Rat Synaptosomes Research Articles

Albiflorin Decreases Glutamate Release from Rat Cerebral Cortex Nerve Terminals (Synaptosomes) through Depressing P/Q-Type Calcium Channels and Protein Kinase A Activity.

The purpose of this study was to investigate whether and how albiflorin, a natural monoterpene glycoside, affects the release of glutamate, one of the most important neurotransmitters involved in neurotoxicity, from cerebrocortical nerve terminals (synaptosomes) in rats. The results showed that albiflorin reduced 4-aminopyridine (4-AP)-elicited glutamate release from synaptosomes, which was abrogated in the absence of extracellular Ca2+ or in the presence of the vesicular glutamate transporter inhibitor or a P/Q-type Ca2+ channel inhibitor, indicating a mechanism of action involving Ca2+-dependent depression of vesicular exocytotic glutamate release. Albiflorin failed to alter the increase in the fluorescence intensity of 3,3-diethylthiacarbocyanine iodide (DiSC3(5)), a membrane-potential-sensitive dye. In addition, the suppression of protein kinase A (PKA) abolished the effect of albiflorin on glutamate release. Albiflorin also reduced the phosphorylation of PKA and synaptosomal-associated protein of 25 kDa (SNAP-25) and synapsin I at PKA-specific residues, which correlated with decreased available synaptic vesicles. The results of transmission electron microscopy (TEM) also observed that albiflorin reduces the release competence of synaptic vesicles evoked by 4-AP in synaptosomes. In conclusion, by studying synaptosomally released glutamate, we suggested that albiflorin reduces vesicular exocytotic glutamate release by decreasing extracellular Ca2+ entry via P/Q-type Ca2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation.

Synaptic Vesicle-Related Proteins and Ubiquilin 2 in Cortical Synaptosomes Mediate Cognitive Impairment in Vascular Dementia Rats.

Synaptic dysfunction is considered the best neuropathological correlate of cognitive decline in vascular dementia (VaD). However, the alterations of synaptic proteins at the synaptosomal level in VaD remain unclear. In this study, a VaD model was established in male rats using bilateral common carotid artery occlusion (2VO). We performed a novel object recognition task to evaluate cognitive impairment. Immunohistochemistry was used to assess the expression of neuron-specific nuclear binding protein (NeuN). Brain synaptosomes were isolated and subjected to label-free proteomic analysis to quantify and identify the synaptic features of differentially expressed proteins (DEPs). Synaptic and hub protein expression was detected in synaptosomes using western blotting. We found that male rats with VaD presented impaired memory and decreased NeuN protein expression in the cortex. Synaptosome proteomic analysis revealed 604 DEPs, with 493 and 111 markedly downregulated and upregulated proteins, respectively. KEGG analysis and SynGO annotation revealed that the synaptic vesicle (SV) cycle may be a key signaling pathway in VaD. Hub protein analysis of the main nodes in the protein network identified UBQLN2 and SV-related proteins, including CLTC, SNAP91, AP2S1, CLTA, VAMP2, EPN1, UBQLN2, AP2B1, AP2A2, and AP2M1. Western blotting showed that the levels of SV2A, CLTC, AP2S1, and VAMP2 decreased in the synaptosomes of 2VO rats, while UBQLN2 expression significantly increased. Our results suggest that the disruption in the presynaptic SV cycle is a key event in male rats with VaD, which could be characterized by the aberrant SV2A expression. SV-related proteins and UBQLN2 may be essential in synaptopathy. Thus, targeting the specific molecular markers in synaptosomes may be critical for the development of mechanism-directed therapies against VaD.

