Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure.

Synaptic refinement is a critical physiological process that removes excess synapses to establish and maintain functional neuronal circuits. Recent studies have shown that focal exposure of phosphatidylserine (PS) on synapses acts as an "eat me" signal to mediate synaptic pruning. However, the molecular mechanism underlying PS externalization at synapses remains elusive. Here, we find that murine CDC50A, a chaperone of phospholipid flippases, localizes to synapses, and that its expression depends on neuronal activity. Cdc50a knockdown leads to phosphatidylserine exposure at synapses and subsequent erroneous synapse removal by microglia partly via the GPR56 pathway. Taken together, our data support that CDC50A safeguards synapse maintenance by regulating focal phosphatidylserine exposure at synapses.

Novel Benzofuran Derivatives Induce Monoamine Release and Substitute for the Discriminative Stimulus Effects of 3,4-Methylenedioxymethamphetamine.

3,4-Methylenedioxymethamphetamine (MDMA) has shown efficacy as a medication adjunct for treating post-traumatic stress disorder (PTSD). However, MDMA is also used in nonmedical contexts that pose risk for cardiovascular and neurologic complications. It is well established that MDMA exerts its effects by stimulating transporter-mediated release of the monoamines 5-hydroxytryptamine (5-HT), norepinephrine, and dopamine. Current research efforts are aimed at developing MDMA-like monoamine releasers with better efficacy and safety profiles. To this end, we investigated neurochemical and behavioral effects of novel analogs of the designer drug 5-(2-methylaminopropyl)benzofuran (5-MAPB). We used in vitro transporter assays in rat brain synaptosomes to examine transmitter uptake inhibition and releasing properties for enantiomers of 5-(2-methylaminobutyl)benzofuran (5-MABB) and 6-(2-methylaminobutyl)benzofuran (6-MABB) compared with MDMA. We then tested these same compounds in male Sprague-Dawley rats trained to discriminate MDMA (1.5 mg/kg) from saline. In vitro results revealed that S isomers of 5- and 6-MABB are efficacious releasing agents at transporters for 5-HT (SERT), norepinephrine (NET), and dopamine (DAT). By contrast, R isomers are efficacious releasers at SERT and partial releasers at NET but lack releasing activity at DAT. In vivo results showed that all compounds produce dose-dependent increases in MDMA-lever responding and full substitution at the highest dose tested. The diminished NET and DAT releasing activities for R isomers of 5- and 6-MABB are associated with reduced potency for inducing behavioral effects. Collectively, these findings indicate that the aminoalkyl benzofuran scaffold may be a viable template for developing compounds with MDMA-like properties. SIGNIFICANCE STATEMENT: Despite the clinical utility of 3,4-methylenedioxymethamphetamine (MDMA), the drug is associated with certain cardiovascular risks and metabolic side effects. Developing a therapeutic alternative with MDMA-like monoamine releasing activity is of interest. Our in vitro and in vivo findings indicate that the aminoalkyl benzofuran scaffold may be useful for developing compounds with MDMA-like properties.

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.

Sex and brain region-specific regulation of serotonin transporter activity in synaptosomes in guanine nucleotide-binding protein G(q) alpha knockout mice.

