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Current Microbiology Sep 2016It is well established now that neuronal dysfunction rather than structural damage may be responsible for the development of rabies. In order to explore the underlying...
It is well established now that neuronal dysfunction rather than structural damage may be responsible for the development of rabies. In order to explore the underlying mechanisms in rabies virus (RABV) and synaptic dysfunctions, a quantitative proteome profiling was carried out on synaptosome samples from mice hippocampus. Synaptosome samples from mice hippocampus were isolated and confirmed by Western blot and transmission electron microscopy. Synaptosome protein content changes were quantitatively detected by Nano-LC-MS/MS. Protein functions were classified by the Gene Ontology (GO) and KEGG pathway. PSICQUIC was used to create a network. MCODE algorithm was applied to obtain subnetworks. Of these protein changes, 45 were upregulated and 14 were downregulated following RABV infection relative to non-infected (mock) synaptosomes. 28 proteins were unique to mock treatment and 12 were unique to RABV treatment. Proteins related to metabolism and synaptic vesicle showed the most changes in expression levels. Furthermore, protein-protein interaction (PPI) networks revealed that several key biological processes related to synaptic functions potentially were modulated by RABV, including energy metabolism, cytoskeleton organization, and synaptic transmission. These data will be useful for better understanding of neuronal dysfunction of rabies and provide the foundation for future research.
Topics: Animals; Electrophoresis, Polyacrylamide Gel; Gene Expression Profiling; Hippocampus; Humans; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Proteome; Rabies; Rabies virus; Stress, Physiological; Synaptosomes
PubMed: 27155843
DOI: 10.1007/s00284-016-1061-5 -
Toxins Oct 2019Ciguatera is the term for poisoning resulting from eating fish from tropical or subtropical regions. The causative toxins collectively named ciguatoxins (CTXs) widely...
Ciguatera is the term for poisoning resulting from eating fish from tropical or subtropical regions. The causative toxins collectively named ciguatoxins (CTXs) widely differ in structures depending on their geographic origins, which range from the Pacific Ocean and the Indian Ocean to the Caribbean Sea. Neurotoxic shellfish poisoning (NSP) is caused by the ingestion of bivalve shellfish contaminated with brevetoxins (BTXs). Structurally, both CTXs and BTXs consist of fused ether rings aligned in a ladder shape. Pharmacologically, they bind at the same site (site-5) of voltage-gated sodium channels. However, the great structural diversity and the rare availability of reference toxins hinder LC-MS and ELISA methods, which operate on structure-based recognition. In this study, we prepared a chemiluminescent ligand, acridinium BTXB2 (ABTX), and tested its suitability for use in competitive binding assays to detect CTXs and BTXs. The affinity of ABTX to the rat brain synaptosome estimated by (1.66 pM) was approximately two-fold higher than that of PbTx-3 (BTX3). In addition, the equilibrium dissociation constant () was 0.84 nM, the maximum number of binding was 6.76 pmol toxin/mg protein, and the detection limit was 1.4 amol. The assays performed on samples spiked with CTX3C or BTXB4 (-palmitoylBTXB2) at 0.2-1.0 ng CTX/g fish flesh, and 200-800 ng BTXB4/g shellfish showed a linear relationship between the theoretical and observed toxin amounts.
Topics: Animals; Binding, Competitive; Ciguatoxins; Fishes; Ligands; Luminescence; Luminescent Measurements; Male; Mytilus; Oxocins; Protein Binding; Rats, Sprague-Dawley; Rats, Wistar; Synaptosomes
PubMed: 31600892
DOI: 10.3390/toxins11100580 -
Synapse (New York, N.Y.) Mar 2019Allopregnanolone, an active metabolite of progesterone, has been reported to exhibit neuroprotective activity in several preclinical models. Considering that the...
