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Biochimica Et Biophysica Acta Nov 1996
Review
Topics: Energy Metabolism; Ion Transport; Synaptosomes
PubMed: 8950370
DOI: 10.1016/s0005-2728(96)00103-x -
British Journal of Pharmacology May 2015Methcathinone (MCAT) is a potent monoamine releaser and parent compound to emerging drugs of abuse including mephedrone (4-CH3 MCAT), the para-methyl analogue of MCAT....
BACKGROUND AND PURPOSE
Methcathinone (MCAT) is a potent monoamine releaser and parent compound to emerging drugs of abuse including mephedrone (4-CH3 MCAT), the para-methyl analogue of MCAT. This study examined quantitative structure-activity relationships (QSAR) for MCAT and six para-substituted MCAT analogues on (a) in vitro potency to promote monoamine release via dopamine and serotonin transporters (DAT and SERT, respectively), and (b) in vivo modulation of intracranial self-stimulation (ICSS), a behavioural procedure used to evaluate abuse potential. Neurochemical and behavioural effects were correlated with steric (Es ), electronic (σp ) and lipophilic (πp ) parameters of the para substituents.
EXPERIMENTAL APPROACH
For neurochemical studies, drug effects on monoamine release through DAT and SERT were evaluated in rat brain synaptosomes. For behavioural studies, drug effects were tested in male Sprague-Dawley rats implanted with electrodes targeting the medial forebrain bundle and trained to lever-press for electrical brain stimulation.
KEY RESULTS
MCAT and all six para-substituted analogues increased monoamine release via DAT and SERT and dose- and time-dependently modulated ICSS. In vitro selectivity for DAT versus SERT correlated with in vivo efficacy to produce abuse-related ICSS facilitation. In addition, the Es values of the para substituents correlated with both selectivity for DAT versus SERT and magnitude of ICSS facilitation.
CONCLUSIONS AND IMPLICATIONS
Selectivity for DAT versus SERT in vitro is a key determinant of abuse-related ICSS facilitation by these MCAT analogues, and steric aspects of the para substituent of the MCAT scaffold (indicated by Es ) are key determinants of this selectivity.
Topics: Animals; Biogenic Monoamines; Dopamine Plasma Membrane Transport Proteins; Dose-Response Relationship, Drug; Electric Stimulation; Male; Propiophenones; Quantitative Structure-Activity Relationship; Rats; Self Stimulation; Serotonin Plasma Membrane Transport Proteins; Synaptosomes
PubMed: 25438806
DOI: 10.1111/bph.13030 -
Marine Drugs Mar 2014Brevenal is a ladder frame polyether produced by the dinoflagellate Karenia brevis. This organism is also responsible for the production of the neurotoxic compounds...
Brevenal is a ladder frame polyether produced by the dinoflagellate Karenia brevis. This organism is also responsible for the production of the neurotoxic compounds known as brevetoxins. Ingestion or inhalation of the brevetoxins leads to adverse effects such as gastrointestinal maladies and bronchoconstriction. Brevenal shows antagonistic behavior to the brevetoxins and shows beneficial attributes when administered alone. For example, in an asthmatic sheep model, brevenal has been shown to increase tracheal mucosal velocity, an attribute which has led to its development as a potential treatment for Cystic Fibrosis. The mechanism of action of brevenal is poorly understood and the exact binding site has not been elucidated. In an attempt to further understand the mechanism of action of brevenal and potentially develop a second generation drug candidate, a series of brevenal derivatives were prepared through modification of the aldehyde moiety. These derivatives include aliphatic, aromatic and heteroaromatic hydrazide derivatives. The brevenal derivatives were tested using in vitro synaptosome binding assays to determine the ability of the compounds to displace brevetoxin and brevenal from their native receptors. A sheep inhalation model was used to determine if instillation of the brevenal derivatives resulted in bronchoconstriction. Only small modifications were tolerated, with larger moieties leading to loss of affinity for the brevenal receptor and bronchoconstriction in the sheep model.
Topics: Administration, Inhalation; Animals; Binding Sites; Bronchoconstriction; Dinoflagellida; Disease Models, Animal; Ethers; Female; Marine Toxins; Oxocins; Polymers; Sheep; Structure-Activity Relationship; Synaptosomes
PubMed: 24686558
DOI: 10.3390/md12041839 -
ACS Chemical Neuroscience Aug 2019The dopamine transporter (DAT) serves a pivotal role in controlling dopamine (DA)-mediated neurotransmission by clearing DA from synaptic and perisynaptic spaces and...
