RESUMO
Common fusion machinery mediates the Ca2+-dependent exocytosis of synaptic vesicles (SVs) and dense-core vesicles (DCVs). Previously, Synapsin Ia (Syn Ia) was found to localize to SVs, essential for mobilizing SVs to the plasma membrane through phosphorylation. However, whether (or how) the phosphoprotein Syn Ia plays a role in regulating DCV exocytosis remains unknown. To answer these questions, we measured the dynamics of DCV exocytosis by using single-vesicle amperometry in PC12 cells (derived from the pheochromocytoma of rats of unknown sex) overexpressing wild-type or phosphodeficient Syn Ia. We found that overexpression of phosphodeficient Syn Ia decreased the DCV secretion rate, specifically via residues previously shown to undergo calmodulin-dependent kinase (CaMK)-mediated phosphorylation (S9, S566, and S603). Moreover, the fusion pore kinetics during DCV exocytosis were found to be differentially regulated by Syn Ia and two phosphodeficient Syn Ia mutants (Syn Ia-S62A and Syn Ia-S9,566,603A). Kinetic analysis suggested that Syn Ia may regulate the closure and dilation of DCV fusion pores via these sites, implying the potential interactions of Syn Ia with certain DCV proteins involved in the regulation of fusion pore dynamics. Furthermore, we predicted the interaction of Syn Ia with several DCV proteins, including Synaptophysin (Syp) and soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. By immunoprecipitation, we found that Syn Ia interacted with Syp via phosphorylation. Moreover, a proximity ligation assay (PLA) confirmed their phosphorylation-dependent, in situ interaction on DCVs. Together, these findings reveal a phosphorylation-mediated regulation of DCV exocytosis by Syn Ia.SIGNIFICANCE STATEMENT Although they exhibit distinct exocytosis dynamics upon stimulation, synaptic vesicles (SVs) and dense-core vesicles (DCVs) may undergo co-release in neurons and neuroendocrine cells through an undefined molecular mechanism. Synapsin Ia (Syn Ia) is known to recruit SVs to the plasma membrane via phosphorylation. Here, we examined whether Syn Ia also affects the dynamics of DCV exocytosis. We showed that Syn Ia regulates the DCV secretion rate and fusion pore kinetics during DCV exocytosis. Moreover, Syn Ia-mediated regulation of DCV exocytosis depends on phosphorylation. We further found that Syn Ia interacts with Synaptophysin (Syp) on DCVs in a phosphorylation-dependent manner. Thus, these results suggest that Syn Ia may regulate the release of DCVs via phosphorylation.
Assuntos
Membrana Celular/metabolismo , Exocitose/fisiologia , Vesículas Secretórias/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Células PC12 , Fosfoproteínas/metabolismo , RatosRESUMO
During the first postnatal week in rodents, cholinergic retinal waves initiate in starburst amacrine cells (SACs), propagating to retinal ganglion cells (RGCs) and visual centers, essential for visual circuit refinement. By modulating exocytosis in SACs, dynamic changes in the protein kinase A (PKA) activity can regulate the spatiotemporal patterns of cholinergic waves. Previously, cysteine string protein-α (CSPα) is found to interact with the core exocytotic machinery by PKA-mediated phosphorylation at serine 10 (S10). However, whether PKA-mediated CSPα phosphorylation may regulate cholinergic waves via SACs remains unknown. Here, we examined how CSPα phosphorylation in SACs regulates cholinergic waves. First, we identified that CSPα1 is the major isoform in developing rat SACs and the inner plexiform layer during the first postnatal week. Using SAC-specific expression, we found that the CSPα1-PKA-phosphodeficient mutant (CSP-S10A) decreased wave frequency, but did not alter the wave spatial correlation compared to control, wild-type CSPα1 (CSP-WT), or two PKA-phosphomimetic mutants (CSP-S10D and CSP-S10E). These suggest that CSPα-S10 phosphodeficiency in SACs dampens the frequency of cholinergic waves. Moreover, the level of phospho-PKA substrates was significantly reduced in SACs overexpressing CSP-S10A compared to control or CSP-WT, suggesting that the dampened wave frequency is correlated with the decreased PKA activity. Further, compared to control or CSP-WT, CSP-S10A in SACs reduced the periodicity of wave-associated postsynaptic currents (PSCs) in neighboring RGCs, suggesting that these RGCs received the weakened synaptic inputs from SACs overexpressing CSP-S10A. Finally, CSP-S10A in SACs decreased the PSC amplitude and the slope to peak PSC compared to control or CSP-WT, suggesting that CSPα-S10 phosphodeficiency may dampen the speed of the SAC-RGC transmission. Thus, via PKA-mediated phosphorylation, CSPα in SACs may facilitate the SAC-RGC transmission, contributing to the robust frequency of cholinergic waves.
