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1.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502320

RESUMO

Retinitis pigmentosa (RP) is a family of inherited disorders caused by the progressive degeneration of retinal photoreceptors. There is no cure for RP, but recent research advances have provided promising results from many clinical trials. All these therapeutic strategies are focused on preserving existing photoreceptors or substituting light-responsive elements. Vision recovery, however, strongly relies on the anatomical and functional integrity of the visual system beyond photoreceptors. Although the retinal structure and optic pathway are substantially preserved at least in early stages of RP, studies describing the visual cortex status are missing. Using a well-established mouse model of RP, we analyzed the response of visual cortical circuits to the progressive degeneration of photoreceptors. We demonstrated that the visual cortex goes through a transient and previously undescribed alteration in the local excitation/inhibition balance, with a net shift towards increased intracortical inhibition leading to improved filtering and decoding of corrupted visual inputs. These results suggest a compensatory action of the visual cortex that increases the range of residual visual sensitivity in RP.


Assuntos
Neurotransmissores/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Retinite Pigmentosa/patologia , Sinaptossomos/patologia , Córtex Visual/fisiopatologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Retinite Pigmentosa/etiologia , Retinite Pigmentosa/metabolismo , Sinaptossomos/metabolismo
2.
Sci Rep ; 11(1): 18583, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545174

RESUMO

Synaptosomes are subcellular fractions prepared from brain tissues that are enriched in synaptic terminals, widely used for the study of neural transmission and synaptic dysfunction. Immunofluorescence imaging is increasingly applied to synaptosomes to investigate protein localization. However, conventional methods for imaging synaptosomes over glass coverslips suffer from formaldehyde-induced aggregation. Here, we developed a facile strategy to capture and image synaptosomes without aggregation artefacts. First, ethylene glycol bis(succinimidyl succinate) (EGS) is chosen as the chemical fixative to replace formaldehyde. EGS/glycine treatment makes the zeta potential of synaptosomes more negative. Second, we modified glass coverslips with 3-aminopropyltriethoxysilane (APTES) to impart positive charges. EGS-fixed synaptosomes spontaneously attach to modified glasses via electrostatic attraction while maintaining good dispersion. Individual synaptic terminals are imaged by conventional fluorescence microscopy or by super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM). We examined tau protein by two-color and three-color dSTORM to understand its spatial distribution within mouse cortical synapses, observing tau colocalization with synaptic vesicles as well postsynaptic densities.


Assuntos
Microscopia de Fluorescência/métodos , Sinaptossomos/metabolismo , Proteínas tau/metabolismo , Animais , Camundongos , Densidade Pós-Sináptica/metabolismo , Eletricidade Estática , Sinapses/metabolismo , Sinaptofisina/metabolismo
3.
Biomolecules ; 11(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34356653

RESUMO

Rosmarinic acid, a major component of rosemary, is a polyphenolic compound with potential neuroprotective effects. Asreducing the synaptic release of glutamate is crucial to achieving neuroprotectant's pharmacotherapeutic effects, the effect of rosmarinic acid on glutamate release was investigated in rat cerebrocortical nerve terminals (synaptosomes). Rosmarinic acid depressed the 4-aminopyridine (4-AP)-induced glutamate release in a concentration-dependent manner. The removal of extracellular calcium and the blockade of vesicular transporters prevented the inhibition of glutamate release by rosmarinic acid. Rosmarinic acid reduced 4-AP-induced intrasynaptosomal Ca2+ elevation. The inhibition of N-, P/Q-type Ca2+ channels and the calcium/calmodulin-dependent kinase II (CaMKII) prevented rosmarinic acid from having effects on glutamate release. Rosmarinic acid also reduced the 4-AP-induced activation of CaMKII and the subsequent phosphorylation of synapsin I, the main presynaptic target of CaMKII. In addition, immunocytochemistry confirmed the presence of GABAA receptors. GABAA receptor agonist and antagonist blocked the inhibitory effect of rosmarinic acid on 4-AP-evoked glutamate release. Docking data also revealed that rosmarinic acid formed a hydrogen bond with the amino acid residues of GABAA receptor. These results suggested that rosmarinic acid activates GABAA receptors in cerebrocortical synaptosomes to decrease Ca2+ influx and CaMKII/synapsin I pathway to inhibit the evoked glutamate release.


Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Ácido Glutâmico/metabolismo , Sinaptossomos/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cinamatos/química , Depsídeos/química , Agonistas de Receptores de GABA-A/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Sinaptossomos/metabolismo
4.
Cells ; 10(7)2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34359897

RESUMO

Phagocytosis is one of the most important physiological functions of the glia directed at maintaining a healthy, homeostatic environment in the brain. Under a homeostatic environment, the phagocytic activities of astrocytes and microglia are tightly coordinated in time and space. In neurodegenerative diseases, both microglia and astrocytes contribute to neuroinflammation and disease pathogenesis, however, whether their phagocytic activities are up- or downregulated in reactive states is not known. To address this question, this current study isolated microglia and astrocytes from C57BL/6J mice infected with prions and tested their phagocytic activities in live-cell imaging assays that used synaptosomes and myelin debris as substrates. The phagocytic uptake by the reactive microglia was found to be significantly upregulated, whereas that of the reactive astrocytes was strongly downregulated. The up- and downregulation of phagocytosis by the two cell types were observed irrespective of whether disease-associated synaptosomes, normal synaptosomes, or myelin debris were used in the assays, indicating that dysregulations are dictated by cell reactive states, not substrates. Analysis of gene expression confirmed dysregulation of phagocytic functions in both cell types. Immunostaining of animal brains infected with prions revealed that at the terminal stage of disease, neuronal cell bodies were subject to engulfment by reactive microglia. This study suggests that imbalance in the phagocytic activities of the reactive microglia and astrocytes, which are dysregulated in opposite directions, is likely to lead to excessive microglia-mediated neuronal death on the one hand, and the inability of astrocytes to clear cell debris on the other hand, contributing to the neurotoxic effects of glia as a whole.


Assuntos
Astrócitos/patologia , Microglia/patologia , Fagocitose , Doenças Priônicas/patologia , Animais , Encéfalo/patologia , Células Cultivadas , Regulação para Baixo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Neurônios/patologia , Sinaptossomos/metabolismo , Sinaptossomos/ultraestrutura , Regulação para Cima
5.
J Neurochem ; 158(5): 1074-1082, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34273193

RESUMO

Imbalance in the metabolic pathway linking excitatory and inhibitory neurotransmission has been implicated in multiple psychiatric and neurologic disorders. Recently, we described enantiomer-specific effects of 2-methylglutamate, which is not decarboxylated to the corresponding methyl analogue of gamma-aminobutyric acid (GABA): 4-aminopentanoic acid (4APA). Here, we tested the hypothesis that 4APA also has enantiomer-specific actions in brain. Mouse cerebral synaptosome uptake (nmol/mg protein over 30 min) of (R)-4APA or (S)-4APA was time and temperature dependent; however, the R enantiomer had greater uptake, reduction of endogenous GABA concentration, and release following membrane depolarization than did the S enantiomer. (S)-4APA exhibited some weak agonist (GABAA α4ß3δ, GABAA α5ß2γ2, and GABAB B1/B2) and antagonist (GABAA α6ß2γ2) activity while (R)-4APA showed weak agonist activity only with GABAA α5ß2γ2. Both 4APA enantiomers (100 mg/kg IP) were detected in mouse brain 10 min after injection, and by 1 hr had reached concentrations that were stable over 6 hr; both enantiomers were cleared rapidly from mouse serum over 6 hr. Two-month-old mice had no mortality following 100-900 mg/kg IP of each 4APA enantiomer but did have similar dose-dependent reduction in distance moved in a novel cage. Neither enantiomer at 30 or 100 mg/kg impacted outcomes in 23 measures of well-being, activity chamber, or withdrawal from hot plate. Our results suggest that enantiomers of 4APA are active in mouse brain, and that (R)-4APA may act as a novel false neurotransmitter of GABA. Future work will focus on disease models and on possible applications as neuroimaging agents.


