RESUMO
Tobacco smoking is a public health problem, with â¼5 million deaths per year, representing a heavy burden for many countries. No effective therapeutic strategies are currently available for nicotine addiction, and it is therefore crucial to understand the etiological and pathophysiological factors contributing to this addiction. The neuronal α5 nicotinic acetylcholine receptor (nAChR) subunit is critically involved in nicotine dependence. In particular, the human polymorphism α5D398N corresponds to the strongest correlation with nicotine dependence risk found to date in occidental populations, according to meta-analysis of genome-wide association studies. To understand the specific contribution of this subunit in the context of nicotine addiction, an efficient screening system for native human nAChRs is needed. We have differentiated human induced pluripotent stem (iPS) cells into midbrain dopaminergic (DA) neurons and obtained a comprehensive characterization of these neurons by quantitative RT-PCR. The functional properties of nAChRs expressed in these human DA neurons, with or without the polymorphism in the α5 subunit, were studied with the patch-clamp electrophysiological technique. Our results in human DA neurons carrying the polymorphism in the α5 subunit showed an increase in EC50, indicating that, in the presence of the polymorphism, more nicotine or acetylcholine chloride is necessary to obtain the same effect. This human cell culturing system can now be used in drug discovery approaches to screen for compounds that interact specifically with human native and polymorphic nAChRs.-Deflorio, C., Blanchard, S., Carisì, M. C., Bohl, D., Maskos, U. Human polymorphisms in nicotinic receptors: a functional analysis in iPS-derived dopaminergic neurons.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores Nicotínicos/metabolismo , Encéfalo/citologia , Linhagem Celular , Humanos , Polimorfismo Genético , Receptores Nicotínicos/genéticaRESUMO
Chronic stress is one of the most relevant triggering factors for major depression. Microglial cells are highly sensitive to stress and, more generally, to environmental challenges. However, the role of these brain immune cells in mediating the effects of stress is still unclear. Fractalkine signaling - which comprises the chemokine CX3CL1, mainly expressed by neurons, and its receptor CX3CR1, almost exclusively present on microglia in the healthy brain - has been reported to critically regulate microglial activity. Here, we investigated whether interfering with microglial function by deleting the Cx3cr1 gene affects the brain's response to chronic stress. To this purpose, we housed Cx3cr1 knockout and wild-type adult mice in either control or stressful environments for 2weeks, and investigated the consequences on microglial phenotype and interactions with synapses, synaptic transmission, behavioral response and corticosterone levels. Our results show that hampering neuron-microglia communication via the CX3CR1-CX3CL1 pathway prevents the effects of chronic unpredictable stress on microglial function, short- and long-term neuronal plasticity and depressive-like behavior. Overall, the present findings suggest that microglia-regulated mechanisms may underlie the differential susceptibility to stress and consequently the vulnerability to diseases triggered by the experience of stressful events, such as major depression.
Assuntos
Comportamento Animal/fisiologia , Receptor 1 de Quimiocina CX3C/deficiência , Transtorno Depressivo Maior/fisiopatologia , Microglia , Plasticidade Neuronal/fisiologia , Neurônios , Transdução de Sinais/fisiologia , Estresse Psicológico/fisiopatologia , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
INTRODUCTION: Fluoxetine is an open channel blocker of fetal muscle acetylcholine (ACh) receptor (AChR) and slow-channel mutant AChRs. It is used commonly to treat patients with slow-channel congenital myasthenic syndromes. Fluoxetine effects on adult wild-type endplate AChR are less characterized, although muscle AChR isoforms are differentially modulated by some drugs. METHODS: Excitotoxicity assays and patch clamp recordings were performed in human embryonic kidney 293 (HEK) cells expressing wild-type or slow-channel mutant human AChRs. RESULTS: Fluoxetine (2-10 µM) abolished ACh-induced death and decreased ACh-activated whole-cell currents in cells expressing all AChR types. In outside-out patches, fluoxetine rapidly curtailed ACh evoked unitary activity and macroscopic currents. The effect was increased if fluoxetine was applied before ACh. CONCLUSIONS: Fluoxetine is an open channel blocker, but it also affects AChR in the closed state. AChR blockade likely underlies the rescue of HEK cells from ACh-induced death.
Assuntos
Acetilcolina/efeitos adversos , Fluoxetina/farmacologia , Placa Motora/efeitos dos fármacos , Neurotoxinas/efeitos adversos , Receptores Colinérgicos/efeitos dos fármacos , Acetilcolina/farmacologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Humanos , Técnicas In Vitro , Placa Motora/fisiologia , Síndromes Miastênicas Congênitas/fisiopatologia , Neurotoxinas/farmacologia , Técnicas de Patch-Clamp , Receptores Colinérgicos/fisiologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: "microtransplantation" of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Músculos/metabolismo , Receptores Colinérgicos/metabolismo , Acetilcolina/farmacologia , Esclerose Lateral Amiotrófica/tratamento farmacológico , Animais , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Músculos/efeitos dos fármacos , Músculos/fisiopatologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Receptores Nicotínicos/fisiologia , Riluzol/farmacologia , Riluzol/uso terapêutico , XenopusRESUMO
A role for chemokines as molecules mediating neuron-glia cross talk has emerged in recent years, both in physiological and pathological conditions. We demonstrate here for the first time that the chemokine CXCL16 and its unique receptor CXCR6 are functionally expressed in the CNS, and induce neuroprotection against excitotoxic damage due to excessive glutamate (Glu) exposure and oxygen glucose deprivation (OGD). In mice and rats we found that, to exert neuroprotection, CXCL16 requires the presence of extracellular adenosine (ADO), and that pharmacological or genetic inactivation of the ADO A(3) receptor, A(3)R, prevents CXCL16 effect. In experiments with astrocytes cocultured with cxcr6(gfp/gfp) hippocampal cells, we demonstrate that CXCL16 acts directly on astrocytes to release soluble factors that are essential to mediate neuroprotection. In particular, we report that (1) upon stimulation with CXCL16 astrocytes release monocyte chemoattractant protein-1/CCL2 and (2) the neuroprotective effect of CXCL16 is reduced in the presence of neutralizing CCL2 antibody. In conclusion, we found that chemokine CXCL16 is able to mediate cross talk between astrocytes and neighboring neurons and, in pathological conditions such as excessive Glu or OGD exposure, is able to counteract neuronal cell death through an ADO-dependent chemokine-induced chemokine-release mechanism.