Assuntos
Células Amácrinas , Proteínas de Choque Térmico HSP40 , Células Amácrinas/metabolismo , Animais , Colinérgicos/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana , Fosforilação , Ratos , Retina/metabolismoRESUMO
Patterned spontaneous activity periodically displays in developing retinas termed retinal waves, essential for visual circuit refinement. In neonatal rodents, retinal waves initiate in starburst amacrine cells (SACs), propagating across retinal ganglion cells (RGCs), further through visual centers. Although these waves are shown temporally synchronized with transiently high PKA activity, the downstream PKA target important for regulating the transmission from SACs remains unidentified. A t-SNARE, synaptosome-associated protein of 25 kDa (SNAP-25/SN25), serves as a PKA substrate, implying a potential role of SN25 in regulating retinal development. Here, we examined whether SN25 in SACs could regulate wave properties and retinogeniculate projection during development. In developing SACs, overexpression of wild-type SN25b, but not the PKA-phosphodeficient mutant (SN25b-T138A), decreased the frequency and spatial correlation of wave-associated calcium transients. Overexpressing SN25b, but not SN25b-T138A, in SACs dampened spontaneous, wave-associated, postsynaptic currents in RGCs and decreased the SAC release upon augmenting the cAMP-PKA signaling. These results suggest that SN25b overexpression may inhibit the strength of transmission from SACs via PKA-mediated phosphorylation at T138. Moreover, knockdown of endogenous SN25b increased the frequency of wave-associated calcium transients, supporting the role of SN25 in restraining wave periodicity. Finally, the eye-specific segregation of retinogeniculate projection was impaired by in vivo overexpression of SN25b, but not SN25b-T138A, in SACs. These results suggest that SN25 in developing SACs dampens the spatiotemporal properties of retinal waves and limits visual circuit refinement by phosphorylation at T138. Therefore, SN25 in SACs plays a profound role in regulating visual circuit refinement.
Assuntos
Sinalização do Cálcio/genética , Retina/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Vias Visuais/fisiologia , Potenciais de Ação/genética , Células Amácrinas/metabolismo , Células Amácrinas/fisiologia , Animais , Animais Recém-Nascidos/genética , Animais Recém-Nascidos/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Patch-Clamp , Fosforilação , Ligação Proteica , Retina/crescimento & desenvolvimento , Retina/fisiologia , Células Ganglionares da Retina/metabolismo , Potenciais Sinápticos/genéticaRESUMO
BACKGROUND INFORMATION: Neuron stem/progenitor cells (NSPCs) of zebrafish central nervous system (CNS) are known to thrive during oxygen recovery after hypoxia, but not all cell types have been fully characterised due to their heterogeneities. In addition, an in vivo model system is not available that can help us to identify what type-specific cell populations that are involved in neural regeneration and to track their cell fate after regeneration. To solve these issues, we employed a zebrafish transgenic line, huORFZ, which harbours an inhibitory upstream open reading frame of human chop mRNA fused downstream with GFP reporter and driven by cytomegalovirus promoter. When huORFZ embryos were exposure to hypoxic stress, followed by oxygen recovery, GFP was exclusively expressed in some particular cells of CNS. Unlike GFP-negative cells, all GFP-expressing cells were not apoptotic, indicating that cell populations that are able to survive after hypoxia can be identified through this approach. RESULTS: When GFP-expressing cells of spinal cord were studied, we found mostly NSPCs and radial glia cells (RGs), along with a few oligodendrocyte progenitor cells and oligodendrocytes, all termed as hypoxia-responsive recovering cells (HrRCs). After hypoxic stress, these GFP-positive HrRCs did not undergo apoptosis, but GFP-negative neurons did. Prolonged recovery time after hypoxia was correlated with higher proportions of GFP(+)-NSPCs and GFP(+)-RGs, in contrast to lower proportions of proliferating/differentiating GFP(-)-NSPCs and GFP(-)-RGs. Among HrRCs subtypes, only GFP(+)-NSPCs and GFP(+)-RGs proliferated, migrated and differentiated into functional neurons during oxygen recovery. When some HrRCs were ablated in the spinal cord of hypoxia-exposed huORFZ embryos, swimming performance was impaired, suggesting that HrRCs are involved in neuronal regeneration. CONCLUSION: We demonstrated type-specific cell populations able to respond sensitively to hypoxic stress in the spinal cord of zebrafish embryos and that these type-specific populations play a role in neural regeneration. SIGNIFICANCE: Among heterogeneous cell types that exist in the spinal cord of zebrafish embryos after hypoxia, the particular cells that are resistant to hypoxia and also involved in neuronal regeneration can be clearly identified and dynamically traced using a transgenic model fish.