Allopregnanolone, an active metabolite of progesterone, has been reported to exhibit neuroprotective activity in several preclinical models. Considering that the excitotoxicity caused by excessive glutamate is implicated in many brain disorders, the effect of allopregnanolone on glutamate release in rat cerebrocortical nerve terminals and possible underlying mechanism were investigated. We observed that allopregnanolone inhibited 4-aminopyridine (4-AP)-evoked glutamate release, and this inhibition was prevented by chelating the extracellular Ca ions and the vesicular transporter inhibitor. Allopregnanolone reduced the elevation of 4-AP-evoked intrasynaptosomal Ca levels, but did not affect the synaptosomal membrane potential. In the presence of N-, P/Q-, and R-type channel blockers, allopregnanolone-mediated inhibition of 4-AP-evoked glutamate release was markedly reduced; however, the intracellular Ca -release inhibitors did not affect the allopregnanolone effect. Furthermore, allopregnanolone-mediated inhibition of 4-AP-evoked glutamate release was completely abolished in the synaptosomes pretreated with inhibitors of Ca /calmodulin, adenylate cyclase, and protein kinase A (PKA), namely calmidazolium, MDL12330A, and H89, respectively. Additionally, the allopregnanolone effect on evoked glutamate release was antagonized by the GABA receptor antagonist SR95531. Our data are the first to suggest that allopregnanolone reduce the Ca influx through N-, P/Q-, and R-type Ca channels, through the activation of GABA receptors present on cerebrocortical nerve terminals, subsequently suppressing the Ca -calmodulin/PKA cascade and decreasing 4-AP-evoked glutamate release.
Topics: 4-Aminopyridine; Animals; Calcium; Calcium Channel Blockers; Cerebral Cortex; Enzyme Inhibitors; Exocytosis; GABA Antagonists; Glutamic Acid; Imidazoles; Imines; Isoquinolines; Male; Neurons; Pregnanolone; Presynaptic Terminals; Protein Kinase Inhibitors; Pyridazines; Rats; Rats, Sprague-Dawley; Sulfonamides; Synaptosomes
PubMed: 30362283
DOI: 10.1002/syn.22076 -
Molecular Neurobiology Mar 2019Purinergic signaling is the main synaptic and non-synaptic signaling system in brain. ATP acts as a fast excitatory transmitter, while adenosine sets a global inhibitory...
Purinergic signaling is the main synaptic and non-synaptic signaling system in brain. ATP acts as a fast excitatory transmitter, while adenosine sets a global inhibitory tone within hippocampal neuronal networks. ATP and adenosine are interconnected by ectonucleotidase enzymes, which convert ATP to adenosine. Existing data point to the converging roles of ovarian steroids and purinergic signaling in synapse formation and refinement and synapse activity in the hippocampus. Therefore, in the present study, we have used enzyme histochemistry and expression analysis to obtain data on spatial distribution and expression of ecto-enzymes NTPDase1, NTPDase2, and ecto-5'-nucleotidase (eN) after removal of ovaries (OVX) and estradiol replacement (E2) in female rat hippocampus. The results show that target ectonucleotidases are predominantly localized in synapse-rich hippocampal layers. The most represented NTPDase in the hippocampal tissue is NTPDase2, being at the same time the mostly affected ectonucleotidase by OVX and E2. Specifically, OVX decreases the expression of NTPDase2 and eN, whereas E2 restores their expression to control level. Impact of OVX and E2 on ectonucleotidase expression was also examined in purified synaptosome (SYN) and gliosome (GLIO) fractions. Data reveal that SYN expresses NTPDase1 and NTPDase2, both of which are reduced following OVX and restored with E2. GLIO exhibits NTPDase2-mediated ATP hydrolysis, which falls in OVX, and recovers by E2. These changes in the activity occur without parallel changes in NTPDase2-protein abundance. The same holds for eN. The lack of correlation between NTPDase2 and eN activities and their respective protein abundances suggest a non-genomic mode of E2 action, which is studied further in primary astrocyte culture. Since ovarian steroids shape hippocampal synaptic networks and regulate ectonucleotidase activities, it is possible that cognitive deficits seen after ovary removal may arise from the loss of E2 modulatory actions on ectonucleotidase expression in the hippocampus.
Topics: 5'-Nucleotidase; Adenosine Triphosphatases; Animals; Antigens, CD; Apyrase; Estradiol; Female; Hippocampus; Neuroglia; Neurons; Ovariectomy; Rats; Rats, Wistar; Synapses; Synaptosomes
PubMed: 29978426
DOI: 10.1007/s12035-018-1217-3 -
Methods in Molecular Biology (Clifton,... 2017Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches....
Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved in isolating synaptosomes, SPMs, and SJCs from brain so that these preparations can be used with new technological advances to address many as yet unanswered questions about the synapse and its remarkable activities in neuronal cell communication.
Topics: Animals; Brain; Cell Fractionation; Membrane Proteins; Post-Synaptic Density; Presynaptic Terminals; Rats; Subcellular Fractions; Synaptic Membranes; Synaptosomes; Ultracentrifugation
PubMed: 27943187
DOI: 10.1007/978-1-4939-6688-2_9 -
Alcohol (Fayetteville, N.Y.) Jun 2019Alcohol hangover (AH) has been associated with oxidative stress and mitochondrial dysfunction. We herein postulate that AH-induced mitochondrial alterations can be due...
Alcohol hangover (AH) has been associated with oxidative stress and mitochondrial dysfunction. We herein postulate that AH-induced mitochondrial alterations can be due to a different pattern of response in synaptosomes and non-synaptic (NS) mitochondria. Mice received intraperitoneal (i.p.) injections of ethanol (3.8 g/kg) or saline and were sacrificed 6 h afterward. Brain cortex NS mitochondria and synaptosomes were isolated by Ficoll gradient. Oxygen consumption rates were measured in NS mitochondria and synaptosomes by high-resolution respirometry. Results showed that NS-synaptic mitochondria from AH animals presented a 26% decrease in malate-glutamate state 3 respiration, a 64% reduction in ATP content, 28-37% decrements in ATP production rates (malate-glutamate or succinate-dependent, respectively), and 44% inhibition in complex IV activity. No changes were observed in mitochondrial transmembrane potential (ΔΨ) or in UCP-2 expression in NS-mitochondria. Synaptosome respiration driving proton leak (in the presence of oligomycin), and spare respiratory capacity (percentage ratio between maximum and basal respiration) were 30% and 15% increased in hangover condition, respectively. Synaptosomal ATP content was 26% decreased, and ATP production rates were 40-55% decreased (malate-glutamate or succinate-dependent, respectively) in AH mice. In addition, a 24% decrease in ΔΨ and a 21% increase in UCP-2 protein expression were observed in synaptosomes from AH mice. Moreover, mitochondrial respiratory complexes I-III, II-III, and IV activities measured in synaptosomes from AH mice were decreased by 18%, 34%, and 50%, respectively. Results of this study reveal that alterations in bioenergetics status during AH could be mainly due to changes in mitochondrial function at the level of synapses.
Topics: Alcoholic Intoxication; Animals; Binge Drinking; Cerebral Cortex; Energy Metabolism; Ethanol; Male; Mice; Mitochondria; Synaptosomes
PubMed: 30385200
DOI: 10.1016/j.alcohol.2018.10.010 -
Cellular and Molecular Life Sciences :... Apr 2022Alzheimer's disease (AD) is characterized by progressive cognitive decline due to accumulating synaptic insults by toxic oligomers of amyloid beta (AβO) and tau (TauO)....
BACKGROUND
Alzheimer's disease (AD) is characterized by progressive cognitive decline due to accumulating synaptic insults by toxic oligomers of amyloid beta (AβO) and tau (TauO). There is growing consensus that preventing these oligomers from interacting with synapses might be an effective approach to treat AD. However, recent clinical trial failures suggest low effectiveness of targeting Aβ in late-stage AD. Researchers have redirected their attention toward TauO as the levels of this species increase later in disease pathogenesis. Here we show that AβO and TauO differentially target synapses and affect each other's binding dynamics.
METHODS
Binding of labeled, pre-formed Aβ and tau oligomers onto synaptosomes isolated from the hippocampus and frontal cortex of mouse and postmortem cognitively intact elderly human brains was evaluated using flow-cytometry and western blot analyses. Binding of labeled, pre-formed Aβ and tau oligomers onto mouse primary neurons was assessed using immunofluorescence assay. The synaptic dysfunction was measured by fluorescence analysis of single-synapse long-term potentiation (FASS-LTP) assay.
RESULTS
We demonstrated that higher TauO concentrations effectively outcompete AβO and become the prevailing synaptic-associated species. Conversely, high concentrations of AβO facilitate synaptic TauO recruitment. Immunofluorescence analyses of mouse primary cortical neurons confirmed differential synaptic binding dynamics of AβO and TauO. Moreover, in vivo experiments using old 3xTgAD mice ICV injected with either AβO or TauO fully supported these findings. Consistent with these observations, FASS-LTP analyses demonstrated that TauO-induced suppression of chemical LTP was exacerbated by AβO. Finally, predigestion with proteinase K abolished the ability of TauO to compete off AβO without affecting the ability of high AβO levels to increase synaptic TauO recruitment. Thus, unlike AβO, TauO effects on synaptosomes are hampered by the absence of protein substrate in the membrane.
CONCLUSIONS
These results introduce the concept that TauO become the main synaptotoxic species at late AD, thus supporting the hypothesis that TauO may be the most effective therapeutic target for clinically manifest AD.
