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Phillip J. Robinson - One of the best experts on this subject based on the ideXlab platform.
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Quantitative analysis of activity-dependent phospho-signalling in Synaptosomes and hippocampal neurons.
2019Co-Authors: Kasper Engholm-keller, Ashley J. Waardenberg, Johannes A. Müller, Jesse R. Wark, Rowena N. Fernando, Jonathan W. Arthur, Phillip J. Robinson, Dirk Dietrich, Susanne Schoch, Mark E. GrahamAbstract:(A) The experimental workflow started with either Synaptosomes from the whole brain of adult rats or cultured primary hippocampal neurons. Shown is a schematic of the stimulation protocol for Synaptosomes and neurons and the time points examined. Only the Synaptosomes were repolarized by including a centrifugation and solution exchange step and were studied for the temporal evolution of phospho-signalling. The lysed samples were digested, enriched for phosphopeptides, analyzed by quantitative phosphoproteomics, and processed using a bioinformatics approach that included determination of inferred protein kinase activity using KinSwing. (B) Principal component analysis of the processed time course data for 76 mM KCl stimulation. All six biological replicates for each time point are shown in the same colour. Counts of the phosphopeptides and phosphoproteins identified are shown for (C) Synaptosomes and (D) primary hippocampal neurons. The counts were calculated with and without filtering for phosphorylation site localization and quantification confidence. The phosphopeptide data are available in S1 Table. The synaptosome data are the result of six independent experiments for each stimulation condition (20 mM and 76 mM KCl). The cultured hippocampal neuron data are from three independent experiments. HILIC, hydrophilic interaction liquid chromatography; HPLC, high-performance liquid chromatography; IMAC, immobilized metal affinity chromatography; LC-MS/MS, liquid chromatography–tandem mass spectrometry; TMT, tandem mass tag.
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Comparison of activity-dependent changes in Synaptosomes and cultured neurons identifies bassoon as a major target of phospho-signalling.
2019Co-Authors: Kasper Engholm-keller, Ashley J. Waardenberg, Johannes A. Müller, Jesse R. Wark, Rowena N. Fernando, Jonathan W. Arthur, Phillip J. Robinson, Dirk Dietrich, Susanne Schoch, Mark E. GrahamAbstract:(A) Plot of log2(76 mM KCl 10-s-stimulated intensity/control intensity) for selected phosphopeptides from cultured hippocampal neurons versus whole-brain Synaptosomes. The phosphopeptides compared were required to be from well-established presynaptic proteins. That is, they were exclusively active zone scaffold proteins or synaptic vesicle–associated proteins. Thus, by default, all postsynaptic and ubiquitous proteins were excluded. Solid circles are phosphopeptides significantly regulated in both types of samples. Open circles are phosphopeptides significantly regulated in at least one sample. Selected solid circles have a red outline and are labelled with a gene name. Two specific synapsin 1 phosphorylation sites are featured. Underlying data for this figure can be found in S1 Data. (B) Heat map of bassoon log2(stimulated intensity/control intensity) values across time for significantly regulated phosphopeptides from Synaptosomes and hippocampal neurons using the indicated colour scale. Note that some log2 fold changes are outside of the colour scale range. The chosen scale was intended to provide greater emphasis for low-moderate fold changes throughout this work, even though some signals will be saturated. See (A) for an example of the data shown on a larger log2 scale. The bassoon domain structure of UniProt accession O88778, obtained from Pfam [34], is shown with accurate phosphorylation site positions. Synaptosome data were required to have significant up-/down-regulation at one or more time points. For hippocampal neurons, “ns” means not significantly regulated. Phosphorylation site positions are from bassoon accessions O8878 and G3V984 (see S1 Table). (C) A protein localization and interaction network for selected protein components of the active zone scaffold, synaptic vesicles, postsynaptic density, and glutamate receptor proteins. The log2 intensities for phosphopeptides after 10 s of 76 mM KCl stimulation of hippocampal cultured neurons were summed for each protein. This value was used to scale the letter size of the gene name in the protein network. Experimentally verified protein interactions (STRING) are shown as red edges. (D) Graphs of the number of significantly regulated phosphorylation sites against the protein length (amino acid residues) for Synaptosomes (upper) and hippocampal neurons (lower). Proteins with relative high numbers of regulated phosphorylation sites are labelled by their gene name. Proteins proposed to be presynaptic signal integrators have blue labels. There was no linear correlation between the parameters in either graph of synaptosome (R2 = 0.06) or neuronal data (R2 = 0.04). The synaptosome data are the result of six independent experiments for each stimulation condition (20 mM and 76 mM KCl). The cultured hippocampal neuron data are from three independent experiments. Underlying data for this figure can be found in S1 Data.