Thiazolium salt mimics the non-coenzyme effects of vitamin B1 in rat synaptosomes

Long-term studies have confirmed a causal relationship between the development of neurodegenerative processes and vitamin B1 (thiamine) deficiency. However, the biochemical mechanisms underlying the high neurotropic activity of thiamine are not fully understood. At the same time, there is increasing evidence that vitamin B1, in addition to its coenzyme functions, may have non-coenzyme activities that are particularly important for neurons. To elucidate which effects of vitamin B1 in neurons are due to its coenzyme function and which are due to its non-coenzyme activity, we conducted a comparative study of the effects of thiamine and its derivative, 3-decyloxycarbonylmethyl-5-(2-hydroxyethyl)-4-methyl-1,3-thiazolium chloride (DMHT), on selected processes in synaptosomes. The ability of DMHT to effectively compete with thiamine for binding to thiamine-binding sites on the plasma membrane of synaptosomes and to participate as a substrate in the thiamine pyrophosphokinase reaction was demonstrated. In experiments with rat brain synaptosomes, unidirectional effects of DMHT and thiamine on the activity of the pyruvate dehydrogenase complex (PDC) and on the incorporation of radiolabeled [2–14C]pyruvate into acetylcholine were demonstrated. The observed effects of thiamine and DMHT on the modulation of acetylcholine synthesis can be explained by suggesting that both compounds, which interact in cells with enzymes of thiamine metabolism, are phosphorylated and exert an inhibitory/activating effect (concentration-dependent) on PDC activity by affecting the regulatory enzymes of the complex. Such effects were not observed in the presence of structural analogues of thiamine and DMHT without a 2-hydroxyethyl substituent at position 5 of the thiazolium cycle. The effect of DMHT on the plasma membrane Ca-ATPase was similar to that of thiamine. At the same time, DMHT showed high cytostatic activity against neuroblastoma cells.

Ex vivo investigation of betaine and boric acid function as preprotective agents on rat synaptosomes to be treated with Aβ (1-42).

Recent evidence suggests that ferroptosis, an iron-dependent cell death process, may be involved in Alzheimer's disease (AD) pathology. The study evaluated the therapeutic potential of betaine and boric acid (BA) pretreatment administered to rats for 21 days in AD. Then, the rats were sacrificed, and morphological and biochemical analyses were performed in brain tissues. Next, an ex vivo AD model was created by applying amyloid-β (Aβ1-42) to synaptosomes isolated from the brain tissues. Synaptosomes were analyzed with micrograph images, and protein and mRNA levels of ferroptotic markers were determined. Betaine and BA pretreatments did not cause any morphological and biochemical differences in the brain tissue. However, Aβ (1-42) administration in synaptosomes increased the levels of acyl-CoA synthetase long chain family member-4 (ACSL4), transferrin receptor-1 protein (TfR1), malondialdehyde (MDA), and 8-hydroxydeoxyguanosine (8-OHdG) and decreased the levels glutathione peroxidase-4 (GPx4) and glutathione (GSH). Moreover, ACSL4, GPx4, and TfR1 mRNA and protein levels were similar to the ELISA results. In contrast, betaine and BA pretreatments decreased the levels of ACSL4, TfR1, MDA, and 8-OHdG in synaptosomes incubated with Aβ1-42, while promoting increased levels of GPx4 and GSH. In addition, betaine and BA pretreatments completely reversed ACSL4, GPx4, and TfR1 mRNA and protein levels. Therefore, betaine and BA pretreatments may contribute to the prevention of neurodegenerative damage by supporting antiferroptotic activities.

Optimization and validation of RP-HPLC method for evaluation of pyrrole -containing hydrazones in isolated rat synaptosomes