The regulation of the serotonin transporter (SERT) by guanine nucleotide-binding protein alpha (Gα) q was investigated using Gαq knockout mice. In the absence of Gαq, SERT-mediated uptake of 5-hydroxytryptamine (5HT) was enhanced in midbrain and frontal cortex synaptosomes, but only in female mice. The mechanisms underlying this sexual dimorphism were investigated using quantitative western blot analysis revealing brain region-specific differences. In the frontal cortex, SERT protein expression was decreased in male knockout mice, seemingly explaining the sex-dependent variation in SERT activity. The differential expression of Gαi1 in female mice contributes to the sex differences in the midbrain. In fact, Gαi1 levels inversely correlate with 5HT uptake rates across both sexes and genotypes. Likely due to differential SERT regulation as well as sex differences in the expression of tryptophan hydroxylase 2, Gαq knockout mice also displayed sex- and genotype-dependent alterations in total 5HT tissue levels as determined by high-performance liquid chromatography. Gαq inhibitors, YM-254890 and BIM-46187, differentially affected SERT activity in both, synaptosomes and cultured cells. YM-254890 treatment mimicked the effect of Gαq knockout in the frontal cortex. BIM-46187, which promotes the nucleotide-free form of Gα proteins, substantially inhibited 5HT uptake, prompting us to hypothesise that Gαq interacts with SERT similarly as with G-protein-coupled receptors and inhibits SERT activity by modulating transport-associated conformational changes. Taken together, our findings reveal a novel mechanism of SERT regulation and impact our understanding of sex differences in diseases associated with dysregulation of serotonin transmission, such as depression and anxiety.

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 1 hr) 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.

Typhaneoside Suppresses Glutamate Release Through Inhibition of Voltage-Dependent Calcium Entry in Rat Cerebrocortical Nerve Terminals.

Glutamate is the major excitatory neurotransmitter in the brain and is involved in many brain functions. In this study, we investigated whether typhaneoside, a flavonoid from Typhae angustifolia pollen, affects endogenous glutamate release from rat cortical synaptosomes. Using a one-line enzyme-coupled fluorometric assay, glutamate release stimulated by the K+ channel blocker 4-aminopyridine was monitored to explore the possible underlying mechanisms. The vesicular transporter inhibitor bafilomycin A1 and chelation of extracellular Ca2+ ions with EGTA suppressed the effect of typhaneoside on the induced glutamate release. Nevertheless, the typhaneoside activity has not been affected by the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate. The synaptosomal plasma membrane potential was assayed using a membrane potential-sensitive dye DiSC3(5), and cytosolic Ca2+ concentrations ([Ca2+]C) was monitored by a Ca2+ indicator Fura-2. Results showed that typhaneoside did not alter the synaptosomal membrane potential but lowered 4-aminopyridine-induced increases in [Ca2+]C. Furthermore, the Cav2.2 (N-type) channel blocker ω-conotoxin GVIA blocked Ca2+ entry and inhibited the effect of typhaneoside on 4-aminopyridine-induced glutamate release. However, the inhibitor of intracellular Ca2+ release dantrolene and the mitochondrial Na+/Ca2+ exchanger blocker 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one have no effect on the suppression of glutamate release mediated by typhaneoside. Moreover, inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) prevented the inhibitory effect of typhaneoside on induced glutamate release. Typhaneoside reduced 4-aminopyridine-induced phosphorylation of ERK1/2 and the major presynaptic ERK target synapsin I, which is a synaptic vesicle-associated protein. In conclusion, these findings suggest a role for typhaneoside in modulating glutamate release by suppressing voltage-dependent Ca2+ channel mediated presynaptic Ca2+ influx and the MAPK/ERK/synapsin I signaling cascade.

Complex I Controls Mitochondrial and Plasma Membrane Potentials in Nerve Terminals.

Reductions in the activities of mitochondrial electron transport chain (ETC) enzymes have been implicated in the pathogenesis of numerous chronic neurodegenerative disorders. Maintenance of the mitochondrial membrane potential (Δψm) is a primary function of these enzyme complexes, and is essential for ATP production and neuronal survival. We examined the effects of inhibition of mitochondrial ETC complexes I, II/III, III and IV activities by titrations of respective inhibitors on Δψm in synaptosomal mitochondria. Small perturbations in the activity of complex I, brought about by low concentrations of rotenone (1-50 nM), caused depolarisation of Δψm. Small decreases in complex I activity caused an immediate and partial Δψm depolarisation, whereas inhibition of complex II/III activity by more than 70% with antimycin A was required to affect Δψm. A similarly high threshold of inhibition was found when complex III was inhibited with myxothiazol, and inhibition of complex IV by more than 90% with KCN was required. The plasma membrane potential (Δψp) had a complex I inhibition threshold of 40% whereas complex III and IV had to be inhibited by more than 90% before changes in Δψp were registered. These data indicate that in synaptosomes, both Δψm and Δψp are more susceptible to reductions in complex I activity than reductions in the other ETC complexes. These findings may be of relevance to the mechanism of neuronal cell death in Parkinson's disease in particular, where such reductions in complex I activity are present.

