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[This corrects the article DOI: 10.1371/journal.pbio.3002483.].
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Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Our studies reveal that SPNs manifest a heterosynaptic, nitric oxide (NO)-dependent form of long-term postsynaptic depression of glutamatergic SPN synapses (NO-LTD) that is preferentially engaged at quiescent synapses. Plasticity is gated by Ca2+ entry through CaV1.3 Ca2+ channels and phosphodiesterase 1 (PDE1) activation, which blunts intracellular cyclic guanosine monophosphate (cGMP) and NO signaling. Both experimental and simulation studies suggest that this Ca2+-dependent regulation of PDE1 activity allows for local regulation of dendritic cGMP signaling. In a mouse model of Parkinson disease (PD), NO-LTD is absent because of impaired interneuronal NO release; re-balancing intrastriatal neuromodulatory signaling restores NO release and NO-LTD. Taken together, these studies provide important insights into the mechanisms governing NO-LTD in SPNs and its role in psychomotor disorders such as PD.
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Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Plasticidad Neuronal , Neuronas , Sinapsis , Animales , Sinapsis/metabolismo , Plasticidad Neuronal/fisiología , Ratones , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Cuerpo Estriado/metabolismo , GMP Cíclico/metabolismo , Ácido Glutámico/metabolismo , Calcio/metabolismo , Ratones Endogámicos C57BL , Masculino , Depresión Sináptica a Largo Plazo/fisiologíaRESUMEN
Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Although considerable attention has been paid to the mechanisms underlying synaptic strengthening and new learning, little scrutiny has been given to those involved in the attenuation of synaptic strength that attends suppression of a previously learned association. Our studies revealed a novel, non-Hebbian, long-term, postsynaptic depression of glutamatergic SPN synapses induced by interneuronal nitric oxide (NO) signaling (NO-LTD) that was preferentially engaged at quiescent synapses. This form of plasticity was gated by local Ca 2+ influx through CaV1.3 Ca 2+ channels and stimulation of phosphodiesterase 1 (PDE1), which degraded cyclic guanosine monophosphate (cGMP) and blunted NO signaling. Consistent with this model, mice harboring a gain-of-function mutation in the gene coding for the pore-forming subunit of CaV1.3 channels had elevated depolarization-induced dendritic Ca 2+ entry and impaired NO-LTD. Extracellular uncaging of glutamate and intracellular uncaging of cGMP suggested that this Ca 2+ -dependent regulation of PDE1 activity allowed for local regulation of dendritic NO signaling. This inference was supported by simulation of SPN dendritic integration, which revealed that dendritic spikes engaged PDE1 in a branch-specific manner. In a mouse model of Parkinson's disease (PD), NO-LTD was absent not because of a postsynaptic deficit in NO signaling machinery, but rather due to impaired interneuronal NO release. Re-balancing intrastriatal neuromodulatory signaling in the PD model restored NO release and NO-LTD. Taken together, these studies provide novel insights into the mechanisms governing NO-LTD in SPN and its role in psychomotor disorders, like PD.
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Synaptic transmission mediated by GABAA receptors (GABAARs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at subthreshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical, and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. In perforated patch recordings, activation of GABAARs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in both juvenile and adult SPNs. Transcriptomic analysis and pharmacological work suggested that this relatively positive GABAAR reversal potential was not attributable to NKCC1 expression, but rather to HCO3- permeability. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to dendritic iGluR signaling and promoted dendritic spikes. Taken together, our results demonstrate that GABAARs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation for the role of intrastriatal GABAergic circuits.
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Interneuronas , Receptores de GABA-A , Ratones , Animales , Cuerpo Estriado/fisiología , Neostriado , Transmisión Sináptica/fisiología , Neuronas GABAérgicas/fisiologíaRESUMEN
Huntington's disease (HD) is a progressive, neurodegenerative disease caused by a CAG triplet expansion in huntingtin. Although corticostriatal dysfunction has long been implicated in HD, the determinants and pathway specificity of this pathophysiology are not fully understood. Here, using a male zQ175+/- knock-in mouse model of HD we carry out optogenetic interrogation of intratelencephalic and pyramidal tract synapses with principal striatal spiny projection neurons (SPNs). These studies reveal that the connectivity of intratelencephalic, but not pyramidal tract, neurons with direct and indirect pathway SPNs increased in early symptomatic zQ175+/- HD mice. This enhancement was attributable to reduced pre-synaptic inhibitory control of intratelencephalic terminals by striatal cholinergic interneurons. Lowering mutant huntingtin selectively in striatal cholinergic interneurons with a virally-delivered zinc finger repressor protein normalized striatal acetylcholine release and intratelencephalic functional connectivity, revealing a node in the network underlying corticostriatal pathophysiology in a HD mouse model.
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Enfermedad de Huntington , Enfermedades Neurodegenerativas , Ratones , Masculino , Animales , Enfermedad de Huntington/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Cuerpo Estriado/metabolismo , Neostriado/metabolismo , Colinérgicos/metabolismo , Modelos Animales de Enfermedad , Ratones Transgénicos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismoRESUMEN
Synaptic transmission mediated by GABA A receptors (GABA A Rs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at sub-threshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. Activation of GABA A Rs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in perforated patch recordings from both juvenile and adult SPNs. Molecular profiling of SPNs suggested that this relatively positive reversal potential was not attributable to NKCC1 expression, but rather to a dynamic equilibrium between KCC2 and Cl-/HCO3-cotransporters. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to coincident glutamatergic input. Taken together, our results demonstrate that GABA A Rs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation of the role intrastriatal GABAergic circuits.
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Background: Pathological accumulation of aggregated α-synuclein (aSYN) is a common feature of Parkinson's disease (PD). However, the mechanisms by which intracellular aSYN pathology contributes to dysfunction and degeneration of neurons in the brain are still unclear. A potentially relevant target of aSYN is the mitochondrion. To test this hypothesis, genetic and physiological methods were used to monitor mitochondrial function in substantia nigra pars compacta (SNc) dopaminergic and pedunculopontine nucleus (PPN) cholinergic neurons after stereotaxic injection of aSYN pre-formed fibrils (PFFs) into the mouse brain. Methods: aSYN PPFs were stereotaxically injected into the SNc or PPN of mice. Twelve weeks later, mice were studied using a combination of approaches, including immunocytochemical analysis, cell- type specific transcriptomic profiling, electron microscopy, electrophysiology and two-photon-laser- scanning microscopy of genetically encoded sensors for bioenergetic and redox status. Results: In addition to inducing a significant neuronal loss, SNc injection of PFFs induced the formation of intracellular, phosphorylated aSYN aggregates selectively in dopaminergic neurons. In these neurons, PFF-exposure decreased mitochondrial gene expression, reduced the number of mitochondria, increased oxidant stress, and profoundly disrupted mitochondrial adenosine triphosphate production. Consistent with an aSