Inhibition of Synaptic Glutamate Exocytosis and Prevention of Glutamate Neurotoxicity by Eupatilin from Artemisia argyi in the Rat Cortex.

The inhibition of synaptic glutamate release to maintain glutamate homeostasis contributes to the alleviation of neuronal cell injury, and accumulating evidence suggests that natural products can repress glutamate levels and associated excitotoxicity. In this study, we investigated whether eupatilin, a constituent of Artemisia argyi, affected glutamate release in rat cortical nerve terminals (synaptosomes). Additionally, we evaluated the effect of eupatilin in an animal model of kainic acid (KA) excitotoxicity, particularly on the levels of glutamate and N-methyl-D-aspartate (NMDA) receptor subunits (GluN2A and GluN2B). We found that eupatilin decreased depolarization-evoked glutamate release from rat cortical synaptosomes and that this effect was accompanied by a reduction in cytosolic Ca2+ elevation, inhibition of P/Q-type Ca2+ channels, decreased synapsin I Ca2+-dependent phosphorylation and no detectable effect on the membrane potential. In a KA-induced glutamate excitotoxicity rat model, the administration of eupatilin before KA administration prevented neuronal cell degeneration, glutamate elevation, glutamate-generating enzyme glutaminase increase, excitatory amino acid transporter (EAAT) decrease, GluN2A protein decrease and GluN2B protein increase in the rat cortex. Taken together, the results suggest that eupatilin depresses glutamate exocytosis from cerebrocortical synaptosomes by decreasing P/Q-type Ca2+ channels and synapsin I phosphorylation and alleviates glutamate excitotoxicity caused by KA by preventing glutamatergic alterations in the rat cortex. Thus, this study suggests that eupatilin can be considered a potential therapeutic agent in the treatment of brain impairment associated with glutamate excitotoxicity.

Soybean Meal Extract Preserves Memory Ability by Increasing Presynaptic Function and Modulating Gut Microbiota in Rats.

Age-related degenerative brain diseases frequently manifest as memory deficits. Dietary interventions or nutraceuticals may provide efficacious treatments through prevention and cure. Soybean meal, a byproduct of soy oil refining, has health benefits, but its effect on memory function is unknown. Therefore, we evaluated the effect of the oral administration of soybean meal extract (SME) for 2 weeks on memory function using the Morris water maze (MWM) test in healthy rats and investigated the possible underlying mechanisms. First, analysis of the composition revealed that SME is rich in isoflavones; SME did not exhibit hepatotoxicity or renal toxicity at the different doses tested. The MWM results revealed that the escape latency and movement distance of rats were significantly shorter in the SME group than in the control group, indicating that SME can help in memory preservation. In addition, SME increased the levels of presynaptic proteins such as synaptophysin, synaptobrevin, synaptotagmin, syntaxin, synapsin I, and 25-kDa synaptosome-associated protein as well as protein kinases and their phosphorylated expression, including extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the hippocampal nerve terminals (synaptosomes). Transmission electron microscopy also indicated that SME increased the number of synaptic vesicles in hippocampal synaptosomes. Furthermore, SME rats exhibited altered microbiota composition compared with control rats. Therefore, our data suggest that SME can increase presynaptic function and modulate gut microbiota, thus aiding in memory preservation in rats.

Parkin-deficient rats are resistant to neurotoxicity of chronic high-dose methamphetamine.

Methamphetamine (METH) is a highly addictive and powerful central nervous system psychostimulant with no FDA-approved pharmacotherapy. Parkin is a neuroprotective protein and its loss of function contributes to Parkinson's disease. This study used 3-month-old homozygous parkin knockout (PKO) rats to determine whether loss of parkin protein potentiates neurotoxicity of chronic METH to the nigrostriatal dopamine pathway. PKO rats were chronically treated with 10 mg/kg METH for 10 consecutive days and assessed for neurotoxicity markers in the striatum on the 5th and 10th day of withdrawal from METH. The PKO rats showed higher METH-induced hyperthermia; however, they did not display augmented deficits in dopaminergic and serotonergic neurotoxicity markers, astrocyte activation or decreased mitochondrial enzyme levels as compared to wild-type (WT) rats. Interestingly, saline-treated PKO rats had lower levels of dopamine (DA) as well as mitochondrial complex I and II levels while having increased basal levels of glial fibrillary acidic protein (GFAP), a marker of gliosis. These results indicate PKO display a certain resistance to METH neurotoxicity, possibly mediated by lowered DA levels and downregulated mitochondria.

Albanin A, Derived from the Root Bark of Morus alba L., Depresses Glutamate Release in the Rat Cerebrocortical Nerve Terminals via Ca2+/Calmodulin/Adenylate Cyclase 1 Suppression.

Decreasing synaptic release of glutamate may counteract glutamate excitotoxicity in many neurological diseases. In this study, we investigated the effect of albanin A, a constituent in the root bark of Morus alba L., on the release of glutamate in rat cerebral cortex nerve endings (synaptosomes). We found that albanin A at 5-30µM suppressed 4-aminopyridine (4-AP)-induced release of glutamate. This phenomenon was abolished by extracellular calcium removal or by vesicular transporter inhibition, and was associated with a decrease in intrasynaptosomal Ca2+ levels. However, albanin A had no effect on the synaptosomal membrane potential. The inhibition of N- and P/Q-type Ca2+ channels, calmodulin, adenylate cyclase (AC), and protein kinase A, abolished the effect of albanin A on the glutamate release evoked by 4-AP. Moreover, the albanin A-mediated inhibition of glutamate release was prevented by the Ca2+/calmodulin-stimulated AC1 inhibitor. Western blot showed that AC1, but not AC8, was presented in the synaptosomes, and albanin A reduced 4-AP-induced increases in synaptosomal cyclic adenosine monophosphate content. In addition, albanin A pretreatment substantially attenuated neuronal damage in a rat model of kainic acid-induced glutamate excitotoxicity. Our data reveal that albanin A suppresses glutamate release by decreasing Ca2+/calmodulin/AC1 activation in synaptosomes and exerts neuroprotective effect in vivo.

Rich fatty acids diet of fish and olive oils modifies membrane properties in striatal rat synaptosomes.

Background: Essential fatty acids (EFAs) and non-essential fatty acids (nEFAs) exert experimental and clinical neuroprotection in neurodegenerative diseases. The main EFAs, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), nEFAs, and oleic acid (OA) contained in olive and fish oils are inserted into the cell membranes, but the exact mechanism through which they exert neuroprotection is still unknown. Objectives and Methods: In this study, we assessed the fatty acids content and membrane fluidity in striatal rat synaptosomes after fatty acid-rich diets (olive- or a fish-oil diet, 15% w/w). Then, we evaluated the effect of enriching striatum synaptosomes with fatty acids on the oxidative damage produced by the prooxidants ferrous sulfate (FeSO4) or quinolinic acid (QUIN). Results and Discussion: Lipid profile analysis in striatal synaptosomes showed that EPA content increased in the fish oil group in comparison with control and olive groups. Furthermore, we found that synaptosomes enriched with fatty acids and incubated with QUIN or FeSO4 showed a significant oxidative damage reduction. Results suggest that EFAs, particularly EPA, improve membrane fluidity and confer antioxidant effect.

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.

TCD, a triterpenoid isolated from wild bitter gourd, reduces synaptosomal release of glutamate and protects against kainic acid-induced neuronal death.

3ß,7ß,25-Trihydroxycucurbita-5,23(E)-dien-19-al (TCD) is a triterpenoid isolated from wild bitter gourd that is a common tropical vegetable with neuroprotective effects. Because excessive glutamate release is a major cause of neuronal damage in various neurological disorders, the aims of this study were to examine the effect of TCD on glutamate release in vitro and to examine the effect of TCD in vivo. In rat cerebrocortical synaptosomes, TCD reduced 4-aminopyridine (4-AP)-stimulated glutamate release and Ca2+ concentration elevation, but had no effect on plasma membrane potential. TCD-mediated inhibition of 4-AP-induced glutamate release was dependent on the presence of extracellular calcium; persisted in the presence of the glutamate transporter inhibitor dl-TBOA, P/Q-type Ca2+ channel blocker ω-agatoxin IVA, and intracellular Ca2+-releasing inhibitors dantrolene and CGP37157; and was blocked by the vesicular transporter inhibitor bafilomycin A1 and the N-type Ca2+ channel blocker ω-conotoxin GVIA. Molecular docking studies have demonstrated that TCD binds to N-type Ca2+ channels. TCD-mediated inhibition of 4-AP-induced glutamate release was abolished by the Ca2+-dependent protein kinase C (PKC) inhibitor Go6976, but was unaffected by the Ca2+-independent PKC inhibitor rottlerin. Furthermore, TCD considerably reduced the phosphorylation of PKC, PKCα, and myristoylated alanine-rich C kinase substrate, a major presynaptic substrate for PKC. In a rat model of kainic acid (KA)-induced excitotoxicity, TCD pretreatment substantially attenuated KA-induced neuronal death in the CA3 hippocampal region. These results suggest that TCD inhibits synaptosomal glutamate release by suppressing N-type Ca2+ channels and PKC activity and exerts protective effects against KA-induced excitotoxicity in vivo.

