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1.
Toxicology ; 440: 152492, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32407874

RESUMO

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.


Assuntos
Antioxidantes/uso terapêutico , Ácido Ascórbico/uso terapêutico , Cerebelo/metabolismo , Metabolismo Energético/efeitos dos fármacos , Intoxicação do Sistema Nervoso por Chumbo/patologia , Sinapses/metabolismo , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Cerebelo/efeitos dos fármacos , Feminino , Glutationa/metabolismo , Chumbo/sangue , Intoxicação do Sistema Nervoso por Chumbo/psicologia , Masculino , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Sinapses/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
2.
Biochem Pharmacol ; 174: 113786, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31887288

RESUMO

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.


Assuntos
Fármacos Neuroprotetores/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Masculino , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/embriologia , Camundongos , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacocinética , Agonistas Nicotínicos/administração & dosagem , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacocinética , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores Nicotínicos/genética , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Xenopus laevis
3.
Biochem Pharmacol ; 174: 113788, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31887290

RESUMO

α6ß2-Containing nicotinic acetylcholine receptors (α6ß2* nAChRs) are predominantly expressed in midbrain dopaminergic neurons, including substantia nigra pars compacta (SNc) neurons and their projections to striatal regions, where they regulate dopamine release and nigrostriatal activity. It is well established that nAChR agonists exert protection against dopaminergic neurotoxicity in cellular assays and parkinsonian animal models. Historically, drug development in the nAChR field has been mostly focused on development of selective agonists and positive allosteric modulators (PAMs) for the predominant neuronal nAChRs, α7 and α4ß2. Here, we report the discovery and characterization of AN6001, a novel selective α6ß2* nAChR PAM. AN6001 mediated increases in both nicotine potency and efficacy at the human α6/α3ß2ß3V9'S nAChR in HEK293 cells, and it positively modulated ACh-evoked currents through both α6/α3ß2ß3V9'S and a concatenated ß3-α6-ß2-α6-ß2 receptor in Xenopus oocytes, displaying EC50 values of 0.58 µM and 0.40 µM, respectively. In contrast, the compound did not display significant modulatory activity at α4ß2, α3ß4, α7 and muscle nAChRs. AN6001 also increased agonist-induced dopamine release from striatal synaptosomes and augmented agonist-induced global cellular responses and inward currents in dopaminergic neurons in SNc slices (measured by Ca2+ imaging and patch clamp recordings, respectively). Finally, AN6001 potentiated the neuroprotective effect of nicotine at MPP+-treated primary dopaminergic neurons. Overall, our studies demonstrate the existence of allosteric sites on α6ß2* nAChRs and that positive modulation of native α6ß2* receptors strengthens DA signaling. Hence, AN6001 represents an important tool for studies of α6ß2* nAChRs and furthermore underlines the therapeutic potential in these receptors in Parkinson's disease.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Dopamina/metabolismo , Fármacos Neuroprotetores/farmacologia , Receptores Nicotínicos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Células HEK293 , Humanos , Camundongos , Fármacos Neuroprotetores/química , Nicotina/farmacologia , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos Sprague-Dawley , Ratos Wistar , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Xenopus laevis
4.
Eur J Pharmacol ; 864: 172725, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31604070

RESUMO

Dehydrocorydaline, is an active alkaloid compound in Corydalis yanhusuo W. T. Wang. We found dehydrocorydaline induced antidepressant-like effects in a chronic unpredictable mild stress mouse model, but the exact mechanisms have not been addressed. We speculated that dehydrocorydaline may have an antidepressant effect via inhibiting monoamine transporters in the brain. We evaluated the mechanism of action of dehydrocorydaline by examining the levels of monoamine transmitters (5-HT, NE and DA) in the prefrontal cortex in chronic unpredictable mild stress mice. Then, we used cell models and the mouse synaptosome to study molecular and cellular mechanisms underlying these behaviors and monoamine alterations by dehydrocorydaline. Our results indicated that dehydrocorydaline affects the concentrations of monoamine transmitters and decreases the turnover ratio, which indicates increased neuronal activity. The possible mechanism is that dehydrocorydaline potently inhibits uptake-2 transporters with the IC50 values of 0.1-4 µM and could inhibit the reuptake of 5-HT/DA/NE in the synaptosome. These data suggest that dehydrocorydaline has an antidepressant effect that is likely related to changing the content of monoamines in the brain by inhibiting uptake-2 transporters.


