RESUMO
A single sub-anesthetic dose of ketamine evokes rapid and long-lasting beneficial effects in patients with a major depressive disorder. However, the mechanisms underlying this effect are unknown. It has been proposed that astrocyte dysregulation of extracellular K+ concentration ([K+]o) alters neuronal excitability, thus contributing to depression. We examined how ketamine affects inwardly rectifying K+ channel Kir4.1, the principal regulator of K+ buffering and neuronal excitability in the brain. Cultured rat cortical astrocytes were transfected with plasmid-encoding fluorescently tagged Kir4.1 (Kir4.1-EGFP) to monitor the mobility of Kir4.1-EGFP vesicles at rest and after ketamine treatment (2.5 or 25 µM). Short-term (30 min) ketamine treatment reduced the mobility of Kir4.1-EGFP vesicles compared with the vehicle-treated controls (p < 0.05). Astrocyte treatment (24 h) with dbcAMP (dibutyryl cyclic adenosine 5'-monophosphate, 1 mM) or [K+]o (15 mM), which increases intracellular cAMP, mimicked the ketamine-evoked reduction of mobility. Live cell immunolabelling and patch-clamp measurements in cultured mouse astrocytes revealed that short-term ketamine treatment reduced the surface density of Kir4.1 and inhibited voltage-activated currents similar to Ba2+ (300 µM), a Kir4.1 blocker. Thus, ketamine attenuates Kir4.1 vesicle mobility, likely via a cAMP-dependent mechanism, reduces Kir4.1 surface density, and inhibits voltage-activated currents similar to Ba2+, known to block Kir4.1 channels.
Assuntos
Transtorno Depressivo Maior , Ketamina , Camundongos , Animais , Ratos , Ketamina/farmacologia , Astrócitos/metabolismo , Transtorno Depressivo Maior/metabolismo , NeurôniosRESUMO
Ketamine, a non-competitive N-methyl-d-aspartate receptor (NMDAR) antagonist, exerts a rapid, potent and long-lasting antidepressant effect, although the cellular and molecular mechanisms of this action are yet to be clarified. In addition to targeting neuronal NMDARs fundamental for synaptic transmission, ketamine also affects the function of astrocytes, the key homeostatic cells of the central nervous system that contribute to pathophysiology of major depressive disorder. Here, I review studies revealing that (sub)anesthetic doses of ketamine elevate intracellular cAMP concentration ([cAMP]i) in astrocytes, attenuate stimulus-evoked astrocyte calcium signaling, which regulates exocytotic secretion of gliosignaling molecules, and stabilize the vesicle fusion pore in a narrow configuration, possibly hindering cargo discharge or vesicle recycling. Next, I discuss how ketamine affects astrocyte capacity to control extracellular K+ by reducing vesicular delivery of the inward rectifying potassium channel (Kir4.1) to the plasmalemma that reduces the surface density of Kir4.1. Modified astroglial K+ buffering impacts upon neuronal firing pattern as demonstrated in lateral habenula in a rat model of depression. Finally, I highlight the discovery that ketamine rapidly redistributes cholesterol in the astrocyte plasmalemma, which may alter the flux of cholesterol to neurons. This structural modification may further modulate a host of processes that synergistically contribute to ketamine's rapid antidepressant action.
RESUMO
Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to biochemically and microscopically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles positive for lysosomal-associated membrane protein 1 (LAMP1) but structurally segregated from dextran-laden and LysoTracker-laden endo-/lysosomes in live cells. As revealed by confocal microscopy, Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Ionomycin-evoked elevation in intracellular free Ca2+ concentration ([Ca2+]i) negligibly affected mobility of Nef.GFP structures but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in the number of Nef.GFP-positive structures was observed in non-stimulated controls (5 ± 1 structures/min), but not in ionomycin-stimulated cells (0 ± 2 structures/min; P < 0.05), indicating that elevated [Ca2+]i inhibits the release of Nef.GFP structures. The latter significantly co-localized with membrane sites immunopositive for the tetraspanins CD9 (36 ± 4%) and CD81 (22 ± 1%). This is the first report to demonstrate that microglial CD9- and CD81-positive plasma membrane-derived compartments are associated with biogenesis and Nef release.
Assuntos
Cálcio/metabolismo , Vesículas Citoplasmáticas/metabolismo , Citosol/metabolismo , Microglia/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Contagem de Células , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/efeitos dos fármacos , Citosol/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ionomicina/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Microglia/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Tetraspanina 28/metabolismo , Tetraspanina 29/metabolismoRESUMO
AIM: Alzheimer's disease (AD) is largely considered a neuron-derived insult, but also involves failure of astroglia. A recent study indicated that mutated presenilin 1 (PS1M146V), a putative endoplasmic reticulum (ER) Ca2+ channel with decreased Ca2+ conductance, impairs the traffic of astroglial peptidergic vesicles. Whether other pathogenically relevant PS1 mutants, such as PS1ΔE9, which code for ER channel with putative increased Ca2+ conductance, similarly affect vesicle traffic, is unknown. METHODS: Here, we cotransfected rat astrocytes with plasmids encoding mutant PS1ΔE9 and atrial natriuretic peptide or vesicular glutamate transporter 1 tagged with fluorescent proteins (pANP.emd or pVGLUT1-EGFP respectively), to microscopically examine whether alterations in vesicle mobility and Ca2+ -regulated release of gliosignalling molecules manifest as a general vesicle-based defect; control cells were transfected to co-express exogenous or native wild-type PS1 and pANP.emd or pVGLUT1-EGFP. The vesicle mobility was analysed at rest and after ATP stimulation that increased intracellular calcium activity. RESULTS: In PS1ΔE9 astrocytes, spontaneous mobility of both vesicle types was reduced (P < .001) when compared to controls. Post-stimulatory recovery of fast vesicle mobility was hampered in PS1ΔE9 astrocytes. The ATP-evoked peptide release was less efficient in PS1ΔE9 astrocytes than in the controls (P < .05), as was the pre-stimulatory mobility of these vesicles. CONCLUSION: Although the PS1 mutants PS1M146V and PS1ΔE9 differently affect ER Ca2+ conductance, our results revealed a common, vesicle-type indiscriminate trafficking defect in PS1ΔE9 astrocytes, indicating that reduced secretory vesicle-based signalling is a general deficit in AD astrocytes.
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Exocitose/fisiologia , Presenilina-1/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico/fisiologia , Células Cultivadas , Feminino , Organelas/metabolismo , Presenilina-1/genética , Ratos WistarRESUMO
Astrocytes are excitable neural cells that contribute to brain information processing via bidirectional communication with neurons. This involves the release of gliosignaling molecules that affect synapses patterning and activity. Mechanisms mediating the release of these molecules likely consist of non-vesicular and vesicular-based mechanisms. It is the vesicle-based regulated exocytosis that is an evolutionary more complex process. It is well established that the release of gliosignaling molecules has profound effects on information processing in different brain regions (e.g., hippocampal astrocytes contribute to long-term potentiation [LTP]), which has traditionally been considered as one of the cellular mechanisms underlying learning and memory. However, the paradigm of vesicle-based regulated release of gliosignaling molecules from astrocytes is still far from being unanimously accepted. One of the most important questions is to what extent can the conclusions obtained from cultured astrocytes be translated to in vivo conditions. Here, we overview the properties of vesicle mobility and their fusion with the plasma membrane in cultured astrocytes and compare these parameters to those recorded in astrocytes from acute brain hippocampal slices. The results from both experimental models are similar, which validates experiments on isolated astrocytes and further supports arguments in favor of in vivo vesicle-based exocytotic release of gliosignaling molecules. © 2017 Wiley Periodicals, Inc.