Assuntos
Quimiocina CCL2/metabolismo , Quimiocina CXCL6/fisiologia , Neurônios/metabolismo , Receptor A3 de Adenosina/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Comunicação Celular/fisiologia , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Quimiocina CXCL16 , Técnicas de Cocultura , Feminino , Técnicas de Introdução de Genes , Ácido Glutâmico/toxicidade , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/citologia , Ratos , Ratos Wistar , Receptor Cross-Talk/fisiologiaRESUMO
Riluzole, the only drug available against amyotrophic lateral sclerosis (ALS), has recently been shown to block muscle ACh receptors (AChRs), raising concerns about possible negative side-effects on neuromuscular transmission in treated patients. In this work we studied riluzole's impact on the function of muscle AChRs in vitro and on neuromuscular transmission in ALS patients, using electrophysiological techniques. Human recombinant AChRs composed of α(1)ß(1)δ subunits plus the γ or ε subunit (γ- or ε-AChR) were expressed in HEK cells or Xenopus oocytes. In both preparations, riluzole at 0.5 µm, a clinically relevant concentration, reversibly reduced the amplitude and accelerated the decay of ACh-evoked current if applied before coapplication with ACh. The action on γ-AChRs was more potent and faster than on ε-AChRs. In HEK outside-out patches, riluzole-induced block of macroscopic ACh-evoked current gradually developed during the initial milliseconds of ACh presence. Single channel recordings in HEK cells and in human myotubes from ALS patients showed that riluzole prolongs channel closed time, but has no effect on channel conductance and open duration. Finally, compound muscle action potentials (CMAPs) evoked by nerve stimulation in ALS patients remained unaltered after a 1 week suspension of riluzole treatment. These data indicate that riluzole, while apparently safe with regard to synaptic transmission, may affect the function of AChRs expressed in denervated muscle fibres of ALS patients, with biological consequences that remain to be investigated.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Antagonistas Colinérgicos/farmacologia , Músculo Esquelético/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores Colinérgicos/fisiologia , Riluzol/farmacologia , Idoso , Animais , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Células Satélites de Músculo Esquelético/fisiologia , Xenopus laevisRESUMO
Denervated muscles undergo fibrillations due to spontaneous activation of voltage-gated sodium (Na(+)) channels generating action potentials. Fibrillations also occur in patients with amyotrophic lateral sclerosis (ALS). Riluzole, the only approved drug for ALS treatment, blocks voltage-gated Na(+) channels, but its effects on muscle Na(+) channels and fibrillations are yet poorly characterized. Using patch-clamp technique, we studied riluzole effect on Na(+) channels in cultured myotubes from ALS patients. Needle electromyography was used to study fibrillation potentials (Fibs) in ALS patients during riluzole treatment and after one week of suspension. Patients were clinically characterized in all recording sessions. In myotubes, riluzole (1 µM, a therapeutic concentration) reduced Na(+) current by 20%. The rate of rise and amplitude of spikes evoked by depolarizing stimuli were also reduced. Fibs were detected in all patients tested during riluzole treatment and riluzole washout had no univocal effect. Our study indicates that, in human myotubes, riluzole partially blocks Na(+) currents and affects action potentials but does not prevent firing. In line with this in vitro finding, muscle Fibs in ALS patients appear to be largely unaffected by riluzole.
RESUMO
In slow-channel congenital myasthenic syndrome, point mutations of the endplate acetylcholine receptor (AChR) prolong channel openings, leading to excessive Ca(2+) entry with ensuing endplate degeneration and myasthenic symptoms. The Ca(2+) permeability of the human endplate AChR-channel is quite high, and is further increased by two slow-channel mutations in its É subunit, worsening the pathological cascade. To gain further support to the hypothesis that the É subunit plays a crucial role in controlling Ca(2+) permeability of endplate AChR-channel, in this work we measured the fractional Ca(2+) current (P(f), i.e., the percentage of the total current carried by Ca(2+) ions) of a panel of AChR carrying slow-channel mutations in the α, ß and É subunits detected in patients (α(N217K), α(S226Y), α(C418W), ß(V266A), ß(V266M), É(I257F), É(V265A) and É(L269F)). We confirm that only mutations in the É subunit altered Ca(2+) permeability of AChR-channels, with É(L269F) increasing P(f) (10% vs. 7% of wild type AChR) and É(I257F) decreasing it (to 4.6%). We also found that, for É(L269F)-AChR, the Ca(2+) permeability and ACh-induced cell death can be normalized by clinically relevant concentrations of salbutamol or verapamil, providing the first evidence that the Ca(2+) permeability of AChR-channels can be modulated and this treatment may provide protection against excitotoxic insults.