In the current study, RP-HPLC method for evaluation of 2 pyrrol-based hydrazones in isolated rat synaptosomes was optimized and validated according to ICH guidelines. The synaptosomes were obtained by multiple centrifugations with Percoll reagent and the selected 2 N-pyrollyl hydrazide-hydrazones were incubated for 2 hours at 37 °C. Subsequently, the purified fraction through protein precipitation was analyzed by an UltiMateDionex 3000 DAD system with Purospher STAR C18 (4.6 x 12.5 cm, 5 µm) column. The mobile phase, consisting of acetonitrile: phosphate buffer pH 3.5: methanol in ratio 42/36/22 (v/v/v), was eluted isocratically with 0.8 mL/min flow rate. Afterwards, the novel and rapid method was applied effectively for identification of biotransformation in isolated rat brain synaptosomes. The analysis results indicated an absence of new peaks and persistent sample concentration which determined the stability of the analyzed ethyl 5-(4-bromophenyl)-1-(2-(2-(2-hydroxybenzylidene) hydrazinyl)-2- oxoethyl)-2-methyl- 1H-pyrrole-3-carboxylate (11b) and ethyl 5-(4-bromophenyl)-1-(3-(2-(2-hydroxybenzylidene)hydrazinyl)-3-oxopropyl)-2-methyl-1H-pyrrole-3-carboxylate (12b) and pointed these structures as promising.

Dopamine-Induced Oligomers of α-Synuclein Inhibit Amyloid Fibril Growth and Show No Toxicity.

Parkinson's disease is characterized by the selective death of dopaminergic neurons in the midbrain and accumulation of amyloid fibrils composed of α-synuclein (αSyn). Current treatment involves approaches that compensate the death of dopaminergic neurons by increasing the dopamine levels in remaining cells. However, dopamine can interact with αSyn and produce oligomeric species which were reported to be toxic in many models. We studied formation of dopamine-induced αSyn oligomers and their effect on the αSyn aggregation. Using the Thioflavin T kinetic assay, we have shown that small oligomers efficiently inhibit αSyn fibrillization by binding to fibril ends and blocking the elongation. Moreover, all the fractions of oligomer species proved to be nontoxic in the differentiated SH-SY5Y cell model and showed negligible neurotoxicity on isolated rat synaptosomes. The observed inhibition is an important insight in understanding of dopamine-enhancing therapy on Parkinson's disease progression and explains the absence of pathology enhancement.

Synapse-specific changes in Arc and BDNF in rat hippocampus following chronic temporal lobe epilepsy

Expression of immediate early genes (IEGs) in the brain is important for synaptic plasticity, and probably also in neurodegenerative conditions. To understand the cellular mechanisms of the underlying neuropathophysiological processes in epilepsy, we need to pinpoint changes in concentration of synaptic plasticity-related proteins at subsynaptic levels. In this study, we examined changes in synaptic expression of Activity-regulated cytoskeleton-associated (Arc) and Brai Derived Neurotrophic Factor (BDNF) in a rat model of kainate-induced temporal lobe epilepsy (TLE). Western blotting showed reduced concentrations of Arc and increased concentrations of BDNF in hippocampal synaptosomes in chronic TLE rats. Then, using quantitative electron microscopy, we found corresponding changes in subsynaptic regions in the hippocampus. Specifically, we detected significant reductions in the concentrations of Arc in the presynaptic terminal of Schaffer collateral glutamatergic synapses in the stratum radiatum of the CA1 area in TLE, as well as in their adjacent postsynaptic spines. In CA3, there was a significant reduction of Arc only in the presynaptic terminal cytoplasm. Conversely, in CA3, there was a significant increase in the expression of BDNF in the presynaptic terminal, but not in the postsynaptic spine. Significant increase in BDNF concentration in the CA1 postsynaptic density was also obtained. We hypothesize that the observed changes in Arc and BDNF may contribute to both cognitive impairment and increased excitotoxic vulnerability in chronic epilepsy.

Fluoxetine Enhances Synaptic Vesicle Trafficking and Energy Metabolism in the Hippocampus of Socially Isolated Rats

Chronic social isolation (CSIS)-induced alternation in synaptic and mitochondrial function of specific brain regions is associated with major depressive disorder (MDD). Despite the wide number of available medications, treating MDD remains an important challenge. Although fluoxetine (Flx) is the most frequently prescribed antidepressant, its mode of action is still unknown. To delineate affected molecular pathways of depressive-like behavior and identify potential targets upon Flx treatment, we performed a comparative proteomic analysis of hippocampal purified synaptic terminals (synaptosomes) of rats exposed to six weeks of CSIS, an animal model of depression, and/or followed by Flx treatment (lasting three weeks of six-week CSIS) to explore synaptic protein profile changes. Results showed that Flx in controls mainly induced decreased expression of proteins involved in energy metabolism and the redox system. CSIS led to increased expression of proteins that mainly participate in Ca2+/calmodulin-dependent protein kinase II (Camk2)-related neurotransmission, vesicle transport, and ubiquitination. Flx treatment of CSIS rats predominantly increased expression of proteins involved in synaptic vesicle trafficking (exocytosis and endocytosis), and energy metabolism (glycolytic and mitochondrial respiration). Overall, these Flx-regulated changes in synaptic and mitochondrial proteins of CSIS rats might be critical targets for new therapeutic development for the treatment of MDD.

In Silico Evaluation and In Vitro Determination of Neuroprotective and MAO-B Inhibitory Effects of Pyrrole-Based Hydrazones: A Therapeutic Approach to Parkinson’s Disease

Parkinson's disease is a huge burden in modern medicinal practice. A serious drawback of current antiparkinsonian therapy is its symptomatic nature. This directed our investigations in the search for new more potent derivatives, affecting not only the loss of dopaminergic neurons but also the oxidative damage of neuronal cells. Thus in vitro neurotoxicity and neuroprotective analysis on a group of N-pyrrolyl hydrazide-hydrazones were performed. The neurotoxicity of the target derivatives was determined on a subcellular level in isolated rat synaptosomes, mitochondria and microsomes determining their effect on cellular vitality, GSH depletion and MDA production. The neuroprotective effects of the evaluated hydrazones were measured in three models of induced oxidative stress: 6-OHDA, t-BuOOH and Fe2+/AA-induced lipid peroxidation. Molecular docking simulations along with in vitro evaluation of MAO-B inhibitory potential of the target molecules were also performed. The results identified the ethyl 5-(4-bromophenyl)-1-(3-hydrazinyl-3-oxopropyl)-2-methyl-1H-pyrrole-3-carboxylate (12) as the most promising compound with the lowest neurotoxicity and highest neuroprotection on all evaluated parameters and inhibiting the hMAOB enzyme by 50%, comparable with the activity of the reference, Selegiline. The compatibility of the in silico and in vitro evaluations is a good prerequisite for these methods to be applied in future assessment of pyrrole-based compounds as anti-Parkinson agents.

The Anti-Aggregative Peptide KLVFF Mimics Aβ1-40 in the Modulation of Nicotinic Receptors: Implications for Peptide-Based Therapy

In recent years, the inhibition of beta-amyloid (Aβ) aggregation has emerged as a potential strategy for Alzheimer’s disease. KLVFF, a small peptide corresponding to the aminoacidic sequence 16-20 of Aβ, reduces Aβ fibrillation dose dependently. Therefore, the toxic and functional characterization of its brain activity is fundamental for clarifying its potential therapeutic role. Accordingly, we studied the modulatory role of KLVFF on the cholinergic receptors regulating dopamine and noradrenaline release in rat synaptosomes. Nicotinic receptors on dopaminergic nerve terminals in the nucleus acccumbens are inhibited by KLVFF, which closely resembles full-length Aβ1-40. Moreover, KLVFF entrapped in synaptosomes does not modify the nicotinic receptor’s function, suggesting that external binding to the receptor is required for its activity. The cholinergic agent desformylflustrabromine counteracts the KLVFF effect. Remarkably, muscarinic receptors on dopaminergic terminals and nicotinic receptors regulating noradrenaline release in the hippocampus are completely insensitive to KLVFF. Based on our findings, KLVFF mimics Aβ1-40 as a negative modulator of specific nicotinic receptor subtypes affecting dopamine transmission in the rat brain. Therefore, new pharmacological strategies using the anti-aggregative properties of KLVFF need to be evaluated for potential interference with nicotinic receptor-mediated transmission.

