking. The firing rate averaged over several hundred ms is used to monitor cellular input. However, plasticity requires the timing of spiking within a few ms; therefore, it is important to understand how phosphorylation governs these events. Here, we investigate whether the GluA1 phosphorylation (p-GluA1) alters the spiking patterns of CA1 cells in vivo. The antidepressant Tianeptine was used for inducing p-GluA1, which resulted in enhanced AMPA-evoked spiking. By comparing the spiking patterns of AMPA-evoked activity with matched firing rates, we show that the spike-trains after Tianeptine application show characteristic features, distinguishing from spike-trains triggered by strong AMPA stimulation. The interspike-interval distributions are different between the two groups, suggesting that neuronal output may differ when new inputs are activated compared to increasing the gain of previously activated receptors. Furthermore, we also show that NMDA evokes spiking with different patterns to AMPA spike-trains. These results support the role of the modulation of NMDAR/AMPAR ratio and p-GluA1 in plasticity and temporal coding.
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A novel method for the rapid determination of beta-amyloid toxicity on acute hippocampal slices using MTT and LDH assays.
Brain Res. Bull.
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It is difficult task to measure precisely the toxic effect of beta-amyloid (A? 1-42) peptides and also the protective effect of novel drug candidates against A?-peptides. The widely used MTT-assay in cell lines or primary cell cultures could be insensitive against A?-peptides. We describe here an easy and relevant method for testing A? 1-42 toxicity on acute hippocampal slices derived from rat. Brain slice viability in different conditions was measured using MTT and LDH assays. The concomitant use of these two assays can give detailed and relevant results on the toxic effect of A? 1-42 in oxygen-glucose deprived (OGD) acute brain slice model. Both assays are capable of quantifying tissue viability by measuring optical density (OD). We found that simultaneous application of OGD and A? 1-42 treatment induced a more intensive decrease in hippocampal slice viability than their separate effects. The use of MTT and LDH assay for quantifying brain slice viability proved to be an easy ex vivo method for investigating A? toxicity. Testing brain slices is more relevant in Alzheimers Disease research than using in vitro cell cultures, due to maintenance of the three dimensional cellular network, the cell variability and intact cell connections.
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