Assuntos
Astrócitos/metabolismo , Exocitose/fisiologia , Hipocampo/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Hipocampo/citologia , Potenciação de Longa Duração/fisiologia , Técnicas de Cultura de Órgãos , Roedores , Vesículas Sinápticas/metabolismoRESUMO
Encoding continuous sensory variables requires sustained synaptic signalling. At several sensory synapses, rapid vesicle supply is achieved via highly mobile vesicles and specialized ribbon structures, but how this is achieved at central synapses without ribbons is unclear. Here we examine vesicle mobility at excitatory cerebellar mossy fibre synapses which sustain transmission over a broad frequency bandwidth. Fluorescent recovery after photobleaching in slices from VGLUT1(Venus) knock-in mice reveal 75% of VGLUT1-containing vesicles have a high mobility, comparable to that at ribbon synapses. Experimentally constrained models establish hydrodynamic interactions and vesicle collisions are major determinants of vesicle mobility in crowded presynaptic terminals. Moreover, models incorporating 3D reconstructions of vesicle clouds near active zones (AZs) predict the measured releasable pool size and replenishment rate from the reserve pool. They also show that while vesicle reloading at AZs is not diffusion-limited at the onset of release, diffusion limits vesicle reloading during sustained high-frequency signalling.
Assuntos
Fibras Nervosas/fisiologia , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Camundongos , Movimento (Física) , Imagem ÓpticaRESUMO
In the brain, astrocytes provide metabolic and trophic support to neurones. Failure in executing astroglial homeostatic functions may contribute to the initiation and propagation of diseases, including Alzheimer disease (AD), characterized by a progressive loss of neurones over years. Here, we examined whether astrocytes from a mice model of AD isolated in the presymptomatic phase of the disease exhibit alterations in vesicle traffic, vesicular peptide release and purinergic calcium signaling. In cultured astrocytes isolated from a newborn wild-type (wt) and 3xTg-AD mouse, secretory vesicles and acidic endosomes/lysosomes were labeled by transfection with plasmid encoding atrial natriuretic peptide tagged with mutant green fluorescent protein (ANP.emd) and by LysoTracker, respectively. The intracellular Ca(2+) concentration ([Ca(2+)]i) was monitored with Fluo-2 and visualized by confocal microscopy. In comparison with controls, spontaneous mobility of ANP- and LysoTracker-labeled vesicles was diminished in 3xTg-AD astrocytes; the track length (TL), maximal displacement (MD) and directionality index (DI) were all reduced in peptidergic vesicles and in endosomes/lysosomes (P < 0.001), as was the ATP-evoked attenuation of vesicle mobility. Similar impairment of peptidergic vesicle trafficking was observed in wt rat astrocytes transfected to express mutated presenilin 1 (PS1M146V). The ATP-evoked ANP discharge from single vesicles was less efficient in 3xTg-AD and PS1M146V-expressing astrocytes than in respective wt controls (P < 0.05). Purinergic stimulation evoked biphasic and oscillatory [Ca(2+)]i responses; the latter were less frequent (P < 0.001) in 3xTg-AD astrocytes. Expression of PS1M146V in astrocytes impairs vesicle dynamics and reduces evoked secretion of the signaling molecule ANP; both may contribute to the development of AD.
Assuntos
Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Presenilina-1/metabolismo , Vesículas Secretórias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Fator Natriurético Atrial/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cátions Bivalentes/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Presenilina-1/genética , Ratos WistarRESUMO
During the last few decades synaptic vesicles have been assigned to a variety of functional and morphological classes or "pools". We have argued in the past (Rizzoli and Betz, 2005) that synaptic activity in several preparations is accounted for by the function of three vesicle pools: the readily releasable pool (docked at active zones and ready to go upon stimulation), the recycling pool (scattered throughout the nerve terminals and recycling upon moderate stimulation), and finally the reserve pool (occupying most of the vesicle clusters and only recycling upon strong stimulation). We discuss here the advancements in the vesicle pool field which took place in the ensuing years, focusing on the behavior of different pools under both strong stimulation and physiological activity. Several new findings have enhanced the three-pool model, with, for example, the disparity between recycling and reserve vesicles being underlined by the observation that the former are mobile, while the latter are "fixed". Finally, a number of altogether new concepts have also evolved such as the current controversy on the identity of the spontaneously recycling vesicle pool.