The Effect of Isosaponarin Derived from Wasabi Leaves on Glutamate Release in Rat Synaptosomes and Its Underlying Mechanism

Excessive glutamate release is known to be involved in the pathogenesis of neurological diseases, and suppression of glutamate release from nerve terminals is considered to be a treatment strategy. In this study, we investigated whether isosaponarin, a flavone glycoside isolated from wasabi leaves, could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). The release of glutamate was evoked by the K+ channel blocker 4-aminopyridine (4-AP) and measured by an online enzyme-coupled fluorimetric assay. Isosaponarin produced a concentration-dependent inhibition of 4-AP-evoked glutamate release with a half-maximum inhibition of release value of 22 μM. The inhibition caused by isosaponarin was prevented by eliminating extracellular Ca2+ or by using bafilomycin A1, an inhibitor of synaptic vesicle exocytosis. Isosaponarin decreased intrasynaptosomal rises in Ca2+ levels that were induced by 4-AP, without affecting the synaptosomal membrane potential. The isosaponarin-induced inhibition of glutamate release was significantly prevented in synaptosomes that were pretreated with a combination of the calcium channel blockers ω-conotoxin GVIA (N-type) and ω-agatoxin IVA (P/Q-types). The protein kinase C (PKC) pan-inhibitor GF109203X and the Ca2+-dependent PKC inhibitor Go6976 abolished the inhibition of glutamate release by isosaponarin, while the Ca2+-independent PKC inhibitor rottlerin did not show any effect. The results from immunoblotting assays also showed that isosaponarin lowered PKC, PKCα, synaptosomal-associated protein of 25 kDa (SNAP-25), and myristoylated alanine-rich C-kinase substrate (MARCKS) phosphorylation induced by 4-AP. In addition, FM1-43-labeled synaptic vesicles in synaptosomes showed that treatment with isosaponarin resulted in an attenuation of the 4-AP-induced decrease in fluorescence intensity that is consistent with glutamate release. Transmission electron microscopy of synaptosomes also provided evidence that isosaponarin altered the number of synaptic vesicles. These results indicate that isosaponarin suppresses the Ca2+-dependent PKC/SNAP-25 and MARCKS pathways in synaptosomes, causing a decrease in the number of available synaptic vesicles, which inhibits vesicular glutamate release from synaptosomes.

Methcathinone decreases dopamine transporter function: Role of protein kinase C.

Methcathinone (MCAT) is a psychostimulant of abuse that can cause both persistent striatal dopaminergic and serotonergic, as well as hippocampal serotonergic, deficits. Evidence suggests that the rapid effects of stimulants that are structurally and mechanistically similar to MCAT on monoamine transporter function may contribute to the abuse liability and/or persistent monoaminergic deficits caused by these agents. Thus, effects of MCAT on 1) striatal dopamine (DA) transporter (DAT); and 2) striatal and hippocampal serotonin transporter (SERT) function, as determined in tissues from adult male rats, were assessed. As reported previously, a single administration of MCAT rapidly (within 1hr) decreases striatal [3 H]DA uptake. Similarly, incubation of rat synaptosomes with MCAT at 37℃ (but not 4˚C) decreased striatal [3 H]DA uptake. Incubation with MCAT likewise decreased [3 H]5HT but not vesicular [3 H]DA uptake. MCAT incubation in vitro was without effect on [3 H]DA uptake in striatal synaptosomes prepared from MCAT-treated rats. The decrease in [3 H]DA uptake caused by MCAT incubation: (a) reflected a decrease in Vmax , with minimal change in Km , and (b) was attenuated by co-incubation with the cell-permeable calcium chelator, N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-1,1'-bis[(acetyloxy)methyl] ester-glycine (BAPTA-AM), as well as the non-selective protein kinase-C (PKC) inhibitors bisindolylmaleimide-1 (BIM-1) and 2-[1-3(Aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide (or Bisindolylmaleimide VIII; Ro-31-7549). Taken together, these results suggest that in vitro MCAT incubation may model important aspects of MCAT administration in vivo, and that calcium and PKC contribute to the in vitro effects of MCAT on DAT.

Protective Effects of Fruit Wines against Hydrogen Peroxide—Induced Oxidative Stress in Rat Synaptosomes

This study aimed to evaluate, in vitro, the antioxidative potential of fruit wines produced from berry fruits (i.e., black chokeberry, blueberry, blackberry, and raspberry), cherry, and apple by different technological processes. For this purpose, the activities of antioxidant enzymes (catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD)) and malondialdehyde (MDA) content as a marker of membrane damage were determined in wine-treated synaptosomes with hydrogen peroxide-induced oxidative stress. All studied wines induced increased antioxidant enzyme activities and decreased MDA levels compared to hydrogen peroxide-treated synaptosomes (i.e., control). The highest SOD activity was observed in synaptosomes treated with blackberry wine (6.81 U/mg), whereas blueberry wine induced the highest catalase and glutathione peroxidase activities (0.058 U/mg and 0.017 U/mg, respectively). Black chokeberry proved to be the best in lipid peroxidation protection with the lowest MDA value (1.42 nmol/mg). Finally, principal component analysis and hierarchical cluster analysis additionally highlighted a higher antioxidant capacity of wines produced from dark-skinned fruits (i.e., blackberry, black chokeberry, and blueberry). The results suggest protective effects of the fruit wines against oxidative damage, and, accordingly, their promising application as functional food.

Ex Vivo Investigation of Bexarotene and Nicotinamide Function as a Protectıve Agent on Rat Synaptosomes Treated with Aβ(1-42).

In this study, we were aimed to investigate the neuroprotective effects of bexarotene and nicotinamide in synaptosomes incubated with amyloid-beta (Aβ). Our study consists of 2 parts, in vivo and in vitro. In the in vivo section, twenty-four Wistar albino male rats were divided into 4 groups (control, dimethyl sulfoxide (DMSO), nicotinamide and bexarotene) with six animals in each group. DMSO(1%), nicotinamide(100mg/kg) and bexarotene(0.1mg/kg) were administered intraperitoneally to animals in the experimental groups for seven days. In the in vitro part of our study, three different isolation methods were used to obtain the synaptosomes from the brain tissue. Total antioxidant capacity(TAS), total oxidant capacity(TOS), cleaved caspase 3(CASP3), cytochrome c(Cyt c), sirtuin 1(SIRT1), peroxisome proliferator-activated receptor gamma(PPARγ) and poly(ADP-ribose) polymerase-1(PARP-1) levels in the synaptosomes incubated with a concentration of 10µM Aβ(1-42) were measured by enzyme-linked immunosorbent assay method. Biochemical analysis and histopathological examinations in serum and brain samples showed that DMSO, nicotinamide and bexarotene treatments did not cause any damage to the rat brain tissue. We found that in vitro Aβ(1-42) administration decreased TAS, SIRT1 and PPARγ levels in synaptosomes while increasing TOS, CASP3, Cyt c, and PARP1 levels. Nicotinamide treatment suppressed oxidative stress and apoptosis by supporting antioxidant capacity and increased PPARγ through SIRT1 activation, causing PARP1 to decrease. On the other hand, bexarotene caused a moderate increase in SIRT1 levels with PPARγ activation. Consequently, we found that nicotinamide can be more effective than bexarotene in AD pathogenesis by regulating mitochondrial functions in synaptosomes.

Protein Phosphorylation in Depolarized Synaptosomes: Dissecting Primary Effects of Calcium from Synaptic Vesicle Cycling