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Validation of stimulus and poststimulus phospho-signalling.
2019Co-Authors: Kasper Engholm-keller, Ashley J. Waardenberg, Johannes A. Müller, Jesse R. Wark, Rowena N. Fernando, Jonathan W. Arthur, Phillip J. Robinson, Dirk Dietrich, Susanne Schoch, Mark E. GrahamAbstract:(A) Heat map of significantly regulated phosphorylation sites for syn1 (upper) and dyn1 (lower) from 20 mM and 76 mM KCl stimulated Synaptosomes (left, n = 6) and a single 10-s time point of 76 mM KCl stimulated hippocampal neurons (right, “ns” means not significantly regulated, n = 3). The log2(stimulated intensity/control intensity) is shown using the same colour scale as Fig 2B. Note: phospho-S662 in syn1 was detected with differential regulation from 1a and 1b isoforms. The synaptosome data are the result of six independent experiments for each stimulation condition (20 mM and 76 mM KCl). The cultured hippocampal neuron data are from three independent experiments. (B) Representative western blots of syn1-pS603, syn1-pS62+S67, dyn1-pS774, and β-actin loading control for 76 mM KCl depolarized and repolarized Synaptosomes. Bar graphs of the densitometry of the western blots, after correction for loading, are shown (below). The intensities were normalized to the control (“Cont.”)/mock stimulation. The bar graphs show the mean and standard deviation of 3 (syn1 pS603), 4 (dyn1 S774), or 5 (syn1 S62+S67) independent experiments. Statistical significance was determined by one-way analysis of variance with Dunnett’s post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; in (B), “ns” means “not significant”. P = 0.0005 using Student t test to compare dyn1-pS774 intensity at 10 s versus control and P = 0.17 when adjusted for multiple comparisons (time points). The heat map rows in (A) for the sites examined in (B) are boxed. Underlying data for this figure can be found in S1 Data. (C) Domain structure of Syn1 using A-E domain naming [39] and Pfam evolutionarily conserved domains [34] with the activity-dependent phosphorylation site positions indicated. dyn1, dynamin 1; syn1, synapsin 1.
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A rapid Percoll gradient procedure for preparation of Synaptosomes
Nature protocols, 2008Co-Authors: Peter R Dunkley, Paula E Jarvie, Phillip J. RobinsonAbstract:Homogenization of fresh brain tissue in isotonic medium shears plasma membranes causing nerve terminals to become separated from their axons and postsynaptic connections. The nerve terminal membranes then reseal to form Synaptosomes. The discontinuous Percoll gradient procedure described here is designed to isolate Synaptosomes from brain homogenates in the minimum time to allow functional experiments to be performed. Synaptosomes are isolated using a medium-speed centrifuge, while maintaining isotonic conditions and minimizing mechanically damaging resuspension steps. This protocol has advantages over other procedures in terms of speed and by producing relatively homogeneous Synaptosomes, minimizing the presence of synaptic and glial plasma membranes and extrasynaptosomal mitochondria. The purified Synaptosomes are viable and take up and release neurotransmitters very efficiently. A typical yield of Synaptosomes is between 2.5 and 4 mg of synaptosomal protein per gram rat brain. The procedure takes approximately 1 h from homogenization of the brain until collection of the synaptosomal suspension from the Percoll gradient.
Mariepaule Roisin - One of the best experts on this subject based on the ideXlab platform.