Assuntos
Comportamento Exploratório/fisiologia , Locomoção/fisiologia , Neurotransmissores/química , Ácidos Pentanoicos/química , Ácido gama-Aminobutírico/química , Animais , Encéfalo/metabolismo , Química Encefálica , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurotransmissores/metabolismo , Ácidos Pentanoicos/metabolismo , Ácidos Pentanoicos/farmacologia , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Estereoisomerismo , Sinaptossomos/metabolismo , Ácido gama-Aminobutírico/metabolismo
6.
J Neurochem ; 159(1): 156-171, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34309872

RESUMO

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


Assuntos
Encéfalo/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Caracteres Sexuais , Sinaptossomos/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Peptídeos Cíclicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Sinaptossomos/efeitos dos fármacos
7.
Cells ; 10(5)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065927

RESUMO

Synaptic plasticity events, including long-term potentiation (LTP), are often regarded as correlates of brain functions of memory and cognition. One of the central players in these plasticity-related phenomena is the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR). Increased levels of AMPARs on postsynaptic membranes thus constitute a biochemical measure of LTP. Isolated synaptic terminals (synaptosomes) are an excellent ex vivo tool to monitor synaptic physiology in healthy and diseased brains, particularly in human research. We herein describe three protocols for chemically-induced LTP (cLTP) in synaptosomes from both rodent and human brain tissues. Two of these chemical stimulation protocols are described for the first time in synaptosomes. A pharmacological block of synaptosomal actin dynamics confirmed the efficiency of the cLTP protocols. Furthermore, the study prototypically evaluated the deficiency of cLTP in cortical synaptosomes obtained from human cases of early-onset Alzheimer's disease (EOAD) and frontotemporal lobar degeneration (FLTD), as well as an animal model that mimics FLTD.


Assuntos
Doença de Alzheimer/metabolismo , Potenciação de Longa Duração/fisiologia , Plasticidade Neuronal/fisiologia , Estimulação Química , Sinapses/metabolismo , Sinaptossomos/metabolismo , Actinas/metabolismo , Idoso , Animais , Encéfalo/fisiologia , Colforsina/administração & dosagem , Feminino , Lobo Frontal/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato/metabolismo , Rolipram/administração & dosagem
8.
Invest Ophthalmol Vis Sci ; 62(7): 20, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34137807

RESUMO

Purpose: Synaptosomal actin dynamics are essential for synaptic structural stability. Whether actin dynamics are involved in structural and functional synaptic plasticity within the primary visual cortex (V1) or behavioral visual acuity in rats has still not been thoroughly investigated. Methods: Synaptosome preparation and western blot analysis were used to analyze synaptosomal actin dynamics. Transmission electron microscopy was used to detect synaptic density and mitochondrial area alterations. A visual water maze task was applied to assess behavioral visual acuity. Microinjection of the actin polymerization inhibitor or stabilizer detected the effect of actin dynamics on visual function. Results: Actin dynamics, the mitochondrial area, and synaptic density within the area of V1 are increased during the critical period for the development of binocularity. Microinjection of the actin polymerization inhibitor cytochalasin D into the V1 decreased the mitochondrial area, synaptic density, and behavioral visual acuity. Long-term monocular deprivation reduced actin dynamics, the mitochondrial area, and synaptic density within the V1 contralateral to the deprived eye compared with those ipsilateral to the deprived eye and impaired visual acuity in the amblyopic eye. In addition, the mitochondrial area, synaptic density, and behavioral visual acuity were improved by stabilization of actin polymerization by jasplakinolide microinjection. Conclusions: During the critical period of visual development of binocularity, synaptosomal actin dynamics regulate synaptic structure and function and play roles in behavioral visual acuity in rats.


Assuntos
Actinas , Plasticidade Neuronal/fisiologia , Sinaptossomos/metabolismo , Acuidade Visual/fisiologia , Córtex Visual/fisiologia , Actinas/química , Actinas/metabolismo , Ambliopia/metabolismo , Ambliopia/fisiopatologia , Animais , Antineoplásicos/farmacologia , Comportamento Animal/fisiologia , Depsipeptídeos/farmacologia , Aprendizagem em Labirinto , Polimerização/efeitos dos fármacos , Ratos , Visão Ocular/fisiologia
9.
Nat Commun ; 12(1): 2603, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972518

