RESUMO
Amyloid beta (Aß) is linked to the pathology of Alzheimer's disease (AD). At physiological concentrations, Aß was proposed to enhance neuroplasticity and memory formation by increasing the neurotransmitter release from presynapse. However, the exact mechanisms underlying this presynaptic effect as well as specific contribution of endogenously occurring Aß isoforms remain unclear. Here, we demonstrate that Aß1-42 and Aß1-16, but not Aß17-42, increased size of the recycling pool of synaptic vesicles (SV). This presynaptic effect was driven by enhancement of endogenous cholinergic signalling via α7 nicotinic acetylcholine receptors, which led to activation of calcineurin, dephosphorylation of synapsin 1 and consequently resulted in reorganization of functional pools of SV increasing their availability for sustained neurotransmission. Our results identify synapsin 1 as a molecular target of Aß and reveal an effect of physiological concentrations of Aß on cholinergic modulation of glutamatergic neurotransmission. These findings provide new mechanistic insights in cholinergic dysfunction observed in AD.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Fragmentos de Peptídeos/farmacologia , Sinapses/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Cálcio/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotransmissores/metabolismo , Nicotina/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Vesículas Sinápticas/fisiologia , Receptor Nicotínico de Acetilcolina alfa7/deficiência , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
The antiepileptic/anticonvulsive action of brivaracetam is considered to occur via modulation of synaptic vesicle protein 2A (SV2A); however, the pharmacological mechanisms of action have not been fully characterised. To explore the antiepileptic/anticonvulsive mechanism of brivaracetam associated with SV2A modulation, this study determined concentration-dependent effects of brivaracetam on astroglial L-glutamate release associated with connexin43 (Cx43), tumour-necrosis factor-α (TNFα) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/glutamate receptor of rat primary cultured astrocytes using ultra-high-performance liquid chromatography. Furthermore, interaction among TNFα, elevated extracellular K+ and brivaracetam on expression of SV2A and Cx43 was determined using capillary immunoblotting. TNFα and elevated extracellular K+ predominantly enhanced astroglial L-glutamate release associated with respective AMPA/glutamate receptor and hemichannel. These effects were enhanced by a synergistic effect of TNFα and elevated extracellular K+ in combination. The activation of astroglial L-glutamate release, and expression of SV2A and Cx43 in the plasma membrane was suppressed by subchronic brivaracetam administration but were unaffected by acute administration. These results suggest that migration of SV2A to the astroglial plasma membrane by hyperexcitability activates astroglial glutamatergic transmission, perhaps via hemichannel activation. Subchronic brivaracetam administration suppressed TNFα-induced activation of AMPA/glutamate receptor and hemichannel via inhibition of ectopic SV2A. These findings suggest that combined inhibition of vesicular and ectopic SV2A functions contribute to the antiepileptic/anticonvulsive mechanism of brivaracetam action.
Assuntos
Astrócitos/metabolismo , Ácido Glutâmico/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Pirrolidinonas/farmacologia , Animais , Animais Recém-Nascidos , Anticonvulsivantes/farmacologia , Astrócitos/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Masculino , Proteínas do Tecido Nervoso/antagonistas & inibidores , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/metabolismo , Receptores de Glutamato/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Due to molecular mimicry, maternal antibacterial antibodies are suspected to promote neurodevelopmental changes in the offspring that finally can cause disorders like autism and schizophrenia. Using a human first trimester prenatal brain multiprotein array (MPA), we demonstrate here that antibodies to the digestive tract bacteria Helicobacter pylori (α-HPy) and Campylobacter jejuni (α-CJe) interact with different synaptic proteins, including the calcium sensor synaptotagmin 5 (Syt5). Interactions of both antisera with Syt5 were confirmed by Western blot with a HEK293-cells overexpression lysate of this protein. Immunofluorescence and Western blotting revealed SiMa cells to express Syt5, which also co-migrated with a band/spot labeled by either α-HPy or α-CJe. Functionally, a 12-h pretreatment of SiMa cells with 10 µg/ml of either α-HPy or α-CJe resulted in a significant reduction of acetylcholine(ACh)-dependent calcium signals as compared to controls. Also ACh-dependent vesicle recycling was significantly reduced in cells pretreated with either α-HPy or α-CJe. Similar effects were observed upon pretreatment of SiMa cells with Syt5-specific antibodies. In conclusion, the present study supports the view that prenatal maternal antibacterial immune responses towards HPy and by this to Syt5 are able to cause functional changes, which in the end might contribute also to neurodevelopmental disorders.