Assuntos
Alcaloides/farmacologia , Antidepressivos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/metabolismo , Alcaloides/uso terapêutico , Animais , Antidepressivos/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Monoaminas Biogênicas/metabolismo , Transporte Biológico/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Doença Crônica , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estresse Psicológico/patologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
5.
PLoS Biol ; 17(9): e3000414, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31479441

RESUMO

Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS.


Assuntos
Síndrome de Bardet-Biedl/patologia , Proteínas do Citoesqueleto/metabolismo , Espinhas Dendríticas/patologia , Animais , Ansiedade , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/fisiopatologia , Síndrome de Bardet-Biedl/psicologia , Giro Denteado/fisiopatologia , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores , Feminino , Masculino , Memória , Camundongos , Receptor IGF Tipo 1/metabolismo , Sinaptossomos/metabolismo
6.
Neurochem Res ; 44(10): 2435-2447, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31535355

RESUMO

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.


Assuntos
Encéfalo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos , Alcaloides de Vinca/farmacologia , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Células Endoteliais/metabolismo , Peróxido de Hidrogênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Ratos Wistar , Sinaptossomos/metabolismo
7.
J Pharmacol Exp Ther ; 371(2): 526-543, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31413138

RESUMO

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.


Assuntos
Comportamento Aditivo/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Inibidores da Captação de Dopamina/administração & dosagem , Locomoção/efeitos dos fármacos , Metanfetamina/administração & dosagem , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , Animais , Comportamento Aditivo/metabolismo , Comportamento Aditivo/psicologia , Encéfalo/metabolismo , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Locomoção/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Autoadministração , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo
8.
Int J Biol Macromol ; 140: 49-58, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31421173

RESUMO

Naja spp. venom is a natural source of active compounds with therapeutic application potential. Phospholipase A2 (PLA2) is abundant in the venom of Naja spp. and can perform neurotoxicity, cytotoxicity, cardiotoxicity, and hematological disorders. The PLA2s from Naja spp. venoms are Asp 49 isoenzymes with the exception of PLA2 Cys 49 from Naja sagittifera. When looking at the functional aspects, the neurotoxicity occurs by PLA2 called ß-toxins that have affinity for phosphatidylcholine in nerve endings and synaptosomes membranes, and by α-toxins that block the nicotinic acetylcholine receptors in the neuromuscular junctions. In addition, these neurotoxins may inhibit K+ and Ca++ channels or even interfere with the Na+/K+/ATPase enzyme. The disturbance in the membrane fluidity also results in inhibition of the release of acetylcholine. The PLA2 can act as anticoagulants or procoagulant. The cytotoxicity exerted by PLA2s result from changes in the cardiomyocyte membranes, triggering cardiac failure and hemolysis. The antibacterial activity, however, is the result of alterations that decrease the stability of the lipid bilayer. Thus, the understanding of the structural and functional aspects of PLA2s can contribute to studies on the toxic and therapeutic mechanisms involved in the envenomation by Naja spp. and in the treatment of pathologies.


Assuntos
Proteínas Neurotóxicas de Elapídeos , Naja , Junção Neuromuscular/metabolismo , Fosfolipases A2 , Sinaptossomos/metabolismo , Animais , Proteínas Neurotóxicas de Elapídeos/química , Proteínas Neurotóxicas de Elapídeos/uso terapêutico , Junção Neuromuscular/patologia , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfolipases A2/química , Fosfolipases A2/uso terapêutico , Relação Estrutura-Atividade , Sinaptossomos/patologia
9.
Int J Mol Sci ; 20(15)2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31349638