Topics: Aged; Alzheimer Disease; Amyloid beta-Peptides; Hippocampus; Humans; Synapses; Synaptosomes
PubMed: 35377002
DOI: 10.1007/s00018-022-04255-9 -
Biochemistry. Biokhimiia Mar 2020Neurodegenerative diseases are accompanied by changes in the activity of thiamine mono- and diphosphate phosphatases, but molecular identification of these mammalian...
Neurodegenerative diseases are accompanied by changes in the activity of thiamine mono- and diphosphate phosphatases, but molecular identification of these mammalian enzymes is incomplete. In this work, the protein fraction of bovine brain synaptosomes displaying phosphatase activity toward thiamine derivatives was subjected to affinity chromatography on thiamine-Sepharose. Protein fractions eluted with thiamine (pH 7.4 or 5.6), NaCl, and urea were assayed for the phosphatase activity against thiamine monophosphate (ThMP), thiamine diphosphate (ThDP), and structurally similar purine nucleotides. Proteins in each fraction were identified by mass spectrometry using the SwissProt database for all organisms because of insufficient annotation of the bovine genome. Peptides of two annotated bacterial phosphatases, alkaline phosphatase L from the DING protein family and exopolyphosphatase, were identified in the acidic thiamine eluate. The abundance of peptides of alkaline phosphatase L and exopolyphosphatase in the eluted fractions correlated with ThMPase and ThDPase activities, respectively. The elution profiles of the ThMPase and ThDPase activities differed from the elution profiles of nucleotide phosphatases, thus indicating the specificity of these enzymes toward thiamine derivatives. The search for mammalian DING phosphatases in the eluates from thiamine-Sepharose revealed X-DING-CD4, mostly eluted by the acidic thiamine solution (pH 5.6). The identified exopolyphosphatase demonstrated structural similarity with apyrases possessing the ThDPase activity. The obtained results demonstrate that mammalian DING proteins and apyrases exhibit ThMPase and ThDPase activity, respectively.
Topics: Animals; Brain; Catalytic Domain; Cattle; Chromatography, Affinity; Diphosphates; Genome; Humans; Hydrogen-Ion Concentration; Phosphoric Monoester Hydrolases; Substrate Specificity; Synaptosomes; Thiamine; Thiamine Monophosphate; Thiamine Pyrophosphate; Urea
PubMed: 32564742
DOI: 10.1134/S000629792003013X -
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 -
Journal of Neurochemistry Mar 2017Food restriction (FR) and obesogenic (OB) diets are known to alter brain dopamine transmission and exert opposite modulatory effects on behavioral responsiveness to...
Food restriction (FR) and obesogenic (OB) diets are known to alter brain dopamine transmission and exert opposite modulatory effects on behavioral responsiveness to psychostimulant drugs of abuse. Mechanisms underlying these diet effects are not fully understood. In this study, we examined diet effects on expression and function of the dopamine transporter (DAT) in caudate-putamen (CPu), nucleus accumbens (NAc), and midbrain regions. Dopamine (DA) uptake by CPu, NAc or midbrain synapto(neuro)somes was measured in vitro with rotating disk electrode voltammetry or with [ H]DA uptake and was found to correlate with DAT surface expression, assessed by maximal [ H](-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane binding and surface biotinylation assays. FR and OB diets were both found to decrease DAT activity in CPu with a corresponding decrease in surface expression but had no effects in the NAc and midbrain. Diet treatments also affected sensitivity to insulin-induced enhancement of DA uptake, with FR producing an increase in CPu and NAc, likely mediated by an observed increase in insulin receptor expression, and OB producing a decrease in NAc. The increased expression of insulin receptor in NAc of FR rats was accompanied by increased DA D receptor expression, and the decreased DAT expression and function in CPu of OB rats was accompanied by decreased DA D receptor expression. These results are discussed as partial mechanistic underpinnings of diet-induced adaptations that contribute to altered behavioral sensitivity to psychostimulants that target the DAT.
Topics: Animals; Biotinylation; Body Weight; Brain Chemistry; Caloric Restriction; Caudate Nucleus; Diet; Dopamine; Dopamine Plasma Membrane Transport Proteins; Hypoglycemic Agents; Insulin; Male; Mesencephalon; Nucleus Accumbens; Obesity; Putamen; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D2; Synaptosomes
PubMed: 27973691
DOI: 10.1111/jnc.13930