Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.450
Filtrar
1.
Int J Mol Sci ; 21(16)2020 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823591

RESUMO

While SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) as the receptor for cell entry, it is important to examine other potential interactions between the virus and other cell receptors. Based on the clinical observation of low prevalence of smoking among hospitalized COVID-19 patients, we examined and identified a "toxin-like" amino acid (aa) sequence in the Receptor Binding Domain of the Spike Glycoprotein of SARS-CoV-2 (aa 375-390), which is homologous to a sequence of the Neurotoxin homolog NL1, one of the many snake venom toxins that are known to interact with nicotinic acetylcholine receptors (nAChRs). We present the 3D structural location of this "toxin-like" sequence on the Spike Glycoprotein and the superposition of the modelled structure of the Neurotoxin homolog NL1 and the SARS-CoV-2 Spike Glycoprotein. We also performed computational molecular modelling and docking experiments using 3D structures of the SARS-CoV-2 Spike Glycoprotein and the extracellular domain of the nAChR α9 subunit. We identified a main interaction between the aa 381-386 of the SARS-CoV-2 Spike Glycoprotein and the aa 189-192 of the extracellular domain of the nAChR α9 subunit, a region which forms the core of the "toxin-binding site" of the nAChRs. The mode of interaction is very similar to the interaction between the α9 nAChR and α-bungarotoxin. A similar interaction was observed between the pentameric α7 AChR chimera and SARS-CoV-2 Spike Glycoprotein. The findings raise the possibility that SARS-CoV-2 may interact with nAChRs, supporting the hypothesis of dysregulation of the nicotinic cholinergic system being implicated in the pathophysiology of COVID-19. Nicotine and other nicotinic cholinergic agonists may protect nAChRs and thus have therapeutic value in COVID-19 patients.


Assuntos
Betacoronavirus/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos/genética , Biologia Computacional , Infecções por Coronavirus/fisiopatologia , Humanos , Simulação de Acoplamento Molecular , Neurotoxinas/genética , Neurotoxinas/metabolismo , Pandemias , Pneumonia Viral/fisiopatologia , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Venenos de Serpentes/genética
2.
Mol Pharmacol ; 98(4): 328-342, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32690626

RESUMO

Epibatidine is a potent analgetic agent with very high affinity for brain nicotinic acetylcholine receptors (nAChR). We determined the activity profiles of three epibatidine derivatives, RTI-36, RTI-76, and RTI-102, which have affinity for brain nAChR equivalent to that of epibatidine but reduced analgetic activity. RNAs coding for nAChR monomeric subunits and/or concatamers were injected into Xenopus oocytes to obtain receptors of defined subunit composition and stoichiometry. The epibatidine analogs produced protracted activation of high sensitivity (HS) α4- and α2-containing receptors with the stoichiometry of 2alpha:3beta subunits but not low sensitivity (LS) receptors with the reverse ratio of alpha and beta subunits. Although not strongly activated by the epibatidine analogs, LS α4- and α2-containing receptors were potently desensitized by the epibatidine analogs. In general, the responses of α4(2)ß2(2)α5 and ß3α4ß2α6ß2 receptors were similar to those of the HS α4ß2 receptors. RTI-36, the analog closest in structure to epibatidine, was the most efficacious of the three compounds, also effectively activating α7 and α3ß4 receptors, albeit with lower potency and less desensitizing effect. Although not the most efficacious agonist, RTI-76 was the most potent desensitizer of α4- and α2-containing receptors. RTI-102, a strong partial agonist for HS α4ß2 receptors, was effectively an antagonist for LS α4ß2 receptors. Our results highlight the importance of subunit stoichiometry and the presence or absence of specific accessory subunits for determining the activity of these drugs on brain nAChR, affecting the interpretation of in vivo studies since in most cases these structural details are not known. SIGNIFICANCE STATEMENT: Epibatidine and related compounds are potent ligands for the high-affinity nicotine receptors of the brain, which are therapeutic targets and mediators of nicotine addiction. Far from being a homogeneous population, these receptors are diverse in subunit composition and vary in subunit stoichiometry. We show the importance of these structural details for drug activity profiles, which present a challenge for the interpretation of in vivo experiments since conventional methods, such as in situ hybridization and immunohistochemistry, cannot illuminate these details.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Agonistas Nicotínicos/farmacologia , Subunidades Proteicas/metabolismo , Piridinas/química , Receptores Nicotínicos/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/química , Humanos , Estrutura Molecular , Complexos Multiproteicos/metabolismo , Agonistas Nicotínicos/química , Subunidades Proteicas/genética , Receptores Nicotínicos/genética , Tropanos/química , Tropanos/farmacologia , Xenopus/genética
3.
Proc Natl Acad Sci U S A ; 117(28): 16283-16291, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32611810

RESUMO

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.


Assuntos
Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Neonicotinoides/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Abelhas/metabolismo , Relação Dose-Resposta a Droga , Drosophila melanogaster/metabolismo , Proteínas de Insetos/agonistas , Proteínas de Insetos/genética , Oócitos/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Xenopus laevis
4.
Am J Physiol Cell Physiol ; 319(2): C321-C330, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32551856

RESUMO

Acetylcholine induces robust electrogenic anion secretion in mammalian intestine and it has long been hypothesized that it mediates the epithelial response through the M3 and, to a lesser extent, the M1 muscarinic receptors in the mouse. However, nicotinic receptors have recently been identified in intestinal enterocytes by quantitative real-time (qRT)-PCR/RNAseq, although any direct influence on intestinal transport has not been identified. We tested the hypothesis that cholinergic-induced anion secretion in the intestine is a result of both muscarinic and nicotinic pathways that are intrinsic to the intestinal epithelia. We developed a method to generate mouse jejunal enteroid monolayers which were used to measure active electrogenic anion secretion by the Ussing chamber/voltage-clamp technique. Here, we show that the cholinergic agonist carbachol (CCh) and the muscarinic agonist bethanechol (BCh) stimulate short-lived, concentration-dependent anion secretion in the epithelial cell-only enteroid monolayers. The muscarinic antagonist atropine completely inhibited CCh- and BCh-induced secretion, while the nicotinic antagonist hexamethonium reduced the CCh response by ~45%. While nicotine alone did not alter anion secretion, it increased the BCh-induced increase in short-circuit current in a concentration-dependent manner; this synergy was prevented by pretreatment with hexamethonium. In addition to being sensitive to hexamethonium, monolayers express both classes of cholinergic receptor by qRT-PCR, including 13 of 16 nicotinic receptor subunits. Our findings indicate that an interaction between muscarinic and nicotinic agonists synergistically stimulates anion secretion in mouse jejunal epithelial cells and identify a role for epithelial nicotinic receptors in anion secretion.


Assuntos
Agonistas Muscarínicos/farmacologia , Sistema Colinérgico não Neuronal/genética , Receptores Muscarínicos/genética , Receptores Nicotínicos/genética , Acetilcolina/farmacologia , Animais , Ânions/metabolismo , Atropina/farmacologia , Agonistas Colinérgicos/farmacologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Hexametônio/farmacologia , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Camundongos , Sistema Colinérgico não Neuronal/efeitos dos fármacos , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo
5.
PLoS One ; 15(6): e0233991, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497060

RESUMO

Neuropeptides are secreted molecules that have conserved roles modulating many processes, including mood, reproduction, and feeding. Dysregulation of neuropeptide signaling is also implicated in neurological disorders such as epilepsy. However, much is unknown about the mechanisms regulating specific neuropeptides to mediate behavior. Here, we report that the expression levels of dozens of neuropeptides are up-regulated in response to circuit activity imbalance in C. elegans. acr-2 encodes a homolog of human nicotinic receptors, and functions in the cholinergic motoneurons. A hyperactive mutation, acr-2(gf), causes an activity imbalance in the motor circuit. We performed cell-type specific transcriptomic analysis and identified genes differentially expressed in acr-2(gf), compared to wild type. The most over-represented class of genes are neuropeptides, with insulin-like-peptides (ILPs) the most affected. Moreover, up-regulation of neuropeptides occurs in motoneurons, as well as sensory neurons. In particular, the induced expression of the ILP ins-29 occurs in the BAG neurons, which were previously shown to function in gas-sensing. We also show that this up-regulation of ins-29 in acr-2(gf) animals is activity-dependent. Our genetic and molecular analyses support cooperative effects for ILPs and other neuropeptides in promoting motor circuit activity in the acr-2(gf) background. Together, this data reveals that a major transcriptional response to motor circuit dysregulation is in up-regulation of multiple neuropeptides, and suggests that BAG sensory neurons can respond to intrinsic activity states to feedback on the motor circuit.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Neuropeptídeos/genética , Receptores Nicotínicos/genética , Transcriptoma , Animais , Caenorhabditis elegans/fisiologia , Perfilação da Expressão Gênica , Neurônios Motores/metabolismo , Mutação , Células Receptoras Sensoriais/metabolismo
6.
J Neurosci ; 40(27): 5214-5227, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32467358