Assuntos
Células-Tronco Neurais/citologia , Neurogênese , Medula Espinal/embriologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Hipóxia Celular , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Humanos , Células-Tronco Neurais/metabolismo , Fases de Leitura Aberta , RNA Mensageiro/genética , Medula Espinal/citologia , Fator de Transcrição CHOP/genética , Peixe-Zebra/genéticaRESUMO
PC12 cells serve as a secretory cell model, especially suitable for studying the molecular mechanisms underlying fusion pore kinetics in regulated exocytosis of dense-core vesicles (DCVs). In this chapter, we describe a series of PC12 cell culture procedures optimized for real-time functional assays such as single-vesicle amperometry. In addition, these conditions have been widely used for single-cell biochemical assays such as the proximity ligation assay with immunostaining.
Assuntos
Neoplasias das Glândulas Suprarrenais , Feocromocitoma , Animais , Exocitose , Cinética , Células PC12 , Ratos , Vesículas SecretóriasRESUMO
Exocytosis-the release of the contents of a vesicle--proceeds by two mechanisms. Full fusion occurs when the vesicle and plasma membranes merge. Alternatively, in what is termed kiss-and-run, vesicles can release transmitter during transient contacts with the plasma membrane. Little is known at the molecular level about how the choice between these two pathways is regulated. Here we report amperometric recordings of catecholamine efflux through individual fusion pores. Transfection with synaptotagmin (Syt) IV increased the frequency and duration of kiss-and-run events, but left their amplitude unchanged. Endogenous Syt IV, induced by forskolin treatment, had a similar effect. Full fusion was inhibited by mutation of a Ca2+ ligand in the C2A domain of Syt I; kiss-and-run was inhibited by mutation of a homologous Ca2+ ligand in the C2B domain of Syt IV. The Ca2+ sensitivity for full fusion was 5-fold higher with Syt I than Syt IV, but for kiss-and-run the Ca2+ sensitivities differed by a factor of only two. Syt thus regulates the choice between full fusion and kiss-and-run, with Ca2+ binding to the C2A and C2B domains playing an important role in this choice.
Assuntos
Proteínas de Ligação ao Cálcio , Membrana Celular/metabolismo , Exocitose , Fusão de Membrana , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Permeabilidade da Membrana Celular , Colforsina/farmacologia , Ligantes , Glicoproteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Células PC12 , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , SinaptotagminasRESUMO
Exocytosis involves the formation of a fusion pore that connects the lumen of secretory vesicles with the extracellular space. Exocytosis from neurons and neuroendocrine cells is tightly regulated by intracellular [Ca2+] and occurs rapidly, but the molecular events that mediate the opening and subsequent dilation of fusion pores remain to be determined. A putative Ca2+ sensor for release, synaptotagmin I (syt), binds directly to syntaxin and SNAP-25, which are components of a conserved membrane fusion complex. Here, we show that Ca2+-triggered syt*SNAP-25 interactions occur rapidly. The tandem C2 domains of syt cooperate to mediate binding to syntaxin/SNAP-25; lengthening the linker that connects C2A and C2B selectively disrupts this interaction. Expression of the linker mutants in PC12 cells results in graded reductions in the stability of fusion pores. Thus, the final step of Ca2+-triggered exocytosis is regulated, at least in part, by direct contacts between syt and SNAP-25/syntaxin.