RESUMO

Synaptic disturbances in excitatory to inhibitory (E/I) balance in forebrain circuits are thought to contribute to the progression of Alzheimer's disease (AD) and dementia, although direct evidence for such imbalance in humans is lacking. We assessed anatomical and electrophysiological synaptic E/I ratios in post-mortem parietal cortex samples from middle-aged individuals with AD (early-onset) or Down syndrome (DS) by fluorescence deconvolution tomography and microtransplantation of synaptic membranes. Both approaches revealed significantly elevated E/I ratios for AD, but not DS, versus controls. Gene expression studies in an independent AD cohort also demonstrated elevated E/I ratios in individuals with AD as compared to controls. These findings provide evidence of a marked pro-excitatory perturbation of synaptic E/I balance in AD parietal cortex, a region within the default mode network that is overly active in the disorder, and support the hypothesis that E/I imbalances disrupt cognition-related shifts in cortical activity which contribute to the intellectual decline in AD.


Assuntos
Doença de Alzheimer/fisiopatologia , Disfunção Cognitiva/fisiopatologia , Síndrome de Down/fisiopatologia , Lobo Parietal/anatomia & histologia , Lobo Parietal/metabolismo , Sinapses/metabolismo , Membranas Sinápticas/fisiologia , Peptídeos beta-Amiloides/metabolismo , Animais , Anuros , Autopsia , Disfunção Cognitiva/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Síndrome de Down/metabolismo , Feminino , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Rede Nervosa/fisiopatologia , Oócitos/fisiologia , Lobo Parietal/fisiopatologia , Sinapses/patologia , Membranas Sinápticas/metabolismo , Sinaptossomos/metabolismo , Sinaptossomos/patologia , Tomografia Óptica , Transcriptoma/genética
10.
Sci Rep ; 11(1): 7395, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795747

RESUMO

Wnt signaling plays a key role in neurodevelopment and neuronal maturation. Specifically, Wnt5a stimulates postsynaptic assemblies, increases glutamatergic neurotransmission and, through calcium signaling, generates nitric oxide (NO). Trying to unveil the molecular pathway triggering these postsynaptic effects, we found that Wnt5a treatment induces a time-dependent increases in the length of the postsynaptic density (PSD), elicits novel synaptic contacts and facilitates F-actin flow both in in vitro and ex vivo models. These effects were partially abolished by the inhibition of the Heme-regulated eukaryotic initiation factor 2α (HRI) kinase, a kinase which phosphorylates the initiation translational factor eIF2α. When phosphorylated, eIF2α normally avoids the translation of proteins not needed during stress conditions, in order to avoid unnecessary energetic expenses. However, phosphorylated eIF2α promotes the translation of some proteins with more than one open reading frame in its 5' untranslated region. One of these proteins targeted by Wnt-HRI-eIF2α mediated translation is the GluN2B subunit of the NMDA receptor. The identified increase in GluN2B expression correlated with increased NMDA receptor function. Considering that NMDA receptors are crucial for excitatory synaptic transmission, the molecular pathway described here contributes to the understanding of the fast and plastic translational mechanisms activated during learning and memory processes.


Assuntos
Hipocampo/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteína Wnt-5a/metabolismo , Regiões 5' não Traduzidas , Actinas/metabolismo , Animais , Meios de Cultivo Condicionados , Regulação da Expressão Gênica , Hipocampo/metabolismo , Aprendizagem , Masculino , Memória , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Óxido Nítrico/metabolismo , Fases de Leitura Aberta , Fosforilação , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sinapses/metabolismo , Sinaptossomos/metabolismo
11.
Sci Rep ; 11(1): 8138, 2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33854131