RESUMO

The limitation of plasticity in the adult brain impedes functional recovery later in life from brain injury or disease. This pressing clinical issue may be resolved by enhancing plasticity in the adult brain. One strategy for triggering robust plasticity in adulthood is to reproduce one of the hallmark physiological events of experience-dependent plasticity observed during the juvenile critical period: to rapidly reduce the activity of parvalbumin (PV)-expressing interneurons and disinhibit local excitatory neurons. This may be achieved through the enhancement of local inhibitory inputs, particularly those of somatostatin (SST)-expressing interneurons. However, to date the means for manipulating SST interneurons for enhancing cortical plasticity in the adult brain are not known. We show that SST interneuron-selective overexpression of Lypd6, an endogenous nicotinic signaling modulator, enhances ocular dominance plasticity in the adult primary visual cortex (V1). Lypd6 overexpression mediates a rapid experience-dependent increase in the visually evoked activity of SST interneurons as well as a simultaneous reduction in PV interneuron activity and disinhibition of excitatory neurons. Recapitulating this transient activation of SST interneurons using chemogenetics similarly enhanced V1 plasticity. Notably, we show that SST-selective Lypd6 overexpression restores visual acuity in amblyopic mice that underwent early long-term monocular deprivation. Our data in both male and female mice reveal selective modulation of SST interneurons and a putative downstream circuit mechanism as an effective method for enhancing experience-dependent cortical plasticity as well as functional recovery in adulthood.SIGNIFICANCE STATEMENT The decline of cortical plasticity after closure of juvenile critical period consolidates neural circuits and behavior, but this limits functional recovery from brain diseases and dysfunctions in later life. Here we show that activation of cortical somatostatin (SST) interneurons by Lypd6, an endogenous modulator of nicotinic acetylcholine receptors, enhances experience-dependent plasticity and recovery from amblyopia in adulthood. This manipulation triggers rapid reduction of PV interneuron activity and disinhibition of excitatory neurons, which are known hallmarks of cortical plasticity during juvenile critical periods. Our study demonstrates modulation of SST interneurons by Lypd6 to achieve robust levels of cortical plasticity in the adult brain and may provide promising targets for restoring brain function in the event of brain trauma or disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Ligadas por GPI/fisiologia , Interneurônios/fisiologia , Plasticidade Neuronal/fisiologia , Somatostatina/fisiologia , Córtex Visual/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Dominância Ocular/genética , Potenciais Evocados Visuais/genética , Potenciais Evocados Visuais/fisiologia , Feminino , Proteínas Ligadas por GPI/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Plasticidade Neuronal/genética , Fosfatidilinositóis/farmacologia , Receptores Nicotínicos/genética , Recuperação de Função Fisiológica/genética , Visão Monocular/genética , Visão Monocular/fisiologia , Acuidade Visual/genética
7.
Curr Top Behav Neurosci ; 45: 101-121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32468493

RESUMO

Nicotine underlies the reinforcing properties of tobacco cigarettes and e-cigarettes. After inhalation and absorption, nicotine binds to various nicotinic acetylcholine receptor (nAChR) subtypes localized on the pre- and postsynaptic membranes of cells, which subsequently leads to the modulation of cellular function and neurotransmitter signaling. In this chapter, we begin by briefly reviewing the current understanding of nicotine's actions on nAChRs and highlight considerations regarding nAChR subtype localization and pharmacodynamics. Thereafter, we discuss the seminal discoveries derived from genetically modified mouse models, which have greatly contributed to our understanding of nicotine's effects on the reward-related mesolimbic pathway and the aversion-related habenulo-interpeduncular pathway. Thereafter, emerging areas of research focusing on modulation of nAChR expression and/or function are considered. Taken together, these discoveries have provided a foundational understanding of various genetic, neurobiological, and behavioral factors underlying the motivation to use nicotine and related dependence processes, which are thereby advancing drug discovery efforts to promote long-term abstinence.


Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Receptores Nicotínicos , Tabagismo , Animais , Camundongos , Camundongos Transgênicos , Nicotina , Receptores Nicotínicos/genética , Tabagismo/genética
8.
PLoS Pathog ; 16(4): e1008396, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32243475

RESUMO

Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs.