Assuntos
Anticorpos Antibacterianos/metabolismo , Neuroblastoma/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagminas/metabolismo , Anticorpos Antibacterianos/farmacologia , Sinalização do Cálcio , Linhagem Celular Tumoral , Células HEK293 , Helicobacter pylori/imunologia , Humanos , Ligação Proteica , Vesículas Sinápticas/efeitos dos fármacosRESUMO
Synaptic vesicles (SVs) can be pooled across multiple synapses, prompting questions about their dynamic allocation for neurotransmission and plasticity. We find that the axonal traffic of recycling vesicles is not supported by ubiquitous microtubule-based motility but relies on actin instead. Vesicles freed from synaptic clusters undergo ~1 µm bouts of active transport, initiated by nearby elongation of actin filaments. Long distance translocation arises when successive bouts of active transport were linked by periods of free diffusion. The availability of SVs for active transport can be promptly increased by protein kinase A, a key player in neuromodulation. Vesicle motion is in turn impeded by shutting off axonal actin polymerization, mediated by nitric oxide-cyclic GMP signaling leading to inhibition of RhoA. These findings provide a potential framework for coordinating post-and pre-synaptic strength, using retrograde regulation of axonal actin dynamics to mobilize and recruit presynaptic SV resources.
Assuntos
Citoesqueleto de Actina/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Óxido Nítrico/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Transporte Axonal/fisiologia , Transporte Biológico Ativo , Células Cultivadas , GMP Cíclico/fisiologia , Feminino , Hipocampo/citologia , Hipocampo/fisiologia , Proteínas Luminescentes/metabolismo , Masculino , Neurônios/fisiologia , Nocodazol/farmacologia , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/efeitos dos fármacosRESUMO
The brain nicotinic acetylcholine receptors (nAChRs) expressed in pre-synaptic nerve terminals regulate neurotransmitter release. However, there is no evidence for the expression of nAChRs in synaptic vesicles, which deliver neurotransmitter to synaptic cleft. The aim of this paper was to investigate the presence of nAChRs in synaptic vesicles purified from the rat brain and to study their possible involvement in vesicles life cycle. According to dynamic light scattering analysis, the antibody against extracellular domain (1-208) of α7 nAChR subunit inhibited synaptic vesicles clustering. Sandwich ELISA with nAChR subunit-specific antibodies demonstrated the presence of α4ß2, α7 and α7ß2nAChR subtypes in synaptic vesicles and showed that α7 and ß2 nAChR subunits are co-localized with synaptic vesicle glycoprotein 2A (SV2A). Pre-incubation with either α7-selective agonist PNU282987 or nicotine did not affect synaptic vesicles clustering but delayed their Ca2+-dependent fusion with the plasma membranes. In contrast, nicotine but not PNU282987 stimulated acidification of isolated synaptic vesicles, indicating that α4ß2 but not α7-containing nAChRs are involved in regulation of proton influx and neurotransmitter refilling. Treatment of rats with levetiracetam, a specific modulator of SV2A, increased the content of α7 nAChRs in synaptic vesicles accompanied by increased clustering but decreased Ca2+-dependent fusion. These data for the first time demonstrate the presence of nAChRs in synaptic vesicles and suggest an active involvement of cholinergic regulation in neurotransmitter release. Synaptic vesicles may be an additional target of nicotine inhaled upon smoking and of α7-specific drugs widely discussed as anti-inflammatory and pro-cognitive tools.