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

In this study, we were aimed to investigate the neuroprotective effects of bexarotene and nicotinamide in synaptosomes incubated with amyloid-beta (Aß). 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(100 mg/kg) and bexarotene(0.1 mg/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 10 µM Aß(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 Aß(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.

Nine prenylated acylphloroglucinols with potential anti-depressive and hepatoprotective activities from Hypericum scabrum.

In our screening program for new biologically active secondary metabolites, nine new polycyclic polyprenyled acylphloroglucinols, hyperscabins D-L, together with three known compounds, were obtained from the aerial parts of Hypericum scabrum. The chemical structures of 1-9 were characterized by extensive spectroscopic analyses, nuclear magnetic resonance calculation with DP4+ probability analysis, and the electronic circular dichroism spectra were calculated. Compound 1 was an unusual prenylated acylphloroglucinol decorated with a 5-oxaspiro [4,5] deca-1,9-dione skeleton. Compound 2 was a newly identified spirocyclic polyprenylated acylphloroglucinol possessing a rare 5,5-spiroketal segment. Compounds 3, 8, and 10 (10 µM) exhibited pronounced hepatoprotective activity against d-galactosamine-induced WB-F344 cell damage in vitro assays. All test compounds (1, 3, and 7-12) demonstrated potential inhibitory effects at 10 µM against noradrenalinet ([3H]-NE) reuptake in rat brain synaptosome.

Dopamine and Methamphetamine Differentially Affect Electron Transport Chain Complexes and Parkin in Rat Striatum: New Insight into Methamphetamine Neurotoxicity.

Methamphetamine (METH) is a highly abused psychostimulant that is neurotoxic to dopaminergic (DAergic) nerve terminals in the striatum and increases the risk of developing Parkinson's disease (PD). In vivo, METH-mediated DA release, followed by DA-mediated oxidative stress and mitochondrial dysfunction in pre- and postsynaptic neurons, mediates METH neurotoxicity. METH-triggered oxidative stress damages parkin, a neuroprotective protein involved in PD etiology via its involvement in the maintenance of mitochondria. It is not known whether METH itself contributes to mitochondrial dysfunction and whether parkin regulates complex I, an enzymatic complex downregulated in PD. To determine this, we separately assessed the effects of METH or DA alone on electron transport chain (ETC) complexes and the protein parkin in isolated striatal mitochondria. We show that METH decreases the levels of selected complex I, II, and III subunits (NDUFS3, SDHA, and UQCRC2, respectively), whereas DA decreases the levels only of the NDUFS3 subunit in our preparations. We also show that the selected subunits are not decreased in synaptosomal mitochondria under similar experimental conditions. Finally, we found that parkin overexpression does not influence the levels of the NDUFS3 subunit in rat striatum. The presented results indicate that METH itself is a factor promoting dysfunction of striatal mitochondria; therefore, it is a potential drug target against METH neurotoxicity. The observed decreases in ETC complex subunits suggest that DA and METH decrease activities of the ETC complexes via oxidative damage to their subunits and that synaptosomal mitochondria may be somewhat "resistant" to DA- and METH-induced disruption in mitochondrial ETC complexes than perikaryal mitochondria. The results also suggest that parkin does not regulate NDUFS3 turnover in rat striatum.

P2X7 receptors exert a permissive effect on the activation of presynaptic AMPA receptors in rat trigeminal caudal nucleus glutamatergic nerve terminals.