The dopamine transporter (DAT) serves a pivotal role in controlling dopamine (DA)-mediated neurotransmission by clearing DA from synaptic and perisynaptic spaces and controlling its action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DAT to mediate their effects by enhancing extracellular DA concentrations. We previously identified a novel allosteric site in the related human serotonin transporter that lies outside the central substrate and inhibitor binding pocket. We used the hybrid structure based (HSB) method to screen for allosteric modulator molecules that target a similar site in DAT. We identified a compound, KM822, that was found to be a selective, noncompetitive inhibitor of DAT. We confirmed the structural determinants of KM822 allosteric binding within the allosteric site by structure/function and substituted cysteine scanning accessibility biotinylation experiments. In the in vitro cell-based assay and ex vivo in both rat striatal synaptosomal and slice preparations, KM822 was found to decrease the affinity of cocaine for DAT. The in vivo effects of KM822 on cocaine were tested on psychostimulant-associated behaviors in a planarian model where KM822 specifically inhibited the locomotion elicited by DAT-interacting stimulants amphetamine and cocaine. Overall, KM822 provides a unique opportunity as a molecular probe to examine allosteric modulation of DAT function.
Topics: Allosteric Regulation; Animals; Cocaine; Corpus Striatum; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; Humans; Male; Motor Activity; Planarians; Rats; Rats, Sprague-Dawley; Synaptosomes
PubMed: 31184115
DOI: 10.1021/acschemneuro.9b00262 -
PloS One 2015Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In...
Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson's disease (PD) and dementia with Lewy body (DLB), αSN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the αSN gene suggest a gain of function associated to aggregated αSN. We have conducted a proteomic screen to identify the αSN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human αSN monomers or oligomers as bait. The isolated αSN binding proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using Windows client application, Skyline Targeted Proteomic Environment. Data are available via ProteomeXchange with identifier PXD001462. To quantify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. We identified 10 proteins preferentially binding monomer, and 76 binding preferentially to oligomer and a group of 92 proteins not displaying any preferred conformation of αSN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer αSN interactors: Abl interactor 1, and myelin proteolipid protein, and oligomer interactors: glutamate decarboxylase 2, synapsin 1, glial fibrillary acidic protein, and VAMP-2. We demonstrate the existence of αSN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological αSN dependent processes.
Topics: Animals; Brain; Humans; Nerve Tissue Proteins; Protein Binding; Protein Multimerization; Proteomics; Swine; Synaptosomes; alpha-Synuclein
PubMed: 25659148
DOI: 10.1371/journal.pone.0116473 -
Biochemical and Biophysical Research... Jan 2023ALS2/alsin, the causative gene product for a number of juvenile recessive motor neuron diseases, acts as a guanine nucleotide exchange factor (GEF) for Rab5, regulating...
ALS2/alsin, the causative gene product for a number of juvenile recessive motor neuron diseases, acts as a guanine nucleotide exchange factor (GEF) for Rab5, regulating early endosome trafficking and maturation. It has been demonstrated that ALS2 forms a tetramer, and this oligomerization is essential for its GEF activity and endosomal localization in established cancer cells. However, despite that ALS2 deficiency is implicated in neurological diseases, neither the subcellular distribution of ALS2 nor the form of its complex in the central nervous system (CNS) has been investigated. In this study, we showed that ALS2 in the brain was enriched both in synaptosomal and cytosolic fractions, while those in the liver were almost exclusively present in cytosolic fraction by differential centrifugation. Gel filtration chromatography revealed that cytosolic ALS2 prepared both from the brain and liver formed a tetramer. Remarkably, synaptosomal ALS2 existed as a high-molecular weight complex in addition to a tetramer. Such complex was also observed not only in embryonic brain but also several neuronal and glial cultures, but not in fibroblast-derived cell lines. Thus, the high-molecular weight ALS2 complex represents a unique form of ALS2-homophilic oligomers in the CNS, which may play a role in the maintenance of neural function.
Topics: Animals; Mice; Amyotrophic Lateral Sclerosis; Brain; Central Nervous System; Endosomes; Guanine Nucleotide Exchange Factors; Molecular Weight; Synaptosomes
PubMed: 36459881
DOI: 10.1016/j.bbrc.2022.11.061 -
Journal of Neurochemistry Feb 2003Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the...
Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.
Topics: Animals; Biogenic Polyamines; Biological Transport; Brain; Brain Chemistry; Cells, Cultured; Hippocampus; In Vitro Techniques; Male; Neuroglia; Rats; Rats, Sprague-Dawley; Spermidine; Spermine; Synaptic Vesicles; Synaptosomes
PubMed: 12558981
DOI: 10.1046/j.1471-4159.2003.01558.x -
Cytometry. Part a : the Journal of the... Mar 2012Amyloid beta (Aβ) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In...