Assuntos
Encéfalo/metabolismo , Membrana Celular/metabolismo , Fusão de Membrana/fisiologia , Vesículas Sinápticas/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Feminino , Concentração de Íons de Hidrogênio , Masculino , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Ratos , Ratos Wistar , Vesículas Sinápticas/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidoresRESUMO
Environmental toxicants have the potential to contribute to the pathophysiology of multiple complex diseases, but the underlying mechanisms remain obscure. One such toxicant is the widely used fungicide ziram, a dithiocarbamate known to have neurotoxic effects and to increase the risk of Parkinson's disease. We have used Drosophila melanogaster as an unbiased discovery tool to identify novel molecular pathways by which ziram may disrupt neuronal function. Consistent with previous results in mammalian cells, we find that ziram increases the probability of synaptic vesicle release by dysregulation of the ubiquitin signaling system. In addition, we find that ziram increases neuronal excitability. Using a combination of live imaging and electrophysiology, we find that ziram increases excitability in both aminergic and glutamatergic neurons. This increased excitability is phenocopied and occluded by null mutant animals of the ether a-go-go (eag) potassium channel. A pharmacological inhibitor of the temperature sensitive hERG (human ether-a-go-go related gene) phenocopies the excitability effects of ziram but only at elevated temperatures. seizure (sei), a fly ortholog of hERG, is thus another candidate target of ziram. Taken together, the eag family of potassium channels emerges as a candidate for mediating some of the toxic effects of ziram. We propose that ziram may contribute to the risk of complex human diseases by blockade of human eag and sei orthologs, such as hERG.
Assuntos
Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Fungicidas Industriais/toxicidade , Neurônios/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Ziram/toxicidade , Animais , Drosophila melanogaster , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Neurônios/metabolismo , Neurotransmissores/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
Acute ethanol treatment induces neurodegeneration in cultured neurons and can lead to brain damage in animal models. Neuronal cells exposed to ethanol showed an increase in reactive oxygen species (ROS), oxidative damage and mitochondrial impairment contributing to synaptic failure. However, the underlying mechanisms of these events are not well understood. Here, we studied the contribution of NADPH oxidase, as a relevant source of ROS production in the brain, to mitochondrial impairment and oxidative stress induced by ethanol. We used primary hippocampal neurons subjected to an acute treatment of ethanol at increasing concentrations (25, 50, and 75 mM, 24 h), and we evaluated ROS production, mitochondrial function, and synaptic vesicle activity. Our studies showed that after ethanol administration, hippocampal neurons presented an increase in ROS levels, mitochondrial dysfunction, calcium handling defects, and synaptic impairment. Interestingly, treatment with the NADPH inhibitor, apocynin, significantly prevented oxidative stress, mitochondrial dysfunction, and the impairment of synaptic vesicle activity induced by ethanol treatment. These results indicate that NADPH oxidase could be a key participant in the molecular mechanism by which alcohol affects the brain.
Assuntos
Intoxicação Alcoólica/enzimologia , Intoxicação Alcoólica/patologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Mitocôndrias/efeitos dos fármacos , NADPH Oxidases , Neurônios/efeitos dos fármacos , Neurônios/patologia , Estresse Oxidativo , Trifosfato de Adenosina/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Gravidez , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Sinapses/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacosRESUMO
Oxidative stress triggers a lethal cascade, leading to Parkinson's disease by causing degeneration of dopaminergic neurons. In this study, eight antioxidants were screened for their neuroprotective effect on PC12 cells (pheochromocytoma cell line) under oxidative stress induced by salsolinol (OSibS). Hydroxytyrosol was found to be the strongest neuroprotective agent; it improved viability of PC12 cells by up to 81.69% under OSibS. Afterward, two synaptic vesicle proteins, synapsin-1 and septin-5, were screened for their neuroprotective role; the overexpression of synapsin-1 and the downregulation of septin-5 separately improved the viability of PC12 cells by up to 71.17% and 67.00%, respectively, compared to PC12 cells only treated with salsolinol (PoTwS) under OSibS. Subsequently, the PC12+syn++sep- cell line was constructed and pretreated with 100 µM hydroxytyrosol, which improved its cell viability by up to 99.03% and led to 14.71- and 6.37-fold reductions in the levels of MDA and H2O2, respectively, and 6.8-, 12.97-, 10.57-, and 7.57-fold increases in the activity of catalase, glutathione reductase, superoxide dismutase, and glutathione peroxidase, respectively, compared to PoTwS under OSibS. Finally, alcohol dehydrogenase-6 from Saccharomyces cerevisiae was expressed in PC12+syn++sep- cells to convert 3,4-dihydroxyphenylacetaldehyde (an endogenous neurotoxin) into hydroxytyrosol. The PC12+syn++sep-+ADH6+ cell line also led to 22.38- and 12.33-fold decreases in the production of MDA and H2O2, respectively, and 7.15-, 13.93-, 12.08-, and 8.11-fold improvements in the activity of catalase, glutathione reductase, superoxide dismutase, and glutathione peroxidase, respectively, compared to PoTwS under OSibS. Herein, we report the endogenous production of a powerful antioxidant, hydroxytyrosol, from 3,4-dihydroxyphenylacetaldehyde, and evaluate its synergistic neuroprotective effect, along with synapsin-1 and septin-5, on PC12 cells under OSibS.