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subcellular fractionation on percoll gradient of mossy fiber Synaptosomes morphological and biochemical characterization in control and degranulated rat hippocampus
Journal of Neurochemistry, 2008Co-Authors: Philippe Taupin, Sylvie Zini, F Cesselin, Yezekiel Benari, Mariepaule RoisinAbstract:A method for preparation of hippocampal mossy fiber Synaptosomes directly from the postnuclear pellet is presented. This method represents an adaptation of that previously described for the isolation of Synaptosomes by centrifugation through Percoll gradients directly from the supernatant fraction. We have characterized by electron microscopy two fractions, PII and PIII, enriched in mossy fiber Synaptosomes; fraction PIII had 75% mossy fiber Synaptosomes with well-preserved morphology (large size 3 microns, complex morphology, high synaptic vesicle density, multisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integrity of Synaptosomes was preserved. Compared with the other synaptosomal fractions, these fractions showed greater levels of dynorphin A (1-8) immunoreactivity and endogenous zinc, which are particularly concentrated in hippocampal mossy fiber terminals. Furthermore, we prepared Synaptosomes from adult hippocampus after neonatal irradiation, which destroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that the rapid Percoll procedure is convenient for the purification of mossy fiber Synaptosomes.
Peter R Dunkley - One of the best experts on this subject based on the ideXlab platform.
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A rapid Percoll gradient procedure for preparation of Synaptosomes
Nature protocols, 2008Co-Authors: Peter R Dunkley, Paula E Jarvie, Phillip J. RobinsonAbstract:Homogenization of fresh brain tissue in isotonic medium shears plasma membranes causing nerve terminals to become separated from their axons and postsynaptic connections. The nerve terminal membranes then reseal to form Synaptosomes. The discontinuous Percoll gradient procedure described here is designed to isolate Synaptosomes from brain homogenates in the minimum time to allow functional experiments to be performed. Synaptosomes are isolated using a medium-speed centrifuge, while maintaining isotonic conditions and minimizing mechanically damaging resuspension steps. This protocol has advantages over other procedures in terms of speed and by producing relatively homogeneous Synaptosomes, minimizing the presence of synaptic and glial plasma membranes and extrasynaptosomal mitochondria. The purified Synaptosomes are viable and take up and release neurotransmitters very efficiently. A typical yield of Synaptosomes is between 2.5 and 4 mg of synaptosomal protein per gram rat brain. The procedure takes approximately 1 h from homogenization of the brain until collection of the synaptosomal suspension from the Percoll gradient.
Allan D Butterfield - One of the best experts on this subject based on the ideXlab platform.
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in vivo protection of Synaptosomes by ferulic acid ethyl ester faee from oxidative stress mediated by 2 2 azobis 2 amidino propane dihydrochloride aaph or fe2 h2o2 insight into mechanisms of neuroprotection and relevance to oxidative stress related n
Neurochemistry International, 2006Co-Authors: Gururaj Joshi, Marzia Perluigi, Rukhsana Sultana, Ravagna Agrippino, Vittorio Calabrese, Allan D ButterfieldAbstract:Ferulic acid ethyl ester (FAEE) is an ester derivative of ferulic acid, the latter known for its anti-inflammatory and antioxidant properties. Previous studies from our laboratory have shown that ferulic acid protects synaptosomal membrane system and neuronal cell culture systems against hydroxyl and peroxyl radical oxidation. FAEE is lipophilic and is able to penetrate lipid bilayer. Previous studies reported that FAEE reduces Alzheimer’s amyloid b peptide Ab(1‐42)-induced oxidative stress and cytotoxicity in neuronal cell culture by direct radical scavenging and by inducing certain antioxidant proteins. In the present study we tested the hypothesis that FAEE would provide neuroprotection against free radical oxidative stress in vivo. Synaptosomes were isolated from the gerbils that were previously injected intraperitoneally (i.p.) with FAEE or DMSO and were treated with oxidants, Fe 2+ /H2O2 or 2,2-azobis(2-amidino-propane)dihydrochloride (AAPH). Synaptosomes isolated from the gerbil previously injected i.p. with FAEE and treated with Fe 2+ /H2O2 and AAPH showed significant reduction in reactive oxygen species (ROS), levels of protein carbonyl, protein bound 4-hydroxynonenal (HNE, a lipid peroxidation product), and 3-nitrotyrosine (3-NT, another marker of protein oxidation formed by reaction of tyrosine residues with peroxynitrite) compared to Fe 2+ /H2O2 or AAPH induced oxidative stress in synapotosomes isolated from the brain of gerbils that were previously injected with DMSO. The Synaptosomes isolated from gerbil pre-injected with FAEE and subsequently treated with AAPH or Fe 2+ /H2O2 showed induction of heme oxygenase (HO-1) and heat shock protein 70 (HSP-70) but reduced inducible nitric oxide synthase (iNOS) levels. These results are discussed with reference to potential use of this lipophilic antioxidant phenolic compound in the treatment of oxidative stress-related neurodegenerative disorders. # 2005 Elsevier Ltd. All rights reserved.