Assuntos
Antinematódeos/farmacologia , Ascaris suum/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Helminto/metabolismo , Faringe/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Ascaris suum/efeitos dos fármacos , Ascaris suum/genética , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Helminto/genética , Faringe/efeitos dos fármacos , Receptores Nicotínicos/genética
9.
J Neurosci ; 40(17): 3465-3477, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32184221

RESUMO

Nicotine addiction, through smoking, is the principal cause of preventable mortality worldwide. Human genome-wide association studies have linked polymorphisms in the CHRNA5-CHRNA3-CHRNB4 gene cluster, coding for the α5, α3, and ß4 nicotinic acetylcholine receptor (nAChR) subunits, to nicotine addiction. ß4*nAChRs have been implicated in nicotine withdrawal, aversion, and reinforcement. Here we show that ß4*nAChRs also are involved in non-nicotine-mediated responses that may predispose to addiction-related behaviors. ß4 knock-out (KO) male mice show increased novelty-induced locomotor activity, lower baseline anxiety, and motivational deficits in operant conditioning for palatable food rewards and in reward-based Go/No-go tasks. To further explore reward deficits we used intracranial self-administration (ICSA) by directly injecting nicotine into the ventral tegmental area (VTA) in mice. We found that, at low nicotine doses, ß4KO self-administer less than wild-type (WT) mice. Conversely, at high nicotine doses, this was reversed and ß4KO self-administered more than WT mice, whereas ß4-overexpressing mice avoided nicotine injections. Viral expression of ß4 subunits in medial habenula (MHb), interpeduncular nucleus (IPN), and VTA of ß4KO mice revealed dose- and region-dependent differences: ß4*nAChRs in the VTA potentiated nicotine-mediated rewarding effects at all doses, whereas ß4*nAChRs in the MHb-IPN pathway, limited VTA-ICSA at high nicotine doses. Together, our findings indicate that the lack of functional ß4*nAChRs result in deficits in reward sensitivity including increased ICSA at high doses of nicotine that is restored by re-expression of ß4*nAChRs in the MHb-IPN. These data indicate that ß4 is a critical modulator of reward-related behaviors.SIGNIFICANCE STATEMENT Human genetic studies have provided strong evidence for a relationship between variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and nicotine addiction. Yet, little is known about the role of ß4 nicotinic acetylcholine receptor (nAChR) subunit encoded by this cluster. We investigated the implication of ß4*nAChRs in anxiety-, food reward- and nicotine reward-related behaviors. Deletion of the ß4 subunit gene resulted in an addiction-related phenotype characterized by low anxiety, high novelty-induced response, lack of sensitivity to palatable food rewards and increased intracranial nicotine self-administration at high doses. Lentiviral vector-induced re-expression of the ß4 subunit into either the MHb or IPN restored a "stop" signal on nicotine self-administration. These results suggest that ß4*nAChRs provide a promising novel drug target for smoking cessation.


Assuntos
Condicionamento Operante/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Nicotina/administração & dosagem , Receptores Nicotínicos/metabolismo , Recompensa , Autocontrole , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Aprendizagem por Discriminação/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Motivação/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Agonistas Nicotínicos/administração & dosagem , Receptores Nicotínicos/genética , Autoadministração
10.
Sci Rep ; 10(1): 1688, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015391

RESUMO

Mirtrons are non-canonical miRNAs arising by splicing and debranching from short introns. A plethora of introns have been inferred by computational analyses as potential mirtrons. Yet, few have been experimentally validated and their functions, particularly in relation to their host genes, remain poorly understood. Here, we found that Drosophila larvae lacking either the mirtron miR-1010 or its binding site in the nicotinic acetylcholine receptor ß2 (nAcRß2) 3'UTR fail to grow properly and pupariate. Increase of cortical nAcRß2 mediated by neural activity elevates the level of intracellular Ca2+, which in turn activates CaMKII and, further downstream, the transcription factor Adf-1. We show that miR-1010 downregulates nAcRß2. We reveal that Adf-1 initiates the expression of SKIP, the host gene of miR-1010. Preventing synaptic potentials from overshooting their optimal range requires both SKIP to temper synaptic potentials (incoherent feedforward loop) and miR-1010 to reduce nAcRß2 mRNA levels (negative feedback loop). Our results demonstrate how a mirtron, in coordination with its host gene, contributes to maintaining appropriate receptor levels, which in turn may play a role in maintaining homeostasis.