New insights in the mode of action of (+)-erythravine and (+)-11α-hydroxy-erythravine alkaloids.

Erythrinian alkaloids ((+)-erythravine and (+)-11-α-hydroxy-erythravine) have been pointed as the main responsible agents for the anticonvulsant and anxiolytic properties of Erythrina mulungu Mart ex Benth. The present work provides a new set of information about the mode of action of these alkaloids by the use of a complementary approach of neurochemical and electrophysiological assays. We propose here that the antiepileptic and anxiolytic properties exhibited by both alkaloids appear not to be related to the inhibition of glutamate binding or GABA uptake, or even to the increase of glutamate uptake or GABA binding, as investigated here by the use of rat cortical synaptosomes. Similarly, and even in a high concentration, (+)-erythravine and (+)-11-α-hydroxy-erythravine did not modulate the main sodium and potassium channel isoforms checked by the use of voltage-clamp studies on Xenopus laevis oocytes. However, unlike (+)-11-α-hydroxy-erythravine, which presented a little effect, it was possible to observe that the (+)-erythravine alkaloid produced a significant inhibitory modulation on α4ß2, α4ß4 and α7 isoforms of nicotinic acetylcholine receptors also checked by the use of voltage-clamp studies, which could explain at least partially its anxiolytic and anticonvulsant properties. Since (+)-11-α-hydroxy-erythravine and (+)-erythravine modulated nicotinic acetylcholine receptors to different extents, it is possible to reinforce that small differences between the chemical structure of these alkaloids can affect the selectivity and affinity of target-ligand interactions, conferring distinct potency and/or pharmacological properties to them, as previously suggested by differential experimental comparison between different erythrinian alkaloids.

Neuroprotective effects of Fraxinus angustifolia Vahl. bark extract against Alzheimer's disease.

Alzheimer disease's (AD) is a neurodegenerative disease induced by amyloid-ß (Aß) aggregation and accumulation of neurotoxic metals in the brain. Fraxinus angustifolia Vahl. (Oleaceae) is a Mediterranean plant traditionally used to treat several human problems as nervous system problems. This study aimed to evaluate the neuroprotective effects of F. angustifolia Vahl. bark extract (FAB) in vitro and in vivo against Aß-aggregation and aluminium induced-neurotoxicity in mice. FAB was characterized by colorimetric methods and its individual compounds were identified and quantified by LC-MS. First, the neuroprotective effect of FAB was evaluated against Aß25-35-aggregation where it was directly incubated with Aß25-35 and the kinetic of aggregation was measured by spectrophotometer at 200 nm. Then, the extract was tested against Aß25-35-induced cytotoxicity on PC12 cells and the cells viability was determined by MTT test. On the other hand, FAB (0.01-0.5 mg/mL) was tested against aluminium-activated lipid peroxidation in mice synaptosomal membranes, and in vivo against aluminium-caused neurotoxicity in male N.M.R.I. (Naval Medical Research Institute) mice; this test consisted of daily co-administration of the extract with Al for 60 days. At the end of the treatment, behavioral and memory tests (locomotor activity, black and white and Morris water maze tests) and histological analysis were realized. The identification and quantification of FAB phenolics revealed the presence of different phenolic classes with high concentration of phenylethanoids and hydroxycoumarins. FAB showed a high Aß25-35 anti-aggregative effect and a dose dependent protective effect on PC12 cells. The extract also demonstrated a significant inhibition of lipid peroxidation and was found to prevent the Al harmful effects where it significantly increased the locomotor activity, decreased the anxiety, improved memory and reduced histological alterations. In conclusion, FAB is rich of bioactive compounds that gave it the ability to inhibit Aß-aggregation and Al-caused neurotoxicity in mice.

Developmental lead (Pb)-induced deficits in redox and bioenergetic status of cerebellar synapses are ameliorated by ascorbate supplementation.