Assuntos
Proteínas de Ligação ao Cálcio , Exocitose/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Membranas Sinápticas/metabolismo , Transmissão Sináptica/genética , Proteínas de Transporte Vesicular , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Exocitose/genética , Cinética , Substâncias Macromoleculares , Fusão de Membrana/genética , Glicoproteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Células PC12 , Terminações Pré-Sinápticas/ultraestrutura , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Qa-SNARE , Ratos , Proteínas SNARE , Membranas Sinápticas/ultraestrutura , Proteína 25 Associada a Sinaptossoma , Sinaptotagmina I , SinaptotagminasRESUMO
Ambient GABA modulates firing patterns in adult neural circuits by tonically activating extrasynaptic GABA(A) receptors. Here, we demonstrate that during a developmental period when activation of GABA(A) receptors causes membrane depolarization, tonic activation of GABA(A) receptors blocks all spontaneous activity recorded in retinal ganglion cells (RGCs) and starburst amacrine cells (SACs). Bath application of the GABA(A) receptor agonist muscimol blocked spontaneous correlated increases in intracellular calcium concentration and compound postsynaptic currents in RGCs associated with retinal waves. In addition, GABA(A) receptor agonists activated a tonic current in RGCs that significantly reduced their excitability. Using a transgenic mouse in which green fluorescent protein is expressed under the metabotropic glutamate receptor subtype 2 promoter to target recordings from SACs, we found that GABA(A) receptor agonists blocked compound postsynaptic currents and also activated a tonic current. GABA(A) receptor antagonists reduced the holding current in SACs but not RGCs, indicating that ambient levels of GABA tonically activate GABA(A) receptors in SACs. GABA(A) receptor antagonists did not block retinal waves but did alter the frequency and correlation structure of spontaneous RGC firing. Interestingly, the drug aminophylline, a general adenosine receptor antagonist used to block retinal waves, induced a tonic GABA(A) receptor antagonist-sensitive current in outside-out patches excised from RGCs, indicating that aminophylline exerts its action on retinal waves by direct activation of GABA(A) receptors. These findings have implications for how various neuroactive drugs and neurohormones known to modulate extrasynaptic GABA(A) receptors may influence spontaneous firing patterns that are critical for the establishment of adult neural circuits.
Assuntos
Receptores de GABA-A/fisiologia , Retina/citologia , Retina/crescimento & desenvolvimento , Vias Visuais/fisiologia , Células Amácrinas/efeitos dos fármacos , Células Amácrinas/fisiologia , Aminofilina/farmacologia , Animais , Animais Recém-Nascidos , Interações Medicamentosas , GABAérgicos/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-Clamp/métodos , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/fisiologia , Vias Visuais/efeitos dos fármacosRESUMO
Recent evidence demonstrates that low-frequency oscillations of intracellular calcium on timescales of seconds to minutes drive distinct aspects of neuronal development, but the mechanisms by which these calcium transients are coupled to signaling cascades are not well understood. Here we test the hypothesis that spontaneous electrical activity activates protein kinase A (PKA). We use live-cell indicators to observe spontaneous and evoked changes in cAMP levels and PKA activity in developing retinal neurons. Expression of cAMP and PKA indicators in neonatal rat retinal explants reveals spontaneous oscillations in PKA activity that are temporally correlated with spontaneous depolarizations associated with retinal waves. In response to short applications of forskolin, dopamine, or high-potassium concentration, we image an increase in cAMP levels and PKA activity, indicating that this second-messenger pathway can be activated quickly by neural activity. Depolarization-evoked increases in PKA activity were blocked by the removal of extracellular calcium, indicating that they are mediated by a calcium-dependent mechanism. These findings demonstrate for the first time that spontaneous activity in developing circuits is correlated with activation of the cAMP/PKA pathway and that PKA activity is turned on and off on the timescale of tens of seconds. These results show a link between neural activity and an intracellular biochemical cascade associated with plasticity, axon guidance, and neural differentiation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Retina/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , AMP Cíclico/análise , Proteínas Quinases Dependentes de AMP Cíclico/análise , Ativação Enzimática/fisiologia , Transferência Ressonante de Energia de Fluorescência , Neurônios/química , Neurônios/metabolismo , Ratos , Retina/químicaRESUMO
A characteristic feature of developing neural networks is spontaneous periodic activity. In the developing retina, retinal ganglion cells fire bursts of action potentials that drive large increases in intracellular calcium concentration with a periodicity of minutes. These periodic bursts of action potentials propagate across the developing inner retina as waves, driving neighboring retinal ganglion cells to fire in a correlated fashion. Here we will review recent progress in elucidating the mechanisms in mammals underlying retinal wave propagation and those regulating the periodicity with which these retinal waves occur. In addition, we will review recent experiments indicating that retinal waves are critical for refining retinal projections to their primary targets in the central visual system and may be involved in driving developmental processes within the retina itself.