Amyloid beta (Aβ) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In cortical AD synapses, p-tau co-localizes with Aβ, but the Aβ and p-tau peptide species responsible for synaptic dysfunction and demise remains unclear. The present experiments were designed to use high-speed cell sorting techniques to purify synaptosome population based on size, and then extend the method to physically isolate Aβ-positive synaptosomes with the goal of understanding the nature of Aβ and tau pathology in AD synapses. To examine the purity of size-gated synaptosomes, samples were first gated on size; particles with sizes between 0.5 and 1.5 microns were collected. Electron microscopy documented a homogenous population of spherical particles with internal vesicles and synaptic densities. Next, size-gated synaptosomes positive for Aβ were collected by fluorescence activated sorting and then analyzed by immunoblotting techniques. Sorted Aβ-positive synaptosomes were enriched for amyloid precursor protein (APP) and for Aβ oligomers and aggregates; immunolabeling for p-tau showed a striking accumulation of p-tau aggregates compared to the original homogenate and purified synaptosomes. These results confirm co-localization of Aβ and p-tau within individual synaptic terminals and provide proof of concept for the utility of flow sorting synaptosomes.
Topics: Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Animals; Cerebral Cortex; Female; Flow Cytometry; Humans; Male; Mice; Microscopy, Electron; Presynaptic Terminals; Synaptosomes; tau Proteins
PubMed: 22213704
DOI: 10.1002/cyto.a.22009 -
Journal of Neurochemistry Dec 2011Mitochondria are key contributors to many forms of cell death including those resulting from neonatal hypoxic-ischemic brain injury. Mice have become increasingly...
Mitochondria are key contributors to many forms of cell death including those resulting from neonatal hypoxic-ischemic brain injury. Mice have become increasingly popular in studies of brain injury, but there are few reports evaluating mitochondrial isolation procedures for the neonatal mouse brain. Using evaluation of respiratory activity, marker enzymes, western blotting and electron microscopy, we have compared a previously published procedure for isolating mitochondria from neonatal mouse brain (method A) with procedures adapted from those for adult rats (method B) and neonatal rats (method C). All three procedures use Percoll density gradient centrifugation as a key step in the isolation but differ in many aspects of the fractionation procedure and the solutions used during fractionation. Methods A and B both produced highly enriched fractions of well-coupled mitochondria with high rates of respiratory activity. The fraction from method C exhibited less preservation of respiratory properties and was more contaminated with other subcellular components. Method A offers the advantage of being more rapid and producing larger mitochondrial yields making it useful for routine applications. However, method B produced mitochondria that were less contaminated with synaptosomes and associated cytosolic components that suits studies that have a requirement for higher mitochondrial purification.
Topics: Adenosine Diphosphate; Animals; Animals, Newborn; Brain; Electron Transport Complex IV; L-Lactate Dehydrogenase; Mice; Mice, Inbred C57BL; Microscopy, Electron; Mitochondria; Subcellular Fractions; Synaptosomes
PubMed: 21985402
DOI: 10.1111/j.1471-4159.2011.07525.x -
European Journal of Biochemistry Aug 2004alpha-Synuclein exists in two different compartments in vivo-- correspondingly existing as two different forms: a membrane-bound form that is predominantly alpha-helical...
alpha-Synuclein exists in two different compartments in vivo-- correspondingly existing as two different forms: a membrane-bound form that is predominantly alpha-helical and a cytosolic form that is randomly structured. It has been suggested that these environmental and structural differences may play a role in aggregation propensity and development of pathological lesions observed in Parkinson's disease (PD). Such effects may be accentuated by mutations observed in familial PD kindreds. In order to test this hypothesis, wild-type and A53T mutant alpha-synuclein interactions with rat brain synaptosomal membranes were examined. Previous data has demonstrated that the A30P mutant has defective lipid binding and therefore was not examined in this study. Electron microscopy demonstrated that wild-type alpha-synuclein fibrillogenesis is accelerated in the presence of synaptosomal membranes whereas the A53T alpha-synuclein fibrillogenesis is inhibited under the same conditions. These results suggested that subtle sequence changes in alpha-synuclein could significantly alter interaction with membrane bilayers. Fluorescence and absorption spectroscopy using environment sensitive probes demonstrated variations in the inherent lipid properties in the presence and absence of alpha-synuclein. Addition of wild-type alpha-synuclein to synaptosomes did not significantly alter the membrane fluidity at either the fatty acyl chains or headgroup space, suggesting that synaptosomes have a high capacity for alpha-synuclein binding. In contrast, synaptosomal membrane fluidity was decreased by A53T alpha-synuclein binding with concomitant packing of the lipid headgroups. These results suggest that alterations in alpha-synuclein-lipid interactions may contribute to physiological changes detected in early onset PD.
Topics: Alanine; Animals; Circular Dichroism; Dimerization; Fatty Acids; Humans; Microscopy, Electron; Mutation; Nerve Tissue Proteins; Protein Binding; Protein Structure, Quaternary; Rats; Spectrometry, Fluorescence; Surface Properties; Synaptic Membranes; Synaptosomes; Synucleins; Tyrosine; alpha-Synuclein
PubMed: 15265037
DOI: 10.1111/j.1432-1033.2004.04250.x