Neurotoxicity induced by exposure to heavy metal lead (Pb) is a concern of utmost importance particularly for countries with industrial-based economies. The developing brain is especially sensitive to exposure to even minute quantities of Pb which can alter neurodevelopmental trajectory with irreversible effects on motor, emotive-social and cognitive attributes even into later adulthood. Chemical synapses form the major pathway of inter-neuronal communications and are prime candidates for higher order brain (motor, memory and behavior) functions and determine the resistance/susceptibility for neurological disorders, including neuropsychopathologies. The synaptic pathways and mechanisms underlying Pb-mediated alterations in neuronal signaling and plasticity are not completely understood. Employing a biochemically isolated synaptosomal fraction which is enriched in synaptic terminals and synaptic mitochondria, this study aimed to analyze the alterations in bioenergetic and redox/antioxidant status of cerebellar synapses induced by developmental exposure to Pb (0.2 %). Moreover, we test the efficacy of vitamin C (ascorbate; 500 mg/kg body weight), a neuroprotective and neuromodulatory antioxidant, in mitigation of Pb-induced neuronal deficits. Our results implicate redox and bioenergetic disruptions as an underlying feature of the synaptic dysfunction observed in developmental Pb neurotoxicity, potentially contributing to consequent deficits in motor, behavioral and psychological attributes of the organisms. In addition, we establish ascorbate as a key ingredient for therapeutic approach against Pb induced neurotoxicity, particularly for early-life exposures.

Topical transient receptor potential ankyrin 1 antagonist treatment attenuates nociception and inflammation in an ultraviolet B radiation-induced burn model in mice.

BACKGROUND: Ultraviolet B (UVB) radiation exposure promotes sunburn and thereby acute and chronic inflammatory processes, contributing to pain development and maintenance. New therapeutic alternatives are necessary because typical treatments can cause adverse effects. An attractive alternative would be to target the transient receptor potential ankyrin 1 (TRPA1), a calcium-permeable, non-selective cation channel, which is involved in a variety of inflammatory pain models. OBJECTIVE: Evaluate the peripheral participation of TRPA1 using a topical treatment (HC030031 gel formulation; a selective TRPA1 antagonist) in nociception and inflammation caused by a UVB radiation-induced burn model in male mice (25-30 g). METHODS: The mice were anaesthetised, and just the right hind paw was exposed to UVB radiation (0.75 J/cm2). Topical treatments were applied immediately after irradiation and once a day for 8 days. RESULTS: HC030031 gel presented suitable pH and spreadability factor, ensuring its quality and the therapeutic effect. HC030031 0.05 % reversed UVB-induced mechanical and cold allodynia, with maximum inhibition (Imax) of 69 ± 13 % and 100 % (on day 4), respectively. HC030031 0.05 % also reduced the paw edema and MPO activity, with Imax of 77 ± 6 % (on day 5) and 69 ± 28 %, respectively. Likewise, UVB radiation increased the H2O2 levels (a TRPA1 agonist) and the Ca2+ influx in mice spinal cord synaptosomes. UVB radiation-induced Ca2+ influx was reduced by HC030031. CONCLUSION: These findings confirm the activation of the TRPA1 channel by UVB radiation, suggesting that topical TRPA1 antagonists can be a new strategy for the adjuvant treatment of sunburn-associated pain and inflammation.

Characterization of AN317, a novel selective agonist of α6ß2-containing nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are crucial mediators of central presynaptic, postsynaptic, and extrasynaptic signaling, and they are implicated in a range of CNS disorders. The numerous nAChR subtypes are differentially expressed and mediate distinct functions throughout the CNS, and thus there is considerable interest in developing subtype-selective nAChR modulators, both for use as pharmacological tools and as putative therapeutics. α6ß2-containing (α6ß2*) nAChRs are highly expressed in and regulate the activity of midbrain dopaminergic neurons, which makes them attractive drug targets in several psychiatric and neurological diseases, including nicotine addiction and Parkinson's disease. This paper presents the preclinical characterization of AN317, a novel α6ß2* agonist exhibiting functional selectivity toward other nAChRs, including α4ß2, α3ß4 and α7 receptors. AN317 induced [3H]dopamine release from rat striatal synaptosomes and augmented dopaminergic neuron activity in substantia nigra pars compacta brain slices in Ca2+ imaging and electrophysiological assays. In line with this, AN317 alleviated the high-frequency tremors arising from reserpine-mediated dopamine depletion in rats. Finally, AN317 mediated significant protective effects on cultured rat mesencephalic neurons treated with the dopaminergic neurotoxin MPP+. AN317 displays good bioavailability and readily crosses the blood-brain barrier, which makes it a unique tool for both in vitro and in vivo studies of native α6ß2* receptors in the nigrostriatal system and other dopaminergic pathways. Altogether, these findings highlight the potential of selective α6ß2* nAChR activation as a treatment strategy for symptoms and possibly even deceleration of disease progression in neurodegenerative diseases such as Parkinson's disease.