Assuntos
Dípteros/genética , Drosophila/genética , Regiões 3' não Traduzidas/genética , Animais , Animais Geneticamente Modificados/genética , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteínas de Drosophila/genética , Íntrons/genética , Larva/genética , MicroRNAs/genética , Processamento de RNA/genética , RNA Mensageiro/genética , Receptores Nicotínicos/genética , Fatores de Transcrição/genética
11.
Pharm Res ; 37(3): 60, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103380

RESUMO

PURPOSE: A patient was denoted to be generic brittle (GB) if they had a negative opinion about generics (e.g. prior history of a switch problem) or took the innovator brand of their most problematic anti-epileptic drug (AED) when generic was available. The aim of this hypothesis-generating study was to assess possible genetic and physiologic differences between GB and not GB patients with epilepsy. METHODS: Patients (n = 148) with epilepsy were previously characterized as being either GB or not GB. Blood was collected from each subject for genotyping and physiologic testing. Genotyping for 24 single nucleotide polymorphisms (SNPs) and two copy number variants (CNVs) was performed across 12 genes in each patient. Forty-four physiologic tests were conducted in each patient. Chi square analysis was performed to assess for associations between genotyping results and GB status, as well as between physiologic test results and GB status. RESULTS: No SNP or CNV discriminated GB status in genetic analysis (genotype or allele frequency). Physiologic test results in this study were not associated with GB status. CONCLUSIONS: Questions from neurologists and patients about generics is frequently based on applicability of generic drug standards to individual subjects. However, findings here in patients with epilepsy did not uncover genetic or physiologic reasons that explained which patients were GB and which were not GB.


Assuntos
Anticonvulsivantes/uso terapêutico , Medicamentos Genéricos/uso terapêutico , Epilepsia/tratamento farmacológico , Epilepsia/genética , Conhecimento do Paciente sobre a Medicação , Anticonvulsivantes/farmacocinética , Comportamento de Escolha , Citocromo P-450 CYP3A/genética , Medicamentos Genéricos/farmacocinética , Frequência do Gene , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , Receptores Nicotínicos/genética , Equivalência Terapêutica
12.
Epileptic Disord ; 22(1): 116-119, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32031532

RESUMO

Sleep-related hypermotor epilepsy, or nocturnal frontal lobe epilepsy, as it was formerly called, is a focal epilepsy with mostly sleep-related seizures of hypermotor, tonic or dystonic semiology. Sleep-related hypermotor epilepsy may be attributed to a monogenetic cause with autosomal dominant inheritance. Mutations are described in different genes, including the genes for three subunits of the nicotinic acetylcholine receptor. We present a family with members over four generations exhibiting sleep-related hypermotor epilepsy. Genetic testing was available for three members from three generations, and revealed two variants in the alpha-4 subunit of the nicotinic acetylcholine receptor (one of them being novel) which are likely to be disease-causing. As these mutations were identified in cis configuration (on the same allele), we do not know whether one of the variants alone or a combination of the two is responsible for the pathogenicity.


Assuntos
Epilepsias Parciais , Receptores Nicotínicos/genética , Transtornos do Despertar do Sono , Adulto , Idoso , Epilepsias Parciais/complicações , Epilepsias Parciais/genética , Epilepsias Parciais/fisiopatologia , Feminino , Humanos , Pessoa de Meia-Idade , Linhagem , Transtornos do Despertar do Sono/etiologia , Transtornos do Despertar do Sono/genética , Transtornos do Despertar do Sono/fisiopatologia , Adulto Jovem
13.
Inflamm Res ; 69(2): 217-231, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31897506

RESUMO

OBJECTIVE: The objective of the study was to test the hypothesis that nicotine guards against endotoxemia-associated renal inflammation and vasoconstrictor dysfunction via the activation of α7-nicotinic acetylcholine receptors (α7-nAChRs)/heme oxygenase-1 (HO-1) cascade. MATERIALS: 91 male and female rats were included in the study. TREATMENTS: Lipopolysaccharide (LPS, 5 mg kg-1), nicotine (0.5-2 mg kg-1), pentoxifylline (PTX, TNFα inhibitor, 3 mg kg-1), methyllycaconitine (MLA, α7-nAChR blocker), zinc protoporphyrin (ZnPP, HO-1 inhibitor), hemin (HO-1 inducer), tricarbonyldichlororuthenium (carbon monoxide-releasing molecule, CORM-2) or bilirubin was administered before LPS. METHODS: Isolated perfused kidney was used to evaluate renal vasoconstriction and immunohistochemistry to assess inflammatory cytokines. RESULTS: LPS reduced renal vasoconstrictions induced by phenylephrine or vasopressin in perfused kidneys of male, but not female, rats. Higher elevations in serum interleukin-1ß and renal expressions of inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) were observed in LPS-treated male rats, whereas greater HO-1 expression was evident in endotoxic female rats. LPS effects were reversed by nicotine or PTX. Further, the favorable nicotine actions were (i) diminished by MLA or ZnPP and (ii) replicated by hemin or CORM-2, but not bilirubin, and (iii) associated with exaggerated and MLA-sensitive increases in HO-1 expression. CONCLUSIONS: α7-nAChR/HO-1/CO signaling mediates nicotine protection against renal inflammation and vasoconstrictor hyporeactivity in endotoxic male rats.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Monóxido de Carbono/metabolismo , Endotoxemia/genética , Endotoxemia/fisiopatologia , Heme Oxigenase (Desciclizante)/genética , Inflamação/genética , Inflamação/fisiopatologia , Nicotina/farmacologia , Receptores Nicotínicos/genética , Transdução de Sinais/efeitos dos fármacos , Vasoconstritores/farmacologia , Animais , Feminino , Técnicas In Vitro , Interleucina-1beta/sangue , Rim/efeitos dos fármacos , Rim/metabolismo , Lipopolissacarídeos , Masculino , Perfusão , Ratos , Ratos Wistar
14.
Nicotine Tob Res ; 22(1): 104-110, 2020 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-30202916