AbstractSynaptic transmission is mediated by the regulated exocytosis of synaptic vesicles. When the presynaptic membrane is depolarized by an incoming action potential, voltage-gated calcium channels open, resulting in the influx of calcium ions that triggers the fusion of synaptic vesicles (SVs) with the plasma membrane. SVs are recycled by endocytosis. Phosphorylation of synaptic proteins plays a major role in these processes, and several studies have shown that the synaptic phosphoproteome changes rapidly in response to depolarization. However, it is unclear which of these changes are directly linked to SV cycling and which might regulate other presynaptic functions that are also controlled by calcium-dependent kinases and phosphatases. To address this question, we analyzed changes in the phosphoproteome using rat synaptosomes in which exocytosis was blocked with botulinum neurotoxins (BoNTs) while depolarization-induced calcium influx remained unchanged. BoNT-treatment significantly alters the response of the synaptic phoshoproteome to depolarization and results in reduced phosphorylation levels when compared with stimulation of synaptosomes by depolarization with KCl alone. We dissect the primary Ca2+-dependent phosphorylation from SV-cycling-dependent phosphorylation and confirm an effect of such SV-cycling-dependent phosphorylation events on syntaxin-1a-T21/T23, synaptobrevin-S75, and cannabinoid receptor-1-S314/T322 on exo- and endocytosis in cultured hippocampal neurons.

The endocannabinoid system dual-target ligand N-cycloheptyl-1,2-dihydro-5-bromo-1-(4-fluorobenzyl)-6-methyl-2-oxo-pyridine-3-carboxamide improves disease severity in a mouse model of multiple sclerosis.

Multiple sclerosis is a chronic inflammatory demyelinating disorder of the central nervous system that eventually leads to progressive neurodegeneration and disability. Recent findings highlighted the emerging role of each target of the endocannabinoid system in controlling the symptoms and disease progression of multiple sclerosis. Therefore, multi-target modulators of the endocannabinoid system could provide a more effective pharmacological strategy as compared to the single target modulation. In this work, N-cycloheptyl-1,2-dihydro-5-bromo-1-(4-fluorobenzyl)-6-methyl-2-oxo-pyridine-3-carboxamide (B2) was identified as the most promising compound with dual agonism at cannabinoid receptors type-1 and cannabinoid receptors type-2 and good drug-like properties. In invitro assays, B2 reduced glutamate release from rat synaptosomes through interaction with cannabinoid receptors type-1 and modulated the production of the pro- and anti-inflammatory cytokines (interleukins IL-1β and IL-6 and interleukin IL-10 respectively) via cannabinoid receptors type-2 activation. Furthermore, B2 demonstrated antinociceptive effects in an animal model of neuropathic pain and efficacy in an experimental autoimmune encephalomyelitis model of multiple sclerosis.

Cell-Based Radiotracer Binding and Uptake Inhibition Assays: A Comparison of In Vitro Methods to Assess the Potency of Drugs That Target Monoamine Transporters.

High-affinity monoamine transporters are targets for prescribed medications and stimulant drugs of abuse. Therefore, assessing monoamine transporter activity for candidate medications and newly-emerging drugs of abuse provides essential information for industry, academia, and public health. Radiotracer binding and uptake inhibition are the gold standard assays for determining drug–transporter interaction profiles. The combined results from such assays yield a unique biochemical fingerprint for each compound. Over time, different assay methods have been developed to assess transporter activity, and the comparability of data across various assay platforms remains largely unclear. Here, we compare the effects of six well-established stimulants in two different cell-based uptake inhibition assays, one method using adherent cells and the other using suspended cells. Furthermore, we compare the data from transfected cell lines derived from different laboratories and data reported from rat synaptosomes. For transporter inhibitors, IC50 values obtained by the two experimental methods were comparable, but using different transfected cell lines yielded disparate results. For transporter substrates, differences between the two cell lines were less pronounced but the drugs displayed different inhibition potencies when evaluated by the two methods. Our study illustrates the inherent limitations when comparing transporter inhibition data from different laboratories and stresses the importance of including appropriate control experiments with reference compounds when investigating new drugs of interest.

Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway

Neurotransmitter release is mediated by ceramide, which is generated by sphingomyelin hydrolysis. In the present study, we examined whether synaptosomal-associated protein 25 (SNAP-25) is involved in ceramide production and exocytosis. Neutral sphingomyelinase 2 (nSMase2) was partially purified from bovine brain and we found that SNAP-25 was enriched in the nSMase2-containing fractions. In rat synaptosomes and PC12 cells, the immunoprecipitation pellet of anti-SNAP-25 antibody showed higher nSMase activity than the immunoprecipitation pellet of anti-nSMase2 antibody. In PC12 cells, SNAP-25 was colocalized with nSMase2. Transfection of SNAP-25 small interfering RNA (siRNA) significantly inhibited nSMase2 translocation to the plasma membrane. A23187-induced ceramide production was concomitantly reduced in SNAP-25 siRNA-transfected PC12 cells compared with that in scrambled siRNA-transfected cells. Moreover, transfection of SNAP-25 siRNA inhibited dopamine release, whereas addition of C6-ceramide to the siRNA-treated cells moderately reversed this inhibition. Additionally, nSMase2 inhibition reduced dopamine release. Collectively, our results indicate that SNAP-25 interacts with nSMase2 during ceramide production, which mediates exocytosis and neurotransmitter release.

Estrogen receptors modulate ectonucleotidases activity in hippocampal synaptosomes of male rats

Extracellular adenine nucleotides and nucleosides, such as adenosine-5'-triphosphate (ATP) and adenosine, are among least investigated signaling factors that participate in 17β-estradiol (E2)-mediated synaptic rearrangements in rodent hippocampus. Their levels in the extrasynaptic space are tightly controlled by ecto-nucleoside triphosphate diphosphohydrolases1-3 (NTPDase1-3)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, the aim of the present study was to get closer insight in the E2-induced decrease in NTPDase and eN activity in the hippocampal synaptic compartment of male rats and to identify estradiol receptors (ERs i.e. ERα, ERβ or GPER1) responsible for the observed effects of E2. In this study we show indiscriminate participation of estradiol receptor α (ERα), -β (ERβ) and G- protein coupled estrogen receptor 1 (GPER1) in the mediation of E2 actions in hippocampal synaptosomes of male rats. Synaptic NTPDase1-3 activities are modulated only through activation of ERβ, while activation of ERα, -β and/or non-classical GPER1 decreases synaptic eN activity. Since both ATP and adenosine function as neuromodulators in the hippocampal networks, influencing its function, profound knowledge of mechanisms by which ectonucleotidases are regulated/modulated is of great importance.

Curcumin Acts as Post-protective Effects on Rat Hippocampal Synaptosomes in a Neuronal Model of Aluminum-Induced Toxicity.

The neurotoxic effects of aluminum are generally associated with reduced antioxidant capacity, increased oxidative stress and apoptosis, which lead to the induction of neurodegenerative processes. Curcumin has a lipophilic polyphenol character and effects of antioxidant and anti-apoptotic. The present study was undertaken to examine possible aluminum exposure in rats brain synaptosomes and to investigate whether protective and therapeutic effects of curcumin on biochemical and morphological changes in both pre- and post-treated groups. Aluminum chloride (AlCl3) at 50µM concentration and curcumin at 5 and 10µg/mL doses were applied to hippocampal synaptosomes of rats according to experimental design. Biochemical effects were evaluated by MTT cytotoxicity, malondialdehyde (MDA) levels, nitric oxide (NO) levels, glutathione (GSH) levels, caspase 3 activities, cytochrome c levels, DNA fragmentation values and protein levels. Morphological examinations were done by TEM analysis. AlCI3 exposure in the synaptosomes enhanced oxidative stress, triggered apoptosis and caused ultrastructural alterations which were well reflected in the TEM images. Curcumin pre-treatment slightly ameliorated the MDA levels, NO levels, cytochrome c levels and caspase 3 activities in AlCI3-exposed synaptosomes, but these results were not statistically significant. Furthermore, curcumin post-treatment significantly improved oxidative damage and morphological alterations, and suppressed cytochrome c and caspase 3 activities. Taken together, our data showed that curcumin had more therapeutic effects than protective effects in AlCI3-induced neurotoxicity. Nevertheless, the therapeutic (post-protective) effects of curcumin should be further investigated in in vivo neurodegenerative models involving behavioral tests.