RESUMO

Synaptosomes are used to decipher the mechanisms involved in chemical transmission, since they permit highlighting the mechanisms of transmitter release and confirming whether the activation of presynaptic receptors/enzymes can modulate this event. In the last two decades, important progress in the field came from the observations that synaptosomes retain changes elicited by both "in vivo" and "in vitro" acute chemical stimulation. The novelty of these studies is the finding that these adaptations persist beyond the washout of the triggering drug, emerging subsequently as functional modifications of synaptosomal performances, including release efficiency. These findings support the conclusion that synaptosomes are plastic entities that respond dynamically to ambient stimulation, but also that they "learn and memorize" the functional adaptation triggered by acute exposure to chemical agents. This work aims at reviewing the results so far available concerning this form of synaptosomal learning, also highlighting the role of these acute chemical adaptations in pathological conditions.


Assuntos
Adaptação Fisiológica , Aprendizagem , Memória , Terminações Pré-Sinápticas/fisiologia , Sinaptossomos/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Animais , Suscetibilidade a Doenças , Ácido Glutâmico/metabolismo , Humanos , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo
10.
J Pharmacol Exp Ther ; 371(1): 36-44, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31320495

RESUMO

Methamphetamine (MA) impairs vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) function and expression, increasing intracellular DA levels that lead to neurotoxicity. The trace amine-associated receptor 1 (TAAR1) is activated by MA, but when the receptor is not activated, MA-induced neurotoxicity is increased. To investigate interactions among TAAR1, VMAT2, and DAT, transporter function and expression were measured in transgenic Taar1 knockout (KO) and wild-type (WT) mice 24 hours following a binge-like regimen (four intraperitoneal injections, 2 hours apart) of MA (5 mg/kg) or the same schedule of saline treatment. Striatal synaptosomes were separated by fractionation to examine the function and expression of VMAT2 localized to cytosolic and membrane-associated vesicles. DAT was measured in whole synaptosomes. VMAT2-mediated [3H]DA uptake inhibition was increased in Taar1 KO mice in synaptosomal and vesicular fractions, but not the membrane-associated fraction, compared with Taar1 WT mice. There was no difference in [3H]dihydrotetrabenazine binding to the VMAT2 or [125I]RTI-55 binding to the DAT between genotypes, indicating activation of TAAR1 does not affect VMAT2 or DAT expression. There was also no difference between Taar1 WT and KO mice in DAT-mediated [3H]DA uptake inhibition following in vitro treatment with MA. These findings provide the first evidence of a TAAR1-VMAT2 interaction, as activation of TAAR1 mitigated MA-induced impairment of VMAT2 function, independently of change in VMAT2 expression. Additionally, the interaction is localized to intracellular VMAT2 on cytosolic vesicles and did not affect expression or function of DAT in synaptosomes or VMAT2 at the plasmalemmal surface, i.e., on membrane-associated vesicles. SIGNIFICANCE STATEMENT: Methamphetamine stimulates the G protein-coupled receptor TAAR1 to affect dopaminergic function and neurotoxicity. Here we demonstrate that a functional TAAR1 protects a specific subcellular fraction of VMAT2, but not the dopamine transporter, from methamphetamine-induced effects, suggesting new directions in pharmacotherapeutic development for neurodegenerative disorders.


Assuntos
Estimulantes do Sistema Nervoso Central/farmacocinética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Metanfetamina/farmacocinética , Síndromes Neurotóxicas/etiologia , Receptores Acoplados a Proteínas-G/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Animais , Estimulantes do Sistema Nervoso Central/toxicidade , Feminino , Masculino , Metanfetamina/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Síndromes Neurotóxicas/metabolismo , Ligação Proteica , Sinaptossomos/metabolismo
11.
Pharmacol Res Perspect ; 7(3): e00484, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31149340