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beneficial effects of dietary restriction on cerebral cortical synaptic terminals preservation of glucose and glutamate transport and mitochondrial function after exposure to amyloid β peptide iron and 3 nitropropionic acid
Journal of Neurochemistry, 2001Co-Authors: Arzu Ersoz, Allan D Butterfield, Mark P MattsonAbstract:Abstract : Recent studies have shown that rats and mice maintained on a dietary restriction (DR) regimen exhibit increased resistance of neurons to excitotoxic, oxidative, and metabolic insults in experimental models of Alzheimer's, Parkinson's, and Huntington's diseases and stroke. Because synaptic terminals are sites where the neurodegenerative process may begin in such neurodegenerative disorders, we determined the effects of DR on synaptic homeostasis and vulnerability to oxidative and metabolic insults. Basal levels of glucose uptake were similar in cerebral cortical Synaptosomes from rats maintained on DR for 3 months compared with Synaptosomes from rats fed ad libitum. Exposure of Synaptosomes to oxidative insults (amyloid β-peptide and Fe2+) and a metabolic insult (the mitochondrial toxin 3-nitropropionic acid) resulted in decreased levels of glucose uptake. Impairment of glucose uptake following oxidative and metabolic insults was significantly attenuated in Synaptosomes from rats maintained on DR. DR was also effective in protecting Synaptosomes against oxidative and metabolic impairment of glutamate uptake. Loss of mitochondrial function caused by oxidative and metabolic insults, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated in Synaptosomes from rats maintained on DR. Levels of the stress proteins HSP-70 and GRP-78 were increased in Synaptosomes from DR rats, consistent with previous data suggesting that the neuroprotective mechanism of DR involves a “preconditioning” effect. Collectively, our data provide the first evidence that DR can alter synaptic homeostasis in a manner that enhances the ability of synapses to withstand adversity.
Susan Wonnacott - One of the best experts on this subject based on the ideXlab platform.
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α bungarotoxin sensitive nicotinic receptors indirectly modulate 3h dopamine release in rat striatal slices via glutamate release
Molecular Pharmacology, 2000Co-Authors: Sergio Kaiser, Susan WonnacottAbstract:Nicotinic agonists elicit the release of dopamine from striatal Synaptosomes by acting on presynaptic nicotinic acetylcholine receptors (nAChRs) on dopamine nerve terminals. Both α3β2* and α4β2 nAChR subtypes (but not α7* nAChRs) have been implicated. Here, we compared nAChR-evoked [3H]dopamine release from rat striatal synaptosome and slice preparations by using the nicotinic agonist anatoxin-a. In the more integral slice preparation, the concentration-response curve for anatoxin-a-evoked [3H]dopamine release was best fitted to a two-site model, giving EC50 values of 241 nM and 5.1 μM, whereas only the higher-affinity component was observed in synaptosome preparations (EC50 = 134 nM). Responses to a high concentration of anatoxin-a (25 μM) in slices (but not in Synaptosomes) were partially blocked by ionotropic glutamate receptor antagonists (kynurenic acid, 6,7-dinitroquinoxaline-2,3-dione) and by α7*-selective nAChR antagonists (α-bungarotoxin, α-conotoxin-ImI, methyllycaconitine) in a nonadditive manner. In contrast, the α3β2-selective nAChR antagonist α-conotoxin-MII partially inhibited [3H]dopamine release from both slice and synaptosome preparations, stimulated with both low (1 μM) and high (25 μM) concentrations of anatoxin-a. Antagonism by α-conotoxin-MII was additive with that of α7*-selective antagonists. These data support a model in which α7* nAChRs on striatal glutamate terminals elicit glutamate release, which in turn acts at ionotropic glutamate receptors on dopamine terminals to stimulate dopamine release. In addition, non-α7* nAChRs on dopamine terminals also stimulate dopamine release. These observations have implications for the complex cholinergic modulation of inputs onto the major efferent neurons of the striatum.