RESUMO

Imbalance of excitatory and inhibitory neurotransmission is implicated in a wide range of psychiatric and neurologic disorders. Here we tested the hypothesis that insertion of a methyl group on the stereogenic alpha carbon of L-Glu or L-Gln would impact the γ-aminobutyric acid (GABA) shunt and the glutamate-glutamine cycle. (S)-2-methylglutamate, or (S)-2MeGlu, was efficiently transported into brain and synaptosomes where it was released by membrane depolarization in a manner equivalent to endogenous L-Glu. (R)-2MeGlu was transported less efficiently into brain and synaptosomes but was not released by membrane depolarization. Each enantiomer of 2MeGlu had limited activity across a panel of over 30 glutamate and GABA receptors. While neither enantiomer of 2MeGlu was metabolized along the GABA shunt, (S)-2MeGlu was selectively converted to (S)-2-methylglutamine, or (S)-2MeGln, which was subsequently slowly hydrolyzed back to (S)-2MeGlu in brain. rac-2MeGln was also transported into brain, with similar efficiency as (S)-2MeGlu. A battery of behavioral tests in young adult wild type mice showed safety with up to single 900 mg/kg dose of (R)-2MeGlu, (S)-2MeGlu, or rac-2MeGln, suppressed locomotor activity with single ≥ 100 mg/kg dose of (R)-2MeGlu or (S)-2MeGlu. No effect on anxiety or hippocampus-dependent learning was evident. Enantiomers of 2MeGlu and 2MeGln show promise as potential pharmacologic agents and imaging probes for cells that produce or transport L-Gln.


Assuntos
Encéfalo/metabolismo , Glutamatos/administração & dosagem , Glutamina/administração & dosagem , Sinaptossomos/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Cromatografia Líquida , Relação Dose-Resposta a Droga , Feminino , Glutamatos/química , Glutamatos/farmacocinética , Glutamina/química , Glutamina/farmacocinética , Masculino , Camundongos , Cultura Primária de Células , Estereoisomerismo , Espectrometria de Massas em Tandem , Ácido gama-Aminobutírico/metabolismo
12.
Front Immunol ; 12: 586521, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717067

RESUMO

Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [3H]D-aspartate ([3H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes with antibodies recognizing the NH2 terminus of the GluA2 and the GluA3 subunits increased, although to a different extent, the GluA2 and 3 densities in synaptosomal membranes, also amplifying the AMPA-evoked glutamate release in a NBQX-dependent fashion. We then analyzed the releasing activity of complement (1:300) from both treated and untreated synaptosomes and found that the complement-induced overflow occurred in a DL-t-BOA-sensitive, NBQX-insensitive fashion. We hypothesized that anti-GluA/GluA complexes in neuronal membranes could trigger the classic pathway of activation of the complement, modifying its releasing activity. Accordingly, the complement-evoked release of [3H]D-Asp from antiGluA2 and anti-GluA3 antibody treated synaptosomes was significantly increased when compared to untreated terminals and facilitation was prevented by omitting the C1q component of the immunocomplex. Antibodies recognizing the NH2 terminus of the GluA1 or the GluA4 subunits failed to affect both the AMPA and the complement-evoked tritium overflow. Our results suggest the presence of GluA2/GluA3-containing release-regulating AMPA autoreceptors in cortical synaptosomes. Incubation of synaptosomes with commercial anti-GluA2 or anti-GluA3 antibodies amplifies the AMPA-evoked exocytosis of glutamate through a complement-independent pathway, involving an excessive insertion of AMPA autoreceptors in plasma membranes but also affects the complement-dependent releasing activity, by promoting the classic pathway of activation of the immunocomplex. Both events could be relevant to the development of autoimmune diseases typified by an overproduction of anti-GluA subunits.


Assuntos
Anticorpos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Receptores de AMPA/antagonistas & inibidores , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Córtex Cerebral/metabolismo , Complemento C1q/imunologia , Imunofluorescência , Masculino , Camundongos , Receptores de AMPA/química , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
13.
J Med Food ; 24(3): 209-217, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33739887

RESUMO

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.


Assuntos
Ácido Glutâmico , Morus , Adenilil Ciclases , Animais , Cálcio/metabolismo , Calmodulina , Córtex Cerebral/metabolismo , Terminações Nervosas/metabolismo , Casca de Planta , Ratos , Ratos Sprague-Dawley , Sinaptossomos/metabolismo
14.
J Biol Chem ; 296: 100266, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33769286