BACKGROUND: Purine receptors play roles in peripheral and central sensitization and are associated with migraine headache. We investigated the possibility that ATP plays a permissive role in the activation of AMPA receptors thus inducing Glu release from nerve terminals isolated from the rat trigeminal caudal nucleus (TCN). METHODS: Nerve endings isolated from the rat TCN were loaded with [3H]D-aspartic acid ([3H]D-ASP), layered into thermostated superfusion chambers, and perfused continuously with physiological medium, alone or with various test drugs. Radioactivity was measured to assess [3H]D-ASP release under different experimental conditions. RESULTS: Synaptosomal [3H]D-ASP spontaneous release was stimulated by ATP and to an even greater extent by the ATP analogue benzoylbenzoylATP (BzATP). The stimulation of [3H]D-ASP basal release by the purinergic agonists was prevented by the selective P2X7 receptor antagonist A438079. AMPA had no effect on basal [3H]D-ASP release, but the release observed when synaptosomes were exposed to AMPA plus a purinoceptor agonist exceeded that observed with ATP or BzATP alone. The selective AMPA receptor antagonist NBQX blocked this "excess" release. Co-exposure to AMPA and BzATP, each at a concentration with no release-stimulating effects, evoked a significant increase in [3H]D-ASP basal release, which was prevented by exposure to a selective AMPA antagonist. CONCLUSIONS: P2X7 receptors expressed on glutamatergic nerve terminals in the rat TCN can mediate Glu release directly and indirectly by facilitating the activation of presynaptic AMPA receptors. The high level of glial ATP that occurs during chronic pain states can promote widespread release of Glu as well as can increase the function of AMPA receptors. In this manner, ATP contributes to the AMPA receptor activation involved in the onset and maintenance of the central sensitization associated with chronic pain.

The Supplement Adulterant ß-Methylphenethylamine Increases Blood Pressure by Acting at Peripheral Norepinephrine Transporters.

ß-Methylphenethylamine [(BMPEA), 2-phenylpropan-1-amine] is a structural isomer of amphetamine (1-phenylpropan-2-amine) that has been identified in preworkout and weight loss supplements, yet little information is available about its pharmacology. Here, the neurochemical and cardiovascular effects of BMPEA and its analogs, N-methyl-2-phenylpropan-1-amine (MPPA) and N,N-dimethyl-2-phenylpropan-1-amine (DMPPA), were compared with structurally related amphetamines. As expected, amphetamine and methamphetamine were potent substrate-type releasing agents at dopamine transporters (DATs) and norepinephrine transporters (NETs) in rat brain synaptosomes. BMPEA and MPPA were also substrates at DATs and NETs, but they were at least 10-fold less potent than amphetamine. DMPPA was a weak substrate only at NETs. Importantly, the releasing actions of BMPEA and MPPA were more potent at NETs than DATs. Amphetamine produced significant dose-related increases in blood pressure (BP), heart rate (HR), and locomotor activity in conscious rats fitted with surgically implanted biotelemetry transmitters. BMPEA, MPPA, and DMPPA produced increases in BP that were similar to the effects of amphetamine, but the compounds failed to substantially affect HR or activity. The hypertensive effect of BMPEA was reversed by the α-adrenergic antagonist prazosin but not the ganglionic blocker chlorisondamine. Radioligand binding at various G protein-coupled receptors did not identify nontransporter sites of action that could account for cardiovascular effects of BMPEA or its analogs. Our results show that BMPEA, MPPA, and DMPPA are biologically active. The compounds are unlikely to be abused due to weak effects at DATs, but they could produce adverse cardiovascular effects via substrate activity at peripheral NET sites.

GZ-11608, a Vesicular Monoamine Transporter-2 Inhibitor, Decreases the Neurochemical and Behavioral Effects of Methamphetamine.