RESUMO

Mutations in leucine-rich repeat kinase 2 (LRRK2) gene have been pathogenically linked to Parkinson's disease, and pharmacological inhibition of LRRK2 is being pursued to tackle nigro-striatal dopaminergic neurodegeneration. However, LRRK2 kinase inhibitors may have manifold actions, affecting not only pathological mechanisms in dopaminergic neurons but also physiological functions in nondopaminergic neurons. Therefore, we investigated whether LRRK2 kinase inhibitors differentially modulate dopamine and glutamate release from the mouse striatum and cerebral cortex. Spontaneous and KCl-evoked [3H]-dopamine and glutamate release from superfused synaptosomes obtained from wild-type and LRRK2 knock-out, kinase-dead or G2019S knock-in mice was measured. Two structurally unrelated inhibitors, LRRK2-IN-1 and GSK2578215A, were tested. LRRK2, phosphoSerine1292 and phosphoSerine935 LRRK2 levels were measured in all genotypes, and target engagement was evaluated by monitoring phosphoSerine935 LRRK2. LRRK2-IN-1 inhibited striatal glutamate but not dopamine release; GSK2578215A inhibited striatal dopamine and cortical glutamate but enhanced striatal glutamate release. LRRK2-IN-1 reduced striatal and cortical phosphoSerine935 levels whereas GSK2578215A inhibited only the former. Neither LRRK2 inhibitor affected neurotransmitter release in LRRK2 knock-out and kinase-dead mice; however, they facilitated dopamine without affecting striatal glutamate in G2019S knock-in mice. GSK2578215A inhibited cortical glutamate release in G2019S knock-in mice. We conclude that LRRK2-IN-1 and GSK2578215A modulate exocytosis by blocking LRRK2 kinase activity, although their effects vary depending on the nerve terminal examined. The G2019S mutation unravels a dopamine-promoting action of LRRK2 inhibitors while blunting their effects on glutamate release, which highlights their positive potential for the treatment of PD, especially of LRRK2 mutation carriers.


Assuntos
Aminopiridinas/farmacologia , Benzamidas/farmacologia , Benzodiazepinonas/farmacologia , Corpo Estriado/citologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Pirimidinas/farmacologia , Córtex Visual/citologia , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/metabolismo , Exocitose , Técnicas de Introdução de Genes , Ácido Glutâmico/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Masculino , Camundongos , Fosforilação , Serina/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Córtex Visual/efeitos dos fármacos , Córtex Visual/metabolismo
12.
Neuroscience ; 411: 185-201, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31158434

RESUMO

Astrocytes regulate extracellular glutamate homeostasis in the central nervous system through the Na+-dependent glutamate transporters glutamate transporter-1 (GLT-1) and glutamate aspartate transporter (GLAST). Impaired astrocyte glutamate uptake could contribute to the development of epilepsy but the regulation of glutamate transporters in epilepsy is not well understood. In this study, we investigate the expression of GLT-1 and GLAST in the mouse intrahippocampal kainic acid (IHKA) model of temporal lobe epilepsy (TLE). We used immunohistochemistry, synaptosomal fractionation and Western blot analysis at 1, 3, 7 and 30 days post-IHKA induced status epilepticus (SE) to examine changes in GLT-1 and GLAST immunoreactivity and synaptosomal expression during the development of epilepsy. We found a significant upregulation in GLT-1 immunoreactivity at 1 and 3 days post-IHKA in the ipsilateral dorsal hippocampus. However, GLT-1 immunoreactivity and synaptosomal protein levels were significantly downregulated at 7 days post-IHKA in the ipsilateral hippocampus, a time point corresponding to the onset of spontaneous seizures in this model. GLAST immunoreactivity was increased in specific layers at 1 and 3 days post-IHKA in the ipsilateral hippocampus. GLAST synaptosomal protein levels were significantly elevated at 30 days compared to 7 days post-IHKA in the ipsilateral hippocampus. Our findings suggest that astrocytic glutamate transporter dysregulation could contribute to the development of epilepsy.


Assuntos
Epilepsia do Lobo Temporal/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Hipocampo/metabolismo , Convulsões/metabolismo , Sinaptossomos/metabolismo , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/induzido quimicamente , Ácido Caínico , Camundongos , Convulsões/induzido quimicamente
13.
eNeuro ; 6(3)2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118206

RESUMO

Dendritic spines are the postsynaptic targets of excitatory synaptic inputs that undergo extensive proliferation and maturation during the first postnatal month in mice. However, our understanding of the molecular mechanisms that regulate spines during this critical period is limited. Previous work has shown that pannexin 1 (Panx1) regulates neurite growth and synaptic plasticity. We therefore investigated the impact of global Panx1 KO on spontaneous cortical neuron activity using Ca2+ imaging and in silico network analysis. Panx1 KO increased both the number and size of spontaneous co-active cortical neuron network ensembles. To understand the basis for these findings, we investigated Panx1 expression in postnatal synaptosome preparations from early postnatal mouse cortex. Between 2 and 4 postnatal weeks, we observed a precipitous drop in cortical synaptosome protein levels of Panx1, suggesting it regulates synapse proliferation and/or maturation. At the same time points, we observed significant enrichment of the excitatory postsynaptic density proteins PSD-95, GluA1, and GluN2a in cortical synaptosomes from global Panx1 knock-out mice. Ex vivo analysis of pyramidal neuron structure in somatosensory cortex revealed a consistent increase in dendritic spine densities in both male and female Panx1 KO mice. Similar findings were observed in an excitatory neuron-specific Panx1 KO line (Emx1-Cre driven; Panx1 cKOE) and in primary Panx1 KO cortical neurons cultured in vitro. Altogether, our study suggests that Panx1 negatively regulates cortical dendritic spine development.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Conexinas/fisiologia , Espinhas Dendríticas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Sinalização do Cálcio , Córtex Cerebral/metabolismo , Conexinas/genética , Conexinas/metabolismo , Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Imagem Óptica , Sinaptossomos/metabolismo , Sinaptossomos/fisiologia
14.
Eur J Pharmacol ; 855: 65-74, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31059709

RESUMO

Neuropathic pain is a common type of chronic pain caused by trauma or chemotherapy. However, this type of pain is undertreated. TsNTxP is a non-toxic protein isolated from the venom of the scorpion Tityus serrulatus, and it is structurally similar to neurotoxins that interact with voltage-gated sodium channels. However, the antinociceptive properties of this protein have not been characterized. The purpose of this study was to investigate the antinociceptive effects of TsNTxP in acute and neuropathic pain models. Male and female Swiss mice (25-30 g) were exposed to different models of acute pain (tail-flick test and nociception caused by capsaicin intraplantar injection) or neuropathic pain (chronic pain syndrome induced by paclitaxel or chronic constriction injury of the sciatic nerve). Hypersensitivity to mechanical or cold stimuli were evaluated in the models of neuropathic pain. The ability of TsNTxP to alter the release of glutamate in mouse spinal cord synaptosomes was also evaluated. The results showed that TsNTxP exerted antinociceptive effects in the tail-flick test to a thermal stimulus and in the intraplantar capsaicin administration model. Furthermore, TsNTxP was non-toxic and exerted antiallodynic effects in neuropathic pain models induced by chronic constriction injury of the sciatic nerve and administration of paclitaxel. TsNTxP reduced glutamate release from mouse spinal cord synaptosomes following stimulation with potassium chloride (KCl) or capsaicin. Thus, this T. serrulatus protein may be a promising non-toxic drug for the treatment of neuropathic pain.


Assuntos
Analgésicos/farmacologia , Proteínas de Artrópodes/farmacologia , Ácido Glutâmico/metabolismo , Venenos de Escorpião/química , Escorpiões , Analgésicos/uso terapêutico , Animais , Proteínas de Artrópodes/uso terapêutico , Feminino , Masculino , Camundongos , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Medula Espinal/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
15.
Neurobiol Dis ; 130: 104482, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31129085