RESUMO

INTRODUCTION: Observational studies have found lower concentrations of plasma bilirubin in current smokers compared with former and never smokers. However, whether there is a causal relationship between smoking and bilirubin is unknown. In a Mendelian randomization analysis, we tested the hypothesis that higher tobacco consumption is causally associated with lower concentrations of plasma bilirubin. METHODS: We genotyped 103 557 individuals aged 20-100 years from the Copenhagen General Population Study for the CHRNA3 rs1051730 genotype, known to be associated with higher tobacco consumption. Tobacco consumption was defined as daily and cumulative tobacco consumption. RESULTS: In observational multivariable-adjusted analyses, a 10 g/day higher daily tobacco consumption was associated with a 0.28 µmol/L (95% confidence interval = 0.20 to 0.35) lower concentration of plasma bilirubin in current smokers, and a 10 pack-year higher cumulative tobacco consumption was associated with a 0.19 µmol/L (0.17 to 0.21) lower concentration of plasma bilirubin in former and current smokers. Using the CHRNA3 rs1051730 genotype as a proxy for daily and cumulative tobacco consumption, the difference in plasma bilirubin per T-allele was -0.12 µmol/L (-0.23 to -0.002) in current smokers and -0.09 µmol/L (-0.15 to -0.01) in current and former smokers combined. Furthermore, observationally bilirubin concentrations increased with time from smoking cessation in former smokers. CONCLUSION: Higher daily and cumulative tobacco consumption were associated with lower concentrations of plasma bilirubin in observational and genetic analyses, suggesting that the association is causal. IMPLICATIONS: Our results are compatible with two possible interpretations of previous observational studies, either that bilirubin is a mediator of smoking-induced respiratory disease or that the association between plasma bilirubin and respiratory disease stems from residual confounding because of smoking. Future studies should examine whether bilirubin is a causal risk factor for respiratory disease, or merely a marker of smoking status.


Assuntos
Bilirrubina/sangue , Polimorfismo de Nucleotídeo Único , Receptores Nicotínicos/genética , Abandono do Hábito de Fumar/estatística & dados numéricos , Fumar/sangue , Fumar/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Dinamarca/epidemiologia , Feminino , Genótipo , Humanos , Masculino , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Risco , Fumantes/psicologia , Fumar/epidemiologia , Adulto Jovem
15.
Nicotine Tob Res ; 22(2): 248-255, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30882151