RESUMO

The accurate retrieval of synaptic vesicle (SV) proteins during endocytosis is essential for the maintenance of neurotransmission. Synaptophysin (Syp) and synaptobrevin-II (SybII) are the most abundant proteins on SVs. Neurons lacking Syp display defects in the activity-dependent retrieval of SybII and a general slowing of SV endocytosis. To determine the role of the cytoplasmic C terminus of Syp in the control of these two events, we performed molecular replacement studies in primary cultures of Syp knockout neurons using genetically encoded reporters of SV cargo trafficking at physiological temperatures. Under these conditions, we discovered, 1) no slowing in SV endocytosis in Syp knockout neurons, and 2) a continued defect in SybII retrieval in knockout neurons expressing a form of Syp lacking its C terminus. Sequential truncations of the Syp C-terminus revealed a cryptic interaction site for the SNARE motif of SybII that was concealed in the full-length form. This suggests that a conformational change within the Syp C terminus is key to permitting SybII binding and thus its accurate retrieval. Furthermore, this study reveals that the sole presynaptic role of Syp is the control of SybII retrieval, since no defect in SV endocytosis kinetics was observed at physiological temperatures.


Assuntos
Neurônios/metabolismo , Vesículas Sinápticas/genética , Sinaptofisina/genética , Proteína 2 Associada à Membrana da Vesícula/genética , Endocitose/genética , Técnicas de Inativação de Genes , Hipocampo/metabolismo , Hipocampo/patologia , Neurônios/química , Cultura Primária de Células , Proteínas SNARE/genética , Transmissão Sináptica/genética , Sinaptofisina/química , Sinaptossomos/química , Sinaptossomos/metabolismo
15.
Mol Brain ; 14(1): 39, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33622379

RESUMO

The SH3 and multiple ankyrin repeat domains 3 (Shank3) protein is a core organizer of the macromolecular complex in excitatory postsynapses, and its defects cause numerous synaptopathies, including autism spectrum disorders. Although the function of Shank3 as a postsynaptic scaffold is adequately established, other potential mechanisms through which Shank3 broadly modulates the postsynaptic proteome remain relatively unexplored. In our previous quantitative proteomic analysis, six up-regulated ribosomal proteins were identified in the striatal synaptosome of Shank3-overexpressing transgenic (TG) mice. In the present study, we validated the increased levels of RPLP1 and RPL36A in synaptosome, but not in whole lysate, of the TG striatum. Moreover, protein synthesis and extracellular signaling-regulated kinase (ERK) activity were enhanced in the TG striatal synaptosome. To understand the potential contribution of increased protein synthesis to the proteomic change in the TG striatal synaptosome, we performed RNA-sequencing analyses on both whole synaptosomal and synaptic polysome-enriched fractions. Comparative analyses showed a positive correlation only between the polysome-associated transcriptome and up-regulated proteome in the TG striatal synaptosome. Our findings suggest a novel mechanism through which Shank3 may remodel the postsynaptic proteome by regulating synaptic protein synthesis, whose dysfunction can be implicated in SHANK3-associated synaptopathies.


Assuntos
Corpo Estriado/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Sinaptossomos/metabolismo , Animais , Sistema de Sinalização das MAP Quinases , Camundongos Transgênicos , Receptores de Dopamina D1/metabolismo
16.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33526688

RESUMO

Chronic stress is one of the most critical factors in the onset of depressive disorders; hence, environmental factors such as psychosocial stress are commonly used to induce depressive-​like traits in animal models of depression. Ventral CA1 (vCA1) in hippocampus and basal lateral amygdala (BLA) are critical sites during chronic stress-induced alterations in depressive subjects; however, the underlying neural mechanisms remain unclear. Here we employed chronic unpredictable mild stress (CUMS) to model depression in mice and found that the activity of the posterior BLA to vCA1 (pBLA-vCA1) innervation was markedly reduced. Mice subjected to CUMS showed reduction in dendritic complexity, spine density, and synaptosomal AMPA receptors (AMPARs). Stimulation of pBLA-vCA1 innervation via chemogenetics or administration of cannabidiol (CBD) could reverse CUMS-induced synaptosomal AMPAR decrease and efficiently alleviate depressive-like behaviors in mice. These findings demonstrate a critical role for AMPARs and CBD modulation of pBLA-vCA1 innervation in CUMS-induced depressive-like behaviors.