RESUMO

In fragile X syndrome, the absence of Fragile X Mental Retardation Protein (FMRP) is known to alter postsynaptic function, although alterations in presynaptic function also occur. We found that the potentiation of glutamate release induced by the ß adrenergic receptor (ßAR) agonist isoproterenol is absent in cerebrocortical nerve terminals (synaptosomes) from mice lacking FMRP (Fmr1 KO), despite the normal cAMP generation. The glutamate release induced by moderate stimulation of synaptosomes with 5 mM KCl was not potentiated in Fmr1 KO synaptosomes by isoproterenol, nor by stimulating the receptor associated signaling pathway with the adenylyl cyclase activator forskolin or with the Epac activator 8-pCPT. Hence, the impairment in the pathway potentiating release is distal to ßARs. Electron microscopy shows that Fmr1 KO cortical synapses have more docked vesicles than WT synapses, consequently occluding the isoproterenol response through which more SVs approach the active zone (AZ) of the plasma membrane. Weak stimulation of synaptosomes with the Ca2+ ionophore ionomycin recovered the release potentiation driven by forskolin and 8-pCPT but not with isoproterenol, revealing an impairment in the efficiency of receptor generated cAMP to activate the release potentiation pathway. Indeed, inhibiting cyclic nucleotide phosphodiesterase PDE2A with BAY 60-7550 reestablished isoproterenol mediated potentiation in Fmr1 KO synaptosomes. Thus, the lack of ß-AR mediated potentiation of glutamate release appears to be the consequence of an impaired capability of the receptor to mobilize SVs to the AZ and because of a decreased efficiency of cAMP to activate the signaling pathway that enhances neurotransmitter release.


Assuntos
Síndrome do Cromossomo X Frágil/metabolismo , Ácido Glutâmico/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sinaptossomos/metabolismo
16.
Mol Neurobiol ; 56(11): 7368-7379, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31037646

RESUMO

When administered as a single subanesthetic dose, the N-methyl-D-aspartate (NMDA) receptor antagonist, ketamine, produces rapid (within hours) and relatively sustained antidepressant actions even in treatment-resistant patients. Preclinical studies have shown that ketamine increases dendritic spine density and synaptic proteins in brain areas critical for the actions of antidepressants, yet the temporal relationship between structural changes and the onset of antidepressant action remains poorly understood. In this study, we examined the effects of a single dose of S-ketamine (15 mg/kg) on dendritic length, dendritic arborization, spine density, and spine morphology in the Flinders Sensitive and Flinders Resistant Line (FSL/FRL) rat model of depression. We found that already 1 h after injection with ketamine, apical dendritic spine deficits in CA1 pyramidal neurons of FSL rats were completely restored. Notably, the observed increase in spine density was attributable to regulation of both mushroom and long-thin spines. In contrast, ketamine had no effect on dendritic spine density in FRL rats. On the molecular level, ketamine normalized elevated levels of phospho-cofilin and the NMDA receptor subunits GluN2A and GluN2B and reversed homer3 deficiency in hippocampal synaptosomes of FSL rats. Taken together, our data suggest that rapid formation of new spines may provide an important structural substrate during the initial phase of ketamine's antidepressant action.


Assuntos
Espinhas Dendríticas/patologia , Hipocampo/patologia , Ketamina/administração & dosagem , Ketamina/farmacologia , Fatores de Despolimerização de Actina/metabolismo , Animais , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Proteínas de Arcabouço Homer/metabolismo , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
17.
Am J Pathol ; 189(8): 1621-1636, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31108099

RESUMO

Apolipoprotein E (apoE) colocalizes with amyloid-ß (Aß) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aß are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aß complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aß complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aß was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)-positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aß concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aß was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aß complex and associated lipids into synaptic terminals, with subsequent Aß clearance in control synapses and accumulation in AD synapses.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E4/metabolismo , Apolipoproteínas E/metabolismo , Córtex Cerebral/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de LDL/metabolismo , Sinapses/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Córtex Cerebral/patologia , Proteína 4 Homóloga a Disks-Large/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sinapses/patologia , Sinaptossomos/metabolismo , Sinaptossomos/patologia
18.
J Med Food ; 22(7): 696-702, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30985232

RESUMO

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.


Assuntos
Cálcio/metabolismo , Córtex Cerebral/metabolismo , Ácido Glutâmico/metabolismo , Terminações Nervosas/metabolismo , Proteína Quinase C/metabolismo , Ácidos Sulfínicos/farmacologia , Animais , Córtex Cerebral/efeitos dos fármacos , Masculino , Terminações Nervosas/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Proteína Quinase C/genética , Ratos , Ratos Sprague-Dawley , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
19.
Neurosci Lett ; 705: 46-50, 2019 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-31005652

RESUMO

Desoxypipradrol (2-DPMP), a new psychoactive substance (NPS), acts as a norepinephrine-dopamine reuptake inhibitor (NDRI). NDRIs can be addictive due to their action mechanisms similar to cocaine and methamphetamine. However, there is a lack of scientific information regarding the exact dependency of 2-DPMP. Thus, the purpose of this study was to evaluate rewarding and reinforcing effects of 2-DPMP in rodents. The effective dose range of 2-DPMP was determined by climbing behavior test. To evaluate rewarding effects of 2-DPMP, conditioned place preference (CPP) test was performed at selected doses in mice. Self-administration (SA) test was then undertaken at two doses that caused the highest effects in the CPP test. Dopamine level changes were analyzed using synaptosomes in order to investigate effects of 2-DPMP on the central nervous system (CNS). Significant responses were observed in the climbing behavior test at doses of 0.1, 0.5, and 1 mg/kg by intraperitoneal injection (i.p.). In the CPP test, mice i.p. administered 2-DPMP at 1 mg/kg showed a significant preference in drug-paired compartment. In the SA test, mice intravenously given 0.1 mg/kg/infusion showed significantly higher active lever responses. Further, dopamine was increased in a dose-dependent manner. Taken together, these results suggest that 2-DPMP may act on the CNS and induce rewarding and reinforcing effects, indicating its dependence liability.


Assuntos
Condicionamento Psicológico/efeitos dos fármacos , Piperidinas/farmacologia , Recompensa , Autoadministração , Animais , Comportamento Animal/efeitos dos fármacos , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Masculino , Camundongos , Sinaptossomos/metabolismo
20.
Food Funct ; 10(5): 2720-2728, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31033966

RESUMO

Piperine is the crucial alkaloid component of black pepper (Piper nigrum Linn.) and has neuroprotective effects. Because inhibition of glutamatergic excitatory neurotransmission is a possible mechanism involved in neuroprotection, we investigated the effect of piperine on the 4-aminopyridine (4-AP)-evoked release of glutamate from rat hippocampal synaptosomes. Piperine inhibited 4-AP-evoked glutamate release, and the inhibition was prevented by the chelation of extracellular Ca2+ ions and a vesicular transporter inhibitor. Piperine reduced the 4-AP-evoked elevation of intrasynaptosomal Ca2+ levels but did not affect the synaptosomal membrane potential. In the presence of ω-conotoxin MVIIC, an N- and P/Q-type channel blocker, the piperine-mediated inhibition of 4-AP-evoked glutamate release was markedly reduced; however, dantrolene and CGP37157, which are intracellular Ca2+-release inhibitors, did not alter the piperine effect. In addition, immunocytochemical analysis confirmed the presence of presynaptic 5-hydroxytryptamine 1A (5-HT1A) receptor proteins. The glutamate release-inhibiting effect of piperine was discovered to be prevented by the 5-HT1A receptor antagonist WAY100635 and the G protein ßγ subunit inhibitor gallein; however, it was unaffected by the adenylate cyclase inhibitor SQ22536 or the protein kinase A inhibitor PKI622. These results suggest that piperine inhibits glutamate release from rat hippocampal nerve terminals by reducing Ca2+ influx through N- and P/Q-type Ca2+ channels and that the activation of presynaptic 5-HT1A receptors and the G protein ßγ subunit is involved in this effect.


Assuntos
Alcaloides/farmacologia , Benzodioxóis/farmacologia , Cálcio/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Receptor 5-HT1A de Serotonina/metabolismo , 4-Aminopiridina/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Hipocampo/efeitos dos fármacos , Masculino , Piper nigrum/química , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1A de Serotonina/genética , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
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