RESUMO

INTRODUCTION: Reducing adverse events from pharmacologic treatment is an important goal of precision medicine and identifying genetic predictors of adverse events is a step toward this goal. In 2012, King et al. reported associations between genetic variants and adverse events in a placebo-controlled smoking cessation trial of varenicline and bupropion. Strong associations were found between gastrointestinal adverse events and 11 variants in the CHRNA5-CHRNA3-CHRNB4 region of chromosome 15, a region repeatedly associated with smoking-related phenotypes. Our goal was to replicate, in an independent sample, the impact of variants in the CHRNA5-CHRNA3-CHRNB4 region on gastrointestinal adverse events and to extend the analyses to adherence and smoking cessation. METHODS: The University of Wisconsin Transdisciplinary Tobacco Use Research Center (TTURC) conducted a multiarmed, placebo-controlled smoking cessation trial of bupropion and nicotine replacement therapy that included 985 genotyped European-ancestry participants. We evaluated relationships between our key variables using logistic regression. RESULTS: Gastrointestinal adverse events were experienced by 31.6% TTURC participants. Each of the CHRNA5-CHRNA3-CHRNB4 associations from the King et al. study was found in TTURC, with the same direction of effect. Neither these variants nor the gastrointestinal adverse events themselves were associated with adherence to medication or successful smoking cessation. CONCLUSIONS: Variants in the CHRNA5-CHRNA3-CHRNB4 region of chromosome 15 are associated with gastrointestinal adverse events in smoking cessation. Additional independent variants in this region strengthen the association. The consistency between the results of these two independent studies supports the conclusion that these findings reflect biological response to the use of smoking cessation medication. IMPLICATIONS: The fact that our findings from the TTURC smoking cessation trial support the independent findings of King et al. suggest that associations of variants in the CHRNA5-CHRNA3-CHRNB4 region of chromosome 15 with gastrointestinal adverse events while taking medications for smoking cessation reflect biology. However, although adherence to medication was a strong predictor of successful smoking cessation in TTURC, neither adverse events nor the genetic variants associated with them predicted either adherence or successful cessation in this study. Thus, although we should strive to minimize adverse events during treatment, we should not expect that to increase successful smoking cessation substantially.


Assuntos
Cromossomos Humanos Par 15/genética , Gastroenteropatias/genética , Proteínas do Tecido Nervoso/genética , Receptores Nicotínicos/genética , Agentes de Cessação do Hábito de Fumar/efeitos adversos , Uso de Tabaco/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bupropiona/efeitos adversos , Feminino , Gastroenteropatias/induzido quimicamente , Variação Genética/genética , Humanos , Masculino , Pessoa de Meia-Idade , Família Multigênica/genética , Valor Preditivo dos Testes , Abandono do Hábito de Fumar/métodos , Uso de Tabaco/terapia , Vareniclina/efeitos adversos , Adulto Jovem
16.
Toxicol In Vitro ; 63: 104741, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31783125

RESUMO

Nicotine from tobacco smoke is absorbed into the bloodstream and transferred into breast milk in breastfeeding mothers. Smoking causes a decrease in breast milk volume, adverse changes to the milk composition, and a shortened lactation period. Breast milk is produced by mammary epithelial cells (MECs) in mammary glands during lactation. However, it remains unclear whether nicotine directly affects milk production in lactating MECs. To address this issue, we prepared a culture model with high milk production ability and less-permeable tight junctions (TJs) by seeding mouse MECs on a cell culture insert. Lactating MECs showed expression of α2, α3, ß2, and ß4 of nicotinic acetylcholine receptors. The high concentration of nicotine at 10-100 µM inhibited ß-casein secretion and caused abnormal localization of TJ proteins. We subsequently investigated whether nicotine at a physiological concentration could affect lactating MECs. Nicotine at 1.0 µM directly inhibited α- and ß-casein secretion in lactating MECs concurrently with inactivation of STAT5 and glucocorticoid receptor without affecting the TJ barrier. Nicotine treatment also induced MEC apoptosis concurrently with inactivation of Akt. These results support the adverse effects of nicotine on breastfeeding in smoking mothers.


Assuntos
Células Epiteliais/efeitos dos fármacos , Lactação/efeitos dos fármacos , Glândulas Mamárias Animais/citologia , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Animais , Caseínas/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Camundongos Endogâmicos ICR , Gravidez , Receptores de Glucocorticoides/metabolismo , Receptores Nicotínicos/genética , Fator de Transcrição STAT5/metabolismo , Proteínas de Junções Íntimas/metabolismo
17.
Seizure ; 74: 60-64, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31835056