Assuntos
Neoplasias das Glândulas Suprarrenais/patologia , Isoquinolinas/toxicidade , Fármacos Neuroprotetores/farmacologia , Álcool Feniletílico/análogos & derivados , Feocromocitoma/patologia , Vesículas Sinápticas/metabolismo , Animais , Catalase/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Dopamina/metabolismo , Sinergismo Farmacológico , Flavonoides/farmacologia , Glutationa Peroxidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Metaboloma , Células PC12 , Álcool Feniletílico/farmacologia , Ratos , Superóxido Dismutase/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Heightened activity of glycogen synthase kinase-3ß (GSK-3ß) is linked to the degeneration of dopaminergic neurons in Parkinson's disease (PD). Phytic acid (PA), a naturally occurring compound with potent antioxidant property, has been shown to confer neuroprotection on dopaminergic neurons in PD. However, the underlying mechanism remains unclear. In the present study, MPTP and MPP+ treatments were used to model PD in mice and SH-SY5Y cells, respectively. We observed reduced tissue dopamine, disrupted synaptic vesicle recycling, and defective neurotransmitter exocytosis. Furthermore, expression of GSK-3ß was upregulated while that of ß-catenin was downregulated, concentration of cytosolic calcium was increased, and expressions of two dopamine carriers, dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) were decreased. PA treatment attenuated the MPTP-induced upregulation of GSK-3ß, increase in cytosolic calcium concentration, decreases in the levels of DAT, VMAT2, tissue dopamine, and synaptic vesicle recycling. Importantly, disturbances in synaptic vesicle recycling are thought to be early events in PD pathology. These findings suggest that PA is a promising therapeutic agent to treat early events in PD.
Assuntos
Antiparkinsonianos/uso terapêutico , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Intoxicação por MPTP/tratamento farmacológico , Ácido Fítico/uso terapêutico , Vesículas Sinápticas/efeitos dos fármacos , Animais , Antiparkinsonianos/farmacologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/biossíntese , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/biossíntese , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Intoxicação por MPTP/metabolismo , Camundongos Endogâmicos C57BL , Neuroblastoma/patologia , Ácido Fítico/farmacologia , Teste de Desempenho do Rota-Rod , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/biossíntese , Proteínas Vesiculares de Transporte de Monoamina/genética , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
The dendrites of retinal ganglion cells (RGCs) synapse with the axon terminals of bipolar cells in the inner plexiform layer (IPL). Changes in the RGC dendrites and synapses between the bipolar cells in the inner retinal layer may critically alter the function of RGCs in glaucoma. The present study attempted to discover changes in the synapse using brain-derived neurotrophic factor (BDNF) after glaucoma induction by chronic intraocular pressure elevation in a rat model. Immunohistochemical staining revealed that the BDNF-injected group had a significant increase in the level of synaptophysin, which is a presynaptic vesicle protein, in the innermost IPL compared with the phosphate-buffered saline (PBS)-injected group. SMI-32, which is a marker of RGCs, was colocalized with synaptophysin in RGC dendrites, and this colocalization significantly increased in the BDNF-injected group. After the induction of glaucoma, the BDNF-injected group exhibited increases in the total number of ribbon synapses, as seen using electron microscopy. Expression of calcium/calmodulin-dependent protein kinase II (CaMKII), cAMP-response element binding protein (CREB) and F-actin, which are key molecules involved in synaptic changes were upregulated after BDNF injection. These initial findings show the capability of BDNF to induce beneficial synaptic changes in glaucoma.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Glaucoma/fisiopatologia , Pressão Intraocular/fisiologia , Plasticidade Neuronal , Sinapses/metabolismo , Actinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Modelos Animais de Doenças , Glaucoma/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Pressão Intraocular/efeitos dos fármacos , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Sinaptofisina/metabolismoRESUMO