Assuntos
Tonsila do Cerebelo/metabolismo , Depressão/genética , Hipocampo/metabolismo , Receptores de AMPA/genética , Estresse Psicológico/genética , Tonsila do Cerebelo/fisiopatologia , Animais , Complexo Nuclear Basolateral da Amígdala/metabolismo , Complexo Nuclear Basolateral da Amígdala/patologia , Canabidiol/farmacologia , Depressão/tratamento farmacológico , Depressão/metabolismo , Depressão/fisiopatologia , Modelos Animais de Doenças , Hipocampo/fisiopatologia , Humanos , Masculino , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/fisiopatologia , Sinaptossomos/metabolismo
17.
Chem Res Toxicol ; 34(5): 1286-1295, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33621091

RESUMO

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


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/metabolismo , Córtex Cerebral/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Glicosídeos/farmacologia , Animais , Córtex Cerebral/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
18.
Eur J Pharmacol ; 898: 173986, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33640406

RESUMO

The high-affinity choline transporter CHT1 mediates choline uptake, the rate-limiting and regulatory step in acetylcholine synthesis at cholinergic presynaptic terminals. CHT1-medated choline uptake is specifically inhibited by hemicholinium-3, which is a type of choline analog that acts as a competitive inhibitor. Although the substrate choline and the inhibitor hemicholinium-3 are well-established ligands of CHT1, few potent ligands other than choline analogs have been reported. Here we show that tetrahydropyrimidine anthelmintics, known as nicotinic acetylcholine receptor agonists, act as competitive inhibitors of CHT1. A ligand-dependent trafficking assay in cell lines expressing human CHT1 was designed to search for CHT1 ligands from a collection of biologically active compounds. We found that morantel as well as other tetrahydropyrimidines, pyrantel and oxantel, potently inhibits the high-affinity choline uptake activity of CHT1 in a competitive manner similar to the inhibitor hemicholinium-3. They also inhibit the high-affinity choline transporter from the nematode Caenorhabditis elegans. Finally, tetrahydropyrimidines potently inhibit the high-affinity choline uptake in rat brain synaptosomes at a low micromolar level, resulting in the inhibition of acetylcholine synthesis. The rank order of potency in synaptosomes is as follows: morantel > pyarantel > oxantel (Ki = 1.3, 5.7, and 8.3 µM, respectively). Our results reveal that tetrahydropyrimidine anthelmintics are novel CHT1 ligands that inhibit the high-affinity choline uptake for acetylcholine synthesis in cholinergic neurons.


Assuntos
Anti-Helmínticos/farmacologia , Encéfalo/efeitos dos fármacos , Proteínas de Transporte de Cátions/antagonistas & inibidores , Colina/metabolismo , Pirimidinas/farmacologia , Simportadores/antagonistas & inibidores , Animais , Anti-Helmínticos/metabolismo , Ligação Competitiva , Transporte Biológico , Encéfalo/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Feminino , Células HEK293 , Humanos , Ligantes , Camundongos , Morantel/metabolismo , Morantel/farmacologia , Ligação Proteica , Transporte Proteico , Pirantel/análogos & derivados , Pirantel/metabolismo , Pirantel/farmacologia , Pirimidinas/metabolismo , Simportadores/genética , Simportadores/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
19.
Neurochem Res ; 46(4): 804-818, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33428094

RESUMO

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


Assuntos
Bexaroteno/farmacologia , Fármacos Neuroprotetores/farmacologia , Niacinamida/farmacologia , Sinaptossomos/efeitos dos fármacos , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/metabolismo , Fragmentos de Peptídeos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Sinaptossomos/metabolismo
20.
Mol Brain ; 14(1): 23, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33494786

RESUMO

N-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2 R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 s than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.


Assuntos
Caderinas/metabolismo , Hipocampo/metabolismo , Aprendizagem Espacial , Sinapses/metabolismo , Análise e Desempenho de Tarefas , Proteína ADAM10/metabolismo , Alelos , Animais , Comportamento Animal , Células CHO , Membrana Celular/metabolismo , Cricetulus , Medo , Técnicas de Introdução de Genes , Memória , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Mutação/genética , Estabilidade Proteica , Células Piramidais/metabolismo , Sinapses/patologia , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia , Sinaptossomos/metabolismo , Sinaptossomos/ultraestrutura
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