Assuntos
Retina/embriologia , Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Visão Ocular/fisiologia , Animais , Humanos , Periodicidade , Percepção Visual/fisiologiaRESUMO
Retinal waves are mediated in part by activation of nicotinic receptors containing the beta2 subunit. Mice deficient in beta2 containing nAChRs have maintained firing of action potentials but do not support correlated waves. As a result, beta2-/- mice have inhibited refinement of circuits within the retina as well as retinal projections to the CNS. Previously, we observed that correlated increases in calcium reminiscent of retinal waves could be induced in beta2-/- retina by pharmacological application of the L-type calcium channel agonist, FPL-64176. Here, we characterize FPL-induced activity patterns in beta2-/- retina using both whole cell and multielectrode array recordings. FPL-induced strong depolarizations in previously non-spiking beta2-/- retinal ganglion cells. Though these strong depolarizations were likely to underlie the FPL-induced calcium transients, they led to highly variable effects on the spiking of individual retinal ganglion cells. In addition, induced spiking activity had significantly weaker nearest-neighbor correlations than WT mice. Initial attempts of intraocular injections of FPL in beta2-/- mice did not rescue eye-specific layer formation. These findings indicate that activity induced by FPL is not sufficient for driving eye-specific segregation in beta2-/- mice.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/fisiologia , Receptores Nicotínicos/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Eletrofisiologia , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Pirróis/farmacologia , Receptores Nicotínicos/deficiência , Células Ganglionares da Retina/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
The activation of GABAA receptors (the type A receptors for γ-aminobutyric acid) produces two distinct forms of responses, phasic (i.e., transient) and tonic (i.e., persistent), that are mediated by synaptic and extrasynaptic GABAA receptors, respectively. During development, the intracellular chloride levels are high so activation of these receptors causes a net outward flow of anions that leads to neuronal depolarization rather than hyperpolarization. Therefore, in developing neural circuits, tonic activation of GABAA receptors may provide persistent depolarization. Recently, it became evident that GABAA receptor-mediated tonic depolarization alters the structure of patterned spontaneous activity, a feature that is common in developing neural circuits and is important for neural circuit refinement. Thus, this persistent depolarization may lead to a long-lasting increase in intracellular calcium level that modulates network properties via calcium-dependent signaling cascades. This article highlights the features of GABAA receptor-mediated tonic depolarization, summarizes the principles for discovery, reviews the current findings in diverse developing circuits, examines the underlying molecular mechanisms and modulation systems, and discusses their functional specializations for each developing neural circuit.
Assuntos
Potenciais de Ação/fisiologia , Encéfalo/crescimento & desenvolvimento , Rede Nervosa/crescimento & desenvolvimento , Inibição Neural/fisiologia , Receptores de GABA-A/fisiologia , Animais , HumanosRESUMO
BACKGROUND: Cysteine string protein-α (CSPα) is a chaperone to ensure protein folding. Loss of CSPα function associates with many neurological diseases. However, its function in modulating regulated exocytosis remains elusive. Although cspα-knockouts exhibit impaired synaptic transmission, overexpression of CSPα in neuroendocrine cells inhibits secretion. These seemingly conflicting results lead to a hypothesis that CSPα may undergo a modification that switches its function in regulating neurotransmitter and hormone secretion. Previous studies implied that CSPα undergoes phosphorylation at Ser10 that may influence exocytosis by altering fusion pore dynamics. However, direct evidence is missing up to date. METHODOLOGY/PRINCIPAL FINDINGS: Using amperometry, we investigated how phosphorylation at Ser10 of CSPα (CSPα-Ser10) modulates regulated exocytosis and if this modulation involves regulating a specific kinetic step of fusion pore dynamics. The real-time exocytosis of single vesicles was detected in PC12 cells overexpressing control vector, wild-type CSPα (WT), the CSPα phosphodeficient mutant (S10A), or the CSPα phosphomimetic mutants (S10D and S10E). The shapes of amperometric signals were used to distinguish the full-fusion events (i.e., prespike feet followed by spikes) and the kiss-and-run events (i.e., square-shaped flickers). We found that the secretion rate was significantly increased in cells overexpressing S10D or S10E compared to WT or S10A. Further analysis showed that overexpression of S10D or S10E prolonged fusion pore lifetime compared to WT or S10A. The fraction of kiss-and-run events was significantly lower but the frequency of full-fusion events was higher in cells overexpressing S10D or S10E compared to WT or S10A. Advanced kinetic analysis suggests that overexpression of S10D or S10E may stabilize open fusion pores mainly by inhibiting them from closing. CONCLUSIONS/SIGNIFICANCE: CSPα may modulate fusion pore dynamics in a phosphorylation-dependent manner. Therefore, through changing its phosphorylated state influenced by diverse cellular signalings, CSPα may have a great capacity to modulate the rate of regulated exocytosis.
Assuntos
Exocitose , Proteínas de Choque Térmico HSP40/genética , Fusão de Membrana , Proteínas de Membrana/genética , Mutação/genética , Animais , Membrana Celular/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Cinética , Proteínas de Membrana/metabolismo , Proteínas Mutantes/metabolismo , Células PC12 , Fosforilação , RatosRESUMO
BACKGROUND: Developing retinas display retinal waves, the patterned spontaneous activity essential for circuit refinement. During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine's regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A(2A) receptor (A(2A)R) regulates retinal waves and whether A(2A)R regulation of retinal waves acts via presynaptic SACs. METHODOLOGY/PRINCIPAL FINDINGS: We showed that A(2A)R was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A(2A)R decreased the frequency of spontaneous Ca²âº transients, suggesting that endogenous A(2A)R may up-regulate wave frequency. To investigate whether A(2A)R acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca²âº transient frequency was increased by expressing wild-type A(2A)R (A2AR-WT) in SACs, suggesting that A(2A)R may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A(2A)R-WT increased the frequency of wave-associated postsynaptic currents (PSCs) or depolarizations compared to the control, without changing the RGC's excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A(2A)R may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A(2A)R mutant (A(2A)R-ΔC) in SACs, the wave frequency was reduced compared to the A(2A)R-WT, but was similar to the control, suggesting that the full-length A(2A)R in SACs is required for A(2A)R up-regulation of retinal waves. CONCLUSIONS/SIGNIFICANCE: A(2A)R up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full-length protein structure. Thus, by coupling with the downstream intracellular signaling, A(2A)R may have a great capacity to modulate patterned spontaneous activity during neural circuit refinement.
Assuntos
Potenciais de Ação , Células Amácrinas/citologia , Receptor A2A de Adenosina/metabolismo , Retina/citologia , Retina/crescimento & desenvolvimento , Regulação para Cima , Adenilil Ciclases/metabolismo , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Imagem Molecular , Mutação , Ratos , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/deficiência , Receptor A2A de Adenosina/genética , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Transdução de Sinais , Potenciais SinápticosRESUMO
In obesity, high levels of tumor necrosis factor α (TNFα) stimulate lipolysis in adipocytes, leading to hyperlipidemia and insulin resistance. Thiazolidinediones (TZDs), the insulin-sensitizing drugs, antagonize TNFα-induced lipolysis in adipocytes, thereby increasing insulin sensitivity in diabetes patients. The cellular target of TZDs is peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor that controls many adipocyte functions. As a transcription factor, PPARγ is closely modulated by coregulators, which include coactivators and corepressors. Previous studies have revealed that in macrophages, the insulin-sensitizing effect of PPARγ may involve suppression of proinflammatory gene expression by recruiting the corepressor complex that contains corepressors and histone deacetylases (HDACs). Therefore, we investigated whether the corepressor complex is involved in TZD-mediated suppression of TNFα-induced lipolysis in 3T3-L1 adipocytes. Trichostatin A (TSA), a pan HDAC inhibitor (HDACI) that inhibits class I and II HDACs, was used to examine the involvement of HDACs in the actions of TZDs. TSA alone increased basal lipolysis and attenuated TZD-mediated suppression of TNFα-induced lipolysis. Increased basal lipolysis may in part result from class I HDAC inhibition because selective class I HDACI treatment had similar results. However, attenuation of TZD-mediated TNFα antagonism may be specific to TSA and related hydroxamate-based HDACI rather than to HDAC inhibition. Consistently, corepressor depletion did not affect TZD-mediated suppression. Interestingly, TSA treatment greatly reduced PPARγ levels in differentiated adipocytes. Finally, extracellular signal-related kinase 1/2 (ERK1/2) mediated TNFα-induced lipolysis, and TZDs suppressed TNFα-induced ERK phosphorylation. We determined that TSA increased basal ERK phosphorylation, and attenuated TZD-mediated suppression of TNFα-induced ERK phosphorylation, consistent with TSA's effects on lipolysis. These studies suggest that TSA, through down-regulating PPARγ, attenuates TZD-mediated suppression of TNFα-induced ERK phosphorylation and lipolysis in adipocytes.
Assuntos
Adipócitos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Hipoglicemiantes/farmacologia , Lipólise/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Diferenciação Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: In neonatal binocular animals, the developing retina displays patterned spontaneous activity termed retinal waves, which are initiated by a single class of interneurons (starburst amacrine cells, SACs) that release neurotransmitters. Although SACs are shown to regulate wave dynamics, little is known regarding how altering the proteins involved in neurotransmitter release may affect wave dynamics. Synaptotagmin (Syt) family harbors two Ca(2+)-binding domains (C2A and C2B) which serve as Ca(2+) sensors in neurotransmitter release. However, it remains unclear whether SACs express any specific Syt isoform mediating retinal waves. Moreover, it is unknown how Ca(2+) binding to C2A and C2B of Syt affects wave dynamics. Here, we investigated the expression of Syt I in the neonatal rat retina and examined the roles of C2A and C2B in regulating wave dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Immunostaining and confocal microscopy showed that Syt I was expressed in neonatal rat SACs and cholinergic synapses, consistent with its potential role as a Ca(2+) sensor mediating retinal waves. By combining a horizontal electroporation strategy with the SAC-specific promoter, we specifically expressed Syt I mutants with weakened Ca(2+)-binding ability in C2A or C2B in SACs. Subsequent live Ca(2+) imaging was used to monitor the effects of these molecular perturbations on wave-associated spontaneous Ca(2+) transients. We found that targeted expression of Syt I C2A or C2B mutants in SACs significantly reduced the frequency, duration, and amplitude of wave-associated Ca(2+) transients, suggesting that both C2 domains regulate wave temporal properties. In contrast, these C2 mutants had relatively minor effects on pairwise correlations over distance for wave-associated Ca(2+) transients. CONCLUSIONS/SIGNIFICANCE: Through Ca(2+) binding to C2A or C2B, the Ca(2+) sensor Syt I in SACs may regulate patterned spontaneous activity to shape network activity during development. Hence, modulating the releasing machinery in presynaptic neurons (SACs) alters wave dynamics.
Assuntos
Cálcio/metabolismo , Domínios e Motivos de Interação entre Proteínas , Retina/metabolismo , Sinaptotagmina I/metabolismo , Animais , Neurônios Colinérgicos/metabolismo , Regulação da Expressão Gênica , Ligação Proteica , Ratos , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Retina/citologia , Retina/fisiologia , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Sinaptotagmina I/química , Sinaptotagmina I/genéticaRESUMO
Synaptotagmin I (Syt I), the putative Ca(2+) sensor in regulated exocytosis, has two Ca(2+)-binding modules, the C2A and C2B domains, and a number of putative effectors to which Syt I binds in a Ca(2+)-dependent fashion. The role of Ca(2+) binding to these domains remains unclear, as efforts to address questions about Ca(2+)-triggered effector interactions have led to conflicting results. We have studied the effects of Ca(2+) on fusion pores using amperometry to follow the exocytosis of single vesicles in real time and analyse the kinetics of fusion pore transitions. Elevating [Ca(2+)] in permeabilized cells reduced the fusion pore lifetime, indicating an action of Ca(2+) during the actual fusion process. Analysing the Ca(2+) dependence of the fusion pore lifetime, together with the frequency of pore openings and the proportion of openings that close without dilating (kiss-and-run events) enabled us to resolve exocytosis into a sequence of kinetic steps representing functional transitions in the fusion pore. Fusion pore opening and dilation were both accelerated by Ca(2+), indicating separate Ca(2+) control over each of these steps. Ca(2+) ligand mutations in either the C2A or C2B domains of Syt I reduced fusion pore opening, but had opposite actions on the rate of fusion pore closure. These studies resolve two separate and distinct Ca(2+)-triggered steps during regulated exocytosis. The C2A and C2B domains of Syt I have different actions during these steps, and these actions may be linked to their distinctive effector interactions.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Sinaptotagmina I/fisiologia , Animais , Cálcio/fisiologia , Interpretação Estatística de Dados , Exocitose/fisiologia , Ligantes , Potenciais da Membrana/fisiologia , Mutação , Células PC12 , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Sinaptotagmina I/química , Sinaptotagmina I/genética , Sinaptotagmina I/farmacologiaRESUMO
The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca2+-triggered exocytosis. Its cytoplasmic domain is composed of tandem C2 domains, C2A and C2B; each C2 domain binds Ca2+. Upon binding Ca2+, positively charged residues within the Ca2+-binding loops are thought to interact with negatively charged phospholipids in the target membrane to mediate docking of the cytoplasmic domain of syt onto lipid bilayers. The C2 domains of syt also interact with syntaxin and SNAP-25, two components of a conserved membrane fusion complex. Here, we have neutralized single positively charged residues at the membrane-binding interface of C2A (R233Q) and C2B (K366Q). Either of these mutations shifted the Ca2+ requirements for syt-liposome interactions from approximately 20 to approximately 40 microm Ca2+. Kinetic analysis revealed that the reduction in Ca2+-sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect sytsyntaxin interactions but resulted in an approximately 50% loss in SNAP-25 binding activity, suggesting that these residues lie at an interface between membranes and SNAP-25. Expression of full-length versions of syt that harbored these mutations reduced the rate of exocytosis in PC12 cells. In both biochemical and functional assays, effects of the R233Q and K366Q mutations were not additive, indicating that mutations in one domain affect the activity of the adjacent domain. These findings indicate that the tandem C2 domains of syt cooperate with one another to trigger release via loop-mediated electrostatic interactions with effector molecules.
Assuntos
Proteínas de Ligação ao Cálcio , Exocitose/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/fisiologia , Animais , Sítios de Ligação , Cálcio , Cinética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , Estrutura Terciária de Proteína , Ratos , Eletricidade Estática , Proteína 25 Associada a Sinaptossoma , Sinaptotagmina I , SinaptotagminasRESUMO
The fusion pore of regulated exocytosis is a channel that connects and spans the vesicle and plasma membranes. The molecular composition of this important intermediate structure of exocytosis is unknown. Here, we found that mutations of some residues within the transmembrane segment of syntaxin (Syx), a plasma membrane protein essential for exocytosis, altered neurotransmitter flux through fusion pores and altered pore conductance. The residues that influenced fusion-pore flux lay along one face of an alpha-helical model. Thus, the fusion pore is formed at least in part by a circular arrangement of 5 to 8 Syx transmembrane segments in the plasma membrane.
Assuntos
Cálcio/metabolismo , Estruturas da Membrana Celular/química , Exocitose , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Neurônios/fisiologia , Animais , Estruturas da Membrana Celular/metabolismo , Capacitância Elétrica , Condutividade Elétrica , Eletrofisiologia , Fusão de Membrana , Proteínas de Membrana/genética , Modelos Biológicos , Mutação , Norepinefrina/metabolismo , Células PC12 , Técnicas de Patch-Clamp , Estrutura Secundária de Proteína , Proteínas Qa-SNARE , Ratos , TransfecçãoRESUMO
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat in the HD gene. We reported recently that complexin II, a protein involved in neurotransmitter release, is depleted from both the brains of mice carrying the HD mutation and from the striatum of post mortem HD brains. Here we show that this loss of complexin II is recapitulated in PC12 cells expressing the HD mutation and is accompanied by a dramatic decline in Ca2+-triggered exocytosis of neurotransmitter. Overexpression of complexin II (but not complexin I) rescued exocytosis, demonstrating that the decline in neurotransmitter release is a direct consequence of complexin II depletion. Complexin II depletion in the brain may account for some of the abnormalities in neurotransmission associated with HD.