Despite escalating methamphetamine use and high relapse rates, pharmacotherapeutics for methamphetamine use disorders are not available. Our iterative drug discovery program had found that R-N-(1,2-dihydroxypropyl)-2,6-cis-di-(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A), a selective vesicular monoamine transporter-2 (VMAT2) inhibitor, specifically decreased methamphetamine's behavioral effects. However, GZ-793A inhibited human-ether-a-go-go-related gene (hERG) channels, suggesting cardiotoxicity and prohibiting clinical development. The current study determined if replacement of GZ-793A's piperidine ring with a phenylalkyl group to yield S-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propan-1-amine (GZ-11608) diminished hERG interaction while retaining pharmacological efficacy. VMAT2 inhibition, target selectivity, and mechanism of GZ-11608-induced inhibition of methamphetamine-evoked vesicular dopamine release were determined. We used GZ-11608 doses that decreased methamphetamine-sensitized activity to evaluate the potential exacerbation of methamphetamine-induced dopaminergic neurotoxicity. GZ-11608-induced decreases in methamphetamine reinforcement and abuse liability were determined using self-administration, reinstatement, and substitution assays. Results show that GZ-11608 exhibited high affinity (Ki = 25 nM) and selectivity (92-1180-fold) for VMAT2 over nicotinic receptors, dopamine transporter, and hERG, suggesting low side-effects. GZ-11608 (EC50 = 620 nM) released vesicular dopamine 25-fold less potently than it inhibited VMAT2 dopamine uptake. GZ-11608 competitively inhibited methamphetamine-evoked vesicular dopamine release (Schild regression slope = 0.9 ± 0.13). GZ-11608 decreased methamphetamine sensitization without altering striatal dopamine content or exacerbating methamphetamine-induced dopamine depletion, revealing efficacy without neurotoxicity. GZ-11608 exhibited linear pharmacokinetics and rapid brain penetration. GZ-11608 decreased methamphetamine self-administration, and this effect was not surmounted by increasing methamphetamine unit doses. GZ-11608 reduced cue- and methamphetamine-induced reinstatement, suggesting potential to prevent relapse. GZ-11608 neither served as a reinforcer nor substituted for methamphetamine, suggesting low abuse liability. Thus, GZ-11608, a potent and selective VMAT2 inhibitor, shows promise as a therapeutic for methamphetamine use disorder. SIGNIFICANCE STATEMENT: GZ-11608 is a potent and selective vesicular monoamine transporter-2 inhibitor that decreases methamphetamine-induced dopamine release from isolated synaptic vesicles from brain dopaminergic neurons. Results from behavioral studies show that GZ-11608 specifically decreases methamphetamine-sensitized locomotor activity, methamphetamine self-administration, and reinstatement of methamphetamine-seeking behavior, without exhibiting abuse liability. Tolerance does not develop to the efficacy of GZ-11608 to decrease the behavioral effects of methamphetamine. In conclusion, GZ-11608 is an outstanding lead in our search for a therapeutic to treat methamphetamine use disorder.

Neurosteroid allopregnanolone inhibits glutamate release from rat cerebrocortical nerve terminals.

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 Ca2+ ions and the vesicular transporter inhibitor. Allopregnanolone reduced the elevation of 4-AP-evoked intrasynaptosomal Ca2+ 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 Ca2+ -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 Ca2+ /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 GABAA receptor antagonist SR95531. Our data are the first to suggest that allopregnanolone reduce the Ca2+ influx through N-, P/Q-, and R-type Ca2+ channels, through the activation of GABAA receptors present on cerebrocortical nerve terminals, subsequently suppressing the Ca2+ -calmodulin/PKA cascade and decreasing 4-AP-evoked glutamate release.

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 50 µM concentration and curcumin at 5 and 10 µg/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.

The Mitochondrial Targets of Neuroprotective Drug Vinpocetine on Primary Neuron Cultures, Brain Capillary Endothelial Cells, Synaptosomes, and Brain Mitochondria.

Vinpocetine is considered as neuroprotectant drug and used for treatment of brain ischemia and cognitive deficiencies for decades. A number of enzymes, channels and receptors can bind vinpocetine, however the mechanisms of many effects' are still not clear. The present study investigated the effects of vinpocetine from the mitochondrial bioenergetic aspects. In primary brain capillary endothelial cells the purinergic receptor-stimulated mitochondrial Ca2+ uptake and efflux were studied. Vinpocetine exerted a partial inhibition on the mitochondrial calcium efflux. In rodent brain synaptosomes vinpocetine (30 µM) inhibited respiration in uncoupler stimulated synaptosomes and decreased H2O2 release from the nerve terminals in resting and in complex I inhibited conditions, respectively. In isolated rat brain mitochondria using either complex I or complex II substrates leak respiration was stimulated, but ADP-induced respiration was inhibited by vinpocetine. The stimulation of oxidation was associated with a small extent of membrane depolarization. Mitochondrial H2O2 production was inhibited by vinpocetine under all conditions investigated. The most pronounced effects were detected with the complex II substrate succinate. Vinpocetine also mitigated both Ca2+-induced mitochondrial Ca2+-release and Ca2+-induced mitochondrial swelling. It lowered the rate of mitochondrial ATP synthesis, while increasing ATPase activity. These results indicate more than a single mitochondrial target of this vinca alkaloid. The relevance of the affected mitochondrial mechanisms in the anti ischemic effect of vinpocetine is discussed.

Inhibition of protein phosphatase-1 and -2A by ellagitannins: structure-inhibitory potency relationships and influences on cellular systems.

Several ellagitannins inhibited the activity of protein phosphatase-1 (PP1) and -2 A (PP2A) catalytic subunits (PP1c and PP2Ac) with preferential suppression of PP1c over PP2Ac. The inhibitory potency for PP1c followed the order of tellimagrandin I > mahtabin A > praecoxin B > 1.2-Di-O-galloyl-4.6-(S)-HHDP-ß-D-glucopyranose > pedunculagin with IC50 values ranging from 0.20 µM to 2.47 µM. The interaction of PP1c and tellimagrandin I was assessed by NMR saturation transfer difference, surface plasmon resonance, isothermal titration calorimetry, and microscale thermophoresis based binding techniques. Tellimagrandin I suppressed viability and phosphatase activity of HeLa cells, while mahtabin A was without effect. Conversely, mahtabin A increased the phosphorylation level of SNAP-25Thr138 and suppressed exocytosis of cortical synaptosomes, whereas tellimagrandin I was without influence. Our results establish ellagitannins as partially selective inhibitors of PP1 and indicate that these polyphenols may act distinctly in cellular systems depending on their membrane permeability and/or their actions on cell membranes.

Revealing the Inhibitory Effect of Ginseng on Mitochondrial Respiration through Synaptosomal Proteomics.

Ginseng, the active ingredients of which are ginsenosides, is the most popular herbal medicine and has potential merit in the treatment of cerebral disorders. To better understand the function of Ginseng in the cerebral system, we examined changes in the protein expression profiles of synaptosomes extracted from the cerebral cortical and hippocampal tissues of rats administered a high or low dose of Ginseng for 2 weeks. More than 5000 proteins belonging to synaptosomes were simultaneously identified and quantitated by an approach combining tandem mass tags with 2D liquid chromatography-mass spectrometry (LC-MS). Regarding differentially expressed proteins, downregulated proteins were much more highly induced than upregulators in the cerebral cortical and hippocampal synaptosomes, regardless of the dose of Ginseng. Bioinformatic analysis indicated the majority of the altered proteins to be located in the mitochondria, directly or indirectly affecting mitochondrial oxidative respiration. Further functional experiments using the substrate-uncoupler inhibitor titration approach confirmed that three representative ginsenosides were able to inhibit oxidative phosphorylation in mitochondria. Our results demonstrate that Ginseng can regulate the function of mitochondria and alter the energy metabolism of cells, which may be useful for the treatment of central nervous disorders.

Pharmacological Rescue of Long-Term Potentiation in Alzheimer Diseased Synapses.

Long-term potentiation (LTP) is an activity-dependent and persistent increase in synaptic transmission. Currently available techniques to measure LTP are time-intensive and require highly specialized expertise and equipment, and thus are not well suited for screening of multiple candidate treatments, even in animal models. To expand and facilitate the analysis of LTP, here we use a flow cytometry-based method to track chemically induced LTP by detecting surface AMPA receptors in isolated synaptosomes: fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). First, we demonstrate that FASS-LTP is simple, sensitive, and models electrically induced LTP recorded in intact circuitries. Second, we conducted FASS-LTP analysis in two well-characterized Alzheimer's disease (AD) mouse models (3xTg and Tg2576) and, importantly, in cryopreserved human AD brain samples. By profiling hundreds of synaptosomes, our data provide the first direct evidence to support the idea that synapses from AD brain are intrinsically defective in LTP. Third, we used FASS-LTP for drug evaluation in human synaptosomes. Testing a panel of modulators of cAMP and cGMP signaling pathways, FASS-LTP identified vardenafil and Bay-73-6691 (phosphodiesterase-5 and -9 inhibitors, respectively) as potent enhancers of LTP in synaptosomes from AD cases. These results indicate that our approach could provide the basis for protocols to study LTP in both healthy and diseased human brains, a previously unattainable goal. SIGNIFICANCE STATEMENT: Learning and memory depend on the ability of synapses to strengthen in response to activity. Long-term potentiation (LTP) is a rapid and persistent increase in synaptic transmission that is thought to be affected in Alzheimer's disease (AD). However, direct evidence of LTP deficits in human AD brain has been elusive, primarily due to methodological limitations. Here, we analyze LTP in isolated synapses from AD brain using a novel approach that allows testing LTP in cryopreserved brain. Our analysis of hundreds of synapses supports the idea that AD-diseased synapses are intrinsically defective in LTP. Further, we identified pharmacological agents that rescue LTP in AD, thus opening up a new avenue for drug screening and evaluation of strategies for alleviating memory impairments.

Presynaptic mGluR5 receptor controls glutamatergic input through protein kinase C-NMDA receptors in paclitaxel-induced neuropathic pain.

Chemotherapeutic drugs such as paclitaxel cause painful peripheral neuropathy in many cancer patients and survivors. Although NMDA receptors (NMDARs) at primary afferent terminals are known to be critically involved in chemotherapy-induced chronic pain, the upstream signaling mechanism that leads to presynaptic NMDAR activation is unclear. Group I metabotropic glutamate receptors (mGluRs) play a role in synaptic plasticity and NMDAR regulation. Here we report that the Group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) significantly increased the frequency of miniature excitatory postsynaptic currents (EPSCs) and the amplitude of monosynaptic EPSCs evoked from the dorsal root. DHPG also reduced the paired-pulse ratio of evoked EPSCs in spinal dorsal horn neurons. These effects were blocked by the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), but not by an mGluR1 antagonist. MPEP normalized the frequency of miniature EPSCs and the amplitude of evoked EPSCs in paclitaxel-treated rats but had no effect in vehicle-treated rats. Furthermore, mGluR5 protein levels in the dorsal root ganglion and spinal cord synaptosomes were significantly higher in paclitaxel- than in vehicle-treated rats. Inhibiting protein kinase C (PKC) or blocking NMDARs abolished DHPG-induced increases in the miniature EPSC frequency of spinal dorsal horn neurons in vehicle- and paclitaxel-treated rats. Moreover, intrathecal administration of MPEP reversed pain hypersensitivity caused by paclitaxel treatment. Our findings suggest that paclitaxel-induced painful neuropathy is associated with increased presynaptic mGluR5 activity at the spinal cord level, which serves as upstream signaling for PKC-mediated tonic activation of NMDARs. mGluR5 is therefore a promising target for reducing chemotherapy-induced neuropathic pain.