Asiatic acid, an active substance of Centella asiatica, presynaptically depresses glutamate release in the rat hippocampus.

Inhibiting glutamate release can reduce neuronal excitability and is recognized as a key mechanism of anti-epileptic drugs. In this study, by using isolated nerve terminal (synaptosome) and slice preparations, we investigated the effect of asiatic acid, a triterpene isolated from Centella asiatica with antiepileptic activity, on glutamate release in the hippocampus of rats. In hippocampal synaptosomes, application of asiatic acid resulted in a concentration-dependent inhibition of 4-aminopyridine-evoked glutamate release. This inhibitory action was dependent on extracellular calcium, blocked by inhibiting the vesicular transporter, but was unaffected by inhibiting the glutamate transporter. In addition, asiatic acid decreased the 4-aminopyridine-induced increase in the intraterminal calcium and failed to alter the synaptosomal potential. Furthermore, the asiatic acid-mediated release inhibition was significantly suppressed by the N- and P/Q-type calcium channel inhibitor ω-conotoxin MVIIC or protein kinase C inhibitor GF109203X. Western blotting data in synaptosomes also revealed that asiatic acid reduced 4-aminopyridine-induced phosphorylation of protein kinase C. In hippocampal slices, asiatic acid decreased the frequencies of spontaneous excitatory postsynaptic currents without changing their amplitudes and glutamate-activated currents in CA3 pyramidal neurons. We also observed that asiatic acid significantly suppressed 4-aminopyridine-induced burst firing. These data suggest that, in rat hippocampal nerve terminals, asiatic acid attenuates the calcium influx via N- and P/Q-type calcium channels, subsequently suppressing protein kinase C activity and decreasing glutamate release.

Allicin Inhibits Glutamate Release from Rat Cerebral Cortex Nerve Terminals Through Suppressing Ca2+ Influx and Protein Kinase C Activity.

Evidence indicates that indirect inhibitory regulation of glutamatergic transmission, via reducing glutamate release, may induce neuroprotection. The present work was designed to examine whether allicin, a major component of garlic with neuroprotective effects, affected the release of glutamate evoked by 4-aminopyridine in rat cerebrocortical nerve terminals (synaptosomes). Allicin caused a potent inhibition on the release of glutamate evoked by 4-aminopyridine, and this inhibitory effect was abolished in the presence of Ca2+-free medium and vesicular transporter inhibitor. Allicin decreased the 4-aminopyridine-evoked elevation of intrasynaptosomal Ca2+ levels, but had no effect on the synaptosomal plasma membrane potential. The allicin-mediated inhibition of glutamate release was prevented by the N- and P/Q-type channel blocker and the protein kinase C (PKC) inhibitor, but was not affected by the intracellular Ca2+-release inhibitors, mitogen-activated protein kinase inhibitor, and protein kinase A inhibitor. Western blotting data also showed that allicin significantly reduced the phosphorylation of PKC. Together, these data indicate that in rat cerebrocortical nerve terminals, allicin depresses glutamate release and appears to decrease N- and P/Q-type Ca2+ channel and PKC activity.

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.

Anabolic-androgen steroids effects on bioenergetics responsiveness of synaptic and extrasynaptic mitochondria.

We hypothesized that supraphysiological administration of the anabolic-androgenic steroids (AAS) like testosterone (TEST) and nandrolone decanoate (NAND) might differentially affect synaptic and extrasynaptic components of mitochondrial bioenergetics, thereby resulting in memory impairment. Oil (VEH), NAND or TEST (15 mg/Kg) were daily administered to male CF-1 albino mice for 19-days. We evaluated in the synaptosomes and extrasynaptic mitochondria, Ca2+ influx/efflux, membrane potential ΔÑ°m, oxidative respiratory states, dehydrogenases activity, H2O2 production, Tau phosphorylation, and spatial memory in the Morris water maze (MWM). In synaptosomes, both AAS increased Ca2+ influx and Na+ dependent efflux. In extrasynaptic mitochondria, NAND increased the Ca2+ influx. NAND prominently impaired ΔÑ°m formation and dissipation in synaptosomal and extrasynaptic mitochondria, while the effect of TEST was less pronounced. TEST increased the Reserve Respiratory Capacity in synaptosomes, and NAND decreased dehydrogenases activity in synaptic and extrasynaptic mitochondria. Also, NAND increased H2O2 production by synaptosomes and extrasynaptic mitochondria. NAND increased pTauSer396 in synaptosomes. Both AAS did not impair memory performance on MWM. We highlight that high doses of NAND cause neurotoxic effects to components of synaptic and extrasynaptic mitochondrial bioenergetics, like calcium influx, membrane potential and H2O2 production. TEST was less neurotoxic to synaptic and extrasynaptic mitochondrial bioenergetics responses.

Alcohol hangover effects on brain cortex non-synaptic mitochondria and synaptosomes bioenergetics.

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.

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.

Near infrared light decreases synaptic vulnerability to amyloid beta oligomers.

Synaptic dysfunction due to the disrupting binding of amyloid beta (Aß) and tau oligomers is one of the earliest impairments in Alzheimer's Disease (AD), driving initial cognitive deficits and clinical manifestation. Consequently, there is ample consensus that preventing early synaptic dysfunction would be an effective therapeutic strategy for AD. With this goal in mind, we investigated the effect of a treatment of mice with near infrared (NIR) light on synaptic vulnerability to Aß oligomers. We found that Aß oligomer binding to CNS synaptosomes isolated from wild type (wt) mice treated with NIR light was significantly reduced and the resulting suppression of long term potentiation (LTP) by Aß oligomers was prevented. Similarly, APP transgenic mice treated with NIR showed a significant reduction of endogenous Aß at CNS synapses. We further found that these phenomena were accompanied by increased synaptic mitochondrial membrane potential in both wt and Tg2576 mice. This study provides evidence that NIR light can effectively reduce synaptic vulnerability to damaging Aß oligomers, thus furthering NIR light therapy as a viable treatment for AD.

Immuno-pharmacological characterization of group II metabotropic glutamate receptors controlling glutamate exocytosis in mouse cortex and spinal cord.

BACKGROUND AND PURPOSE: We recently proposed the existence of mGlu3 -preferring autoreceptors in spinal cord terminals and of mGlu2 -preferring autoreceptors in cortical terminals. This study aims to verify our previous conclusions and to extend their pharmacological characterization. EXPERIMENTAL APPROACH: We studied the effect of LY566332, an mGlu2 receptor positive allosteric modulator (PAM), and of LY2389575, a selective mGlu3 receptor negative allosteric (NAM) modulator, on the mGlu2/3 agonist LY379268-mediated inhibition of glutamate exocytosis [measured as KCl-evoked release of preloaded [3 H]-D-aspartate]. The mGlu2 PAM BINA and the mGlu3 NAM ML337, as well as selective antibodies recognizing the N-terminal of the receptor proteins, were used to confirm the pharmacological characterization of the native receptors. KEY RESULTS: Cortical synaptosomes possess LY566332-sensitive autoreceptors that are slightly, although significantly, susceptible to LY2389575. In contrast, LY566332-insensitive and LY2389575-sensitive autoreceptors are present in spinal cord terminals. BINA and ML337 mimicked LY566332 and LY2389575, respectively, in controlling LY379268-mediated inhibition of glutamate exocytosis from both cortical and spinal cord synaptosomes. Incubation of cortical synaptosomes with anti-mGlu2 antibody prevented the LY379268-induced inhibition of glutamate exocytosis, and this response was partially reduced by the anti-mGlu3 antibody. Incubation of spinal cord synaptosomes with the anti-mGlu3 antibody abolished LY379268-mediated reduction of glutamate exocytosis from these terminals, while the anti-mGlu2 antibody was inactive. Western blot analysis and confocal microscopy data were largely consistent with these functional observations. CONCLUSIONS AND IMPLICATIONS: We confirmed that mGlu3 -preferring autoreceptors exist in spinal cord terminals. Differently, cortical glutamatergic terminals possess mGlu2 /mGlu3 heterodimers, whose inhibitory effect is largely mediated by mGlu2 receptors.