Hydrogen peroxide (H2O2) is one of the reactive oxygen species (ROS), endogenously produced during metabolism, which acts as a second messenger. In skeletal muscles, hypoxia- or hyperthermia-induced increase in H2O2 might affect synaptic transmission by targeting the most redox-sensitive presynaptic compartment (Giniatullin et al., 2006). However, the effects of H2O2 as a signal molecule have not previously been studied in different patterns of the synaptic activity. Here, using optical and microelectrode recording of synaptic vesicle exocytosis, we studied the use-dependent action of low concentrations of H2O2 and other oxidants in the mouse neuromuscular junction. We found that: (i) H2O2 at low micromole concentrations inhibited both spontaneous and evoked transmitter releases from the motor nerve terminals in a use-dependent manner, (ii) the antioxidant N-acetylcysteine (NAC) eliminated these depressant effects, (iii) the influence of H2O2 was not associated with lipid oxidation suggesting a pure signaling action, (iv) the intracellular oxidant Chloramine-T or (v) the glutathione depletion produced similar to H2O2 depressant effects. Taken together, our data revealed the effective inhibition of neurotransmitter release by ROS, which was proportional to the intensity of synaptic activity at the neuromuscular junction. The combination of various oxidants suggested an intracellular location for redox-sensitive sites responsible for modulation of the synaptic transmission in the skeletal muscle.
Assuntos
Peróxido de Hidrogênio/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Oxidantes/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Cloraminas/metabolismo , Diafragma/efeitos dos fármacos , Diafragma/inervação , Diafragma/metabolismo , Relação Dose-Resposta a Droga , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Feminino , Glutationa/metabolismo , Masculino , Lipídeos de Membrana/metabolismo , Camundongos , Junção Neuromuscular/fisiologia , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/fisiologia , Técnicas de Cultura de Tecidos , Compostos de Tosil/metabolismoRESUMO
Accumulating evidence suggests that the lesions of Parkinson's disease (PD) expand due to transneuronal spreading of fibrils composed of misfolded alpha-synuclein (a-syn), over the course of 5-10 years. However, the precise mechanisms and the processes underlying the spread of these fibril seeds have not been clarified in vivo. Here, we investigated the speed of a-syn transmission, which has not been a focus of previous a-syn transmission experiments, and whether a-syn pathologies spread in a neural circuit-dependent manner in the mouse brain. We injected a-syn preformed fibrils (PFFs), which are seeds for the propagation of a-syn deposits, either before or after callosotomy, to disconnect bilateral hemispheric connections. In mice that underwent callosotomy before the injection, the propagation of a-syn pathology to the contralateral hemisphere was clearly reduced. In contrast, mice that underwent callosotomy 24 h after a-syn PFFs injection showed a-syn pathology similar to that seen in mice without callosotomy. These results suggest that a-syn seeds are rapidly disseminated through neuronal circuits immediately after seed injection, in a prion-like seeding experiment in vivo, although it is believed that clinical a-syn pathologies take years to spread throughout the brain. In addition, we found that botulinum toxin B blocked the transsynaptic transmission of a-syn seeds by specifically inactivating the synaptic vesicle fusion machinery. This study offers a novel concept regarding a-syn propagation, based on the Braak hypothesis, and also cautions that experimental transmission systems may be examining a unique type of transmission, which differs from the clinical disease state.
Assuntos
Encéfalo/patologia , Rede Nervosa/patologia , Neurônios/metabolismo , Doença de Parkinson/patologia , Doenças Priônicas/patologia , alfa-Sinucleína/metabolismo , Amiloide/toxicidade , Animais , Toxinas Botulínicas Tipo A/metabolismo , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Lateralidade Funcional , Humanos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Neurônios/patologia , Transporte Proteico , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/fisiologia , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Riluzole is a potent neuroprotective agent which primarily inhibits excitatory neurotransmission interfering with presynaptic release, uptake and postsynaptic actions of glutamate by mechanisms that are not well understood. Riluzole and related prodrugs with improved blood brain barrier penetrance, are shown to be effective for the treatment of amyotrophic lateral sclerosis, ataxias, epilepsy and mood disorders. Our study was undertaken to decipher molecular and subcellular mechanisms of riluzole's antiglutamatergic effect, particularly focusing on presynaptic active zone structure and function. Applying multifarious live cell imaging techniques and amperometric glutamate recordings, we measured the impact of riluzole on presynaptic activity, synaptic vesicle recycling and glutamate release. Our in vitro and in vivo data revealed a unique mechanism whereby riluzole reduces the efficacy of glutamatergic transmission by selectively lowering the size of the readily releasable pool. This effect was correlated with the inhibition of protein kinase C-dependent Munc18-1 phosphorylation which is known to interfere with neurotransmitter release.
Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Fármacos Neuroprotetores/farmacologia , Riluzol/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Animais , Antígeno CD146/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Proteína Quinase C/metabolismo , Ratos Wistar , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismoRESUMO
Our lab has previously shown that nitric oxide (NO) can alter the synaptic response properties of amacrine cells by releasing Cl- from internal acidic compartments. This alteration in the Cl- gradient brings about a positive shift in the reversal potential of the GABA-gated current, which can convert inhibitory synapses into excitatory synapses. Recently, we have shown that the cystic fibrosis transmembrane regulator (CFTR) Cl- channel is involved in the Cl- release. Here, we test the hypothesis that (acidic) synaptic vesicles are a source of NO-releasable Cl- in chick retinal amacrine cells. If SVs are a source of Cl-, then depleting synaptic vesicles should decrease the nitric oxide-dependent shift in the reversal potential of the GABA-gated current. The efficacy of four inhibitors of dynamin (dynasore, Dyngo 4a, Dynole 34-2, and MiTMAB) were evaluated. In order to deplete synaptic vesicles, voltage-steps were used to activate V-gated Ca2+ channels and stimulate the synaptic vesicle cycle either under control conditions or after treatment with the dynamin inhibitors. Voltage-ramps were used to measure the NO-dependent shift in the reversal potential of the GABA-gated currents under both conditions. Our results reveal that activating the synaptic vesicle cycle in the presence of dynasore or Dyngo 4a blocked the NO-dependent shift in EGABA. However, we also discovered that some dynamin inhibitors reduced Ca2+ signaling and L-type Ca2+ currents. Conversely, dynasore also increased neurotransmitter release at autaptic sites. To further resolve the mechanism underlying the inhibition of the NO-dependent shift in the reversal potential for the GABA-gated currents, we also tested the effects of the clathrin assembly inhibitor Pitstop 2 and found that this compound also inhibited the shift. These data provide evidence that dynamin inhibitors have multiple effects on amacrine cell synaptic transmission. These data also suggest that inhibition of endocytosis disrupts the ability of NO to elicit Cl- release from internal stores which may in part be due to depletion of synaptic vesicles.
Assuntos
Células Amácrinas/metabolismo , Cetilpiridínio/metabolismo , Dinaminas/metabolismo , Endocitose , Óxido Nítrico/metabolismo , Vesículas Sinápticas/metabolismo , Actinas/metabolismo , Células Amácrinas/efeitos dos fármacos , Animais , Ânions/metabolismo , Proteínas Aviárias/metabolismo , Canais de Cálcio/metabolismo , Técnicas de Cultura de Células , Embrião de Galinha , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dinaminas/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Vesículas Sinápticas/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
(±)-cis-4,4'-Dimethylaminorex (4,4'-DMAR) is a new psychoactive substance (NPS) that has been associated with 31 fatalities and other adverse events in Europe between June 2013 and February 2014. We used in vitro uptake inhibition and transporter release assays to determine the effects of 4,4'-DMAR on human high-affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). In addition, we assessed its binding affinities to monoamine receptors and transporters. Furthermore, we investigated the interaction of 4,4'-DMAR with the vesicular monoamine transporter 2 (VMAT2) in rat phaeochromocytoma (PC12) cells and synaptic vesicles prepared from human striatum. 4,4'-DMAR inhibited uptake mediated by human DAT, NET or SERT, respectively in the low micromolar range (IC50 valuesâ¯<â¯2⯵M). Release assays identified 4,4'-DMAR as a substrate type releaser, capable of inducing transporter-mediated reverse transport via DAT, NET and SERT. Furthermore, 4,4'-DMAR inhibited both the rat and human isoforms of VMAT2 at a potency similar to 3,4-methylenedioxymethylamphetamine (MDMA). This study identified 4,4'-DMAR as a potent non-selective monoamine releasing agent. In contrast to the known effects of aminorex and 4-methylaminorex, 4,4'-DMAR exerts profound effects on human SERT. The latter finding is consistent with the idea that fatalities associated with its abuse may be linked to monoaminergic toxicity including serotonin syndrome. The activity at VMAT2 suggests that chronic abuse of 4,4'-DMAR may result in long-term neurotoxicity.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Oxazóis/farmacologia , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Animais , Estimulantes do Sistema Nervoso Central/química , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Células HEK293 , Humanos , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Oxazóis/química , Células PC12 , Ratos , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidoresRESUMO
We previously reported that l-Cysteine, an H2S donor, significantly alleviated brain injury after hypoxia-ischemic (HI) injury in neonatal mice. However, the mechanisms underlying this neuroprotective effect of l-Cysteine against HI insult remain unknown. In the present study, we tested the hypothesis that the protective effects of l-Cysteine are associated with glial responses and autophagy, and l-Cysteine attenuates synaptic injury as well as behavioral deficits resulting from HI. Consistent with our previous findings, we found that treatment with l-Cysteine after HI reduced early brain injury, improved behavioral deficits and synaptic damage, effects which were associated with an up-regulation of synaptophysin and postsynaptic density protein 95 expression in the lesioned cortex. l-Cysteine attenuated the accumulation of CD11b+/CD45high cells, activation of microglia and astrocytes and diminished HI-induced increases in reactive oxygen species and malondialdehyde within the lesioned cortex. In addition, l-Cysteine increased microtubule associated protein 1 light chain 3-II and Beclin1 expression, decreased p62 expression and phosphor-mammalian target of rapamycin and phosphor-signal transducer and activator of transcription 3. Further support for a critical role of l-Cysteine was revealed from results demonstrating that treatment with an inhibitor of the H2S-producing enzyme, amino-oxyacetic acid, reversed the beneficial effects of l-Cysteine described above. These results demonstrate that l-Cysteine effectively alleviates HI injury and improves behavioral outcomes by inhibiting reactive glial responses and synaptic damage and an accompanying triggering of autophagic flux. Accordingly, l-Cysteine may provide a new a therapeutic approach for the treatment of HI via the formation of H2S.
Assuntos
Cisteína/farmacologia , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/metabolismo , Ácido Amino-Oxiacético/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Autofagia/efeitos dos fármacos , Cisteína/metabolismo , Sulfeto de Hidrogênio , Hipóxia , Camundongos , Microglia/metabolismo , Neuroglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Vesículas Sinápticas/efeitos dos fármacos , Sinaptofisina/análiseRESUMO
Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology.
Assuntos
Comportamento Animal , Proteínas de Transporte/metabolismo , Endocitose , Cinesinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Comportamento Social , Animais , Células COS , Chlorocebus aethiops , Cognição , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Dineínas/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Ácido Glutâmico/farmacologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Proteínas de Membrana , Camundongos Knockout , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismoRESUMO
The relative contributions of glycolysis and oxidative phosphorylation to neuronal presynaptic energy demands are unclear. In rat hippocampal neurons, ATP production by either glycolysis or oxidative phosphorylation alone sustained basal evoked synaptic transmission for up to 20 min. However, combined inhibition of both ATP sources abolished evoked transmission. Neither action potential propagation failure nor depressed Ca2+ influx explained loss of evoked synaptic transmission. Rather, inhibition of ATP synthesis caused massive spontaneous vesicle exocytosis, followed by arrested endocytosis, accounting for the disappearance of evoked postsynaptic currents. In contrast to its weak effects on basal transmission, inhibition of oxidative phosphorylation alone depressed recovery from vesicle depletion. Local astrocytic lactate shuttling was not required. Instead, either ambient monocarboxylates or neuronal glycolysis was sufficient to supply requisite substrate. In summary, basal transmission can be sustained by glycolysis, but strong presynaptic demands are met preferentially by oxidative phosphorylation, which can be maintained by bulk but not local monocarboxylates or by neuronal glycolysis.SIGNIFICANCE STATEMENT Neuronal energy levels are critical for proper CNS function, but the relative roles for the two main sources of ATP production, glycolysis and oxidative phosphorylation, in fueling presynaptic function in unclear. Either glycolysis or oxidative phosphorylation can fuel low-frequency synaptic function and inhibiting both underlies loss of synaptic transmission via massive vesicle release and subsequent failure to endocytose lost vesicles. Oxidative phosphorylation, fueled by either glycolysis or endogenously released monocarboxylates, can fuel more metabolically demanding tasks such as vesicle recovery after depletion. Our work demonstrates the flexible nature of fueling presynaptic function to maintain synaptic function.
Assuntos
Trifosfato de Adenosina/metabolismo , Neurônios/fisiologia , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células Cultivadas , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Inibidores Enzimáticos/farmacologia , Glicólise/efeitos dos fármacos , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Potenciais Sinápticos/efeitos dos fármacos , Potenciais Sinápticos/fisiologia , Vesículas Sinápticas/efeitos dos fármacosRESUMO
Variance analysis of postsynaptic current amplitudes suggests the presence of distinct docking sites (also called release sites) where vesicles pause before exocytosis. Docked vesicles participate in the readily releasable pool (RRP), but the relation between docking site number and RRP size remains unclear. It is also unclear whether all vesicles of the RRP are equally release competent, and what cellular mechanisms underlie RRP renewal. We address here these questions at single glutamatergic synapses, counting released vesicles using deconvolution. We find a remarkably low variance of cumulative vesicle counts during action potential trains. This, combined with Monte Carlo simulations, indicates that vesicles transit through two successive states before exocytosis, so that the RRP is up to 2-fold higher than the docking site number. The transition to the second state has a very rapid rate constant, and is specifically inhibited by latrunculin B and blebbistatin, suggesting the involvement of actin and myosin.
Assuntos
Actinas/metabolismo , Exocitose , Miosinas/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Actinas/antagonistas & inibidores , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cerebelo/fisiologia , Ácido Egtázico/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Fusão de Membrana/efeitos dos fármacos , Modelos Neurológicos , Método de Monte Carlo , Miosinas/antagonistas & inibidores , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Vesículas Sinápticas/efeitos dos fármacos , Tiazolidinas/farmacologiaRESUMO
Post mortem studies on familial and sporadic Parkinson's disease patient striatal tissue have shown that nearly 90% of α-synuclein deposited in Lewy-bodies is phosphorylated at serine-129 (pSyn-129) as opposed to only 4% in normal human brain. We aimed to find the influence of endogenous neurotoxin 6-hydroxydopamine (6-OHDA) on α-synuclein phosphorylation, resting vesicles, and vesicular dopamine release. The relative distribution of pSyn-129+ cells in apoptotic and non-apoptotic populations at different 6-OHDA concentrations was assessed along with changes in oxidant-antioxidant system, mitochondrial membrane-potential, and intracellular-Ca2+ . Exposing SH-SY5Y cells to different concentrations of 6-OHDA for 48 h showed cell-death and apoptosis. Immunocytochemical analysis indicated an increase in pSyn-129 with increasing 6-OHDA concentration, and ELISA-estimation showed a significant increase in the pSyn-129 to α-synuclein ratio. FACS analysis also showed a significant increase in pSyn-129; and at sub-lethal 6-OHDA concentrations, pSyn-129+ cells were primarily distributed in the non-apoptotic population, suggesting that phosphorylation of α-synuclein precedes apoptosis. At higher 6-OHDA concentrations, the pSyn-129+ cell count significantly increased in the apoptotic population and decreased in the non-apoptotic population. Cytosolic co-localization of α-synuclein and ubiquitin was noticed at higher doses of 6-OHDA. FACS analysis showed decrease in vesicular monoamine transporter-2 (VMAT2) expression in 6-OHDA-treated cells, confirmed by reduction in functional dopamine-release on KCl and ATP stimulation. Significant decrease in VMAT2 expression and vesicular dopamine-release were observed with the lower 6-OHDA concentration, together with mild occurrence of apoptosis and significant increase in phosphorylated α-synuclein. This suggests that at sub-lethal 6-OHDA concentrations, the decrease in resting vesicles (VMAT2) and vesicular dopamine release are not attributable to apoptotic cell death and occur concomitantly with the phosphorylation of α-synuclein. J. Cell. Biochem. 117: 2719-2736, 2016. © 2016 Wiley Periodicals, Inc.