RESUMO

PURPOSE: Genetics of Sleep-related Hypermotor Epilepsy (SHE) includes mutations in several genes that cumulatively account for 30 % of families. This approximate estimate comes from different case-series, each focused on the screening of a single gene. We systematically investigated a large cohort of SHE patients to estimate the frequency of pathogenic variants in the main genes thus far implicated in this epilepsy syndrome. METHODS: We selected familial and isolated cases diagnosed with clinical/confirmed SHE who underwent genetic analysis by comparable next generation sequencing (NGS) techniques (WES/ multigene epilepsy panel). The identified heterozygous variants were classified according to the American College of Medical Genetics and Genomics guidelines. RESULTS: We included 103 SHE patients (M/F:61/42) who underwent NGS. Sixteen (15.5 %) were familial cases, 16.5 % had focal cortical dysplasia (FCD). We identified three pathogenic variants in CHRNA4 (2.9 %, CI: 0.6-8.3 %), two of whom novel; one pathogenic variant in KCNT1 (1 %, CI: 0.02-5.29 %); four loss-of-function variants in DEPDC5 (3.9 %, CI: 1.1-9.7 %), one of whom never reported; finally, one missense change in NPRL2 (1 %, CI: 0.02-5.29 %), already reported as pathogenic. Three out of the four patients with DEPDC5 variants had FCD. CONCLUSIONS: The overall frequency of pathogenic variants in our SHE cohort was 8.7 %, 19 % and 7 % considering familial and sporadic cases, respectively. Pathogenic variants in the GATOR1-complex genes account for 5 % of the cases. DEPDC5 shows the highest variants frequency, especially in patients with genetic-structural etiology. From a practical perspective, analysis of this gene is recommended even in isolated cases, because of possible implications for patient management.


Assuntos
Epilepsia Reflexa/genética , Síndromes Epilépticas/genética , Proteínas Ativadoras de GTPase/genética , Transtornos do Sono-Vigília/genética , Adolescente , Criança , Epilepsia Reflexa/diagnóstico , Síndromes Epilépticas/diagnóstico , Feminino , Humanos , Itália , Masculino , Proteínas do Tecido Nervoso/genética , Linhagem , Canais de Potássio Ativados por Sódio/genética , Receptores Nicotínicos/genética , Transtornos do Sono-Vigília/diagnóstico
18.
J Clin Invest ; 130(1): 345-358, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31793909

RESUMO

Axon regeneration failure causes neurological deficits and long-term disability after spinal cord injury (SCI). Here, we found that the α2δ2 subunit of voltage-gated calcium channels negatively regulates axon growth and regeneration of corticospinal neurons, the cells that originate the corticospinal tract. Increased α2δ2 expression in corticospinal neurons contributed to loss of corticospinal regrowth ability during postnatal development and after SCI. In contrast, α2δ2 pharmacological blockade through gabapentin administration promoted corticospinal structural plasticity and regeneration in adulthood. Using an optogenetic strategy combined with in vivo electrophysiological recording, we demonstrated that regenerating corticospinal axons functionally integrate into spinal circuits. Mice administered gabapentin recovered upper extremity function after cervical SCI. Importantly, such recovery relies on reorganization of the corticospinal pathway, as chemogenetic silencing of injured corticospinal neurons transiently abrogated recovery. Thus, targeting α2δ2 with a clinically relevant treatment strategy aids repair of motor circuits after SCI.


Assuntos
Axônios/metabolismo , Gabapentina/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Traumatismos da Coluna Vertebral/tratamento farmacológico , Animais , Axônios/patologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Regeneração Nervosa/genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Traumatismos da Coluna Vertebral/genética , Traumatismos da Coluna Vertebral/metabolismo , Traumatismos da Coluna Vertebral/patologia
19.
Oncol Rep ; 43(1): 159-168, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31789411

RESUMO

Nicotinic acetylcholine receptor (nAChR) subunit α5 (α5­nAChR) is involved in tumor cell proliferation, inhibition of apoptosis, progression of metastasis, and induction of angiogenesis in certain solid tumors. However, the role of α5­nAChR in prostate cancer cell growth and metastasis is unclear. In the present study, the role of α5­nAChR in cell proliferation, migration, invasion and apoptosis was investigated by silencing the expression levels of α5­nAChR in the prostate cancer cell lines DU145 and PC3. A siRNA oligonucleotide targeting α5­nAChR was designed. The cell proliferation of DU145 and PC3 cell lines was analyzed by the Cell Counting Kit­8 (CCK­8) assay. Cell migratory and invasive activities were determined using wound healing and Transwell assays, respectively. Western blot analysis was used to quantify α5­nAChR, p­AKT and p­ERK1/2 levels in DU145 and PC3 cells. Knockdown of α5­nAChR was associated with decreased cell proliferation, migration, invasion and increased apoptosis. In addition, decreased phosphorylation levels of AKT and ERK1/2 were revealed following α5­nAChR knockdown in DU145 and PC3 cells compared with those observed in the scramble control samples. The expression levels of the apoptosis­related proteins were altered following silencing of α5­nAChR. In summary, the data indicated that α5­nAChR was involved in the proliferation and invasion of human prostate cancer cells.


Assuntos
Neoplasias da Próstata/patologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Regulação para Cima , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Células PC-3 , Fosforilação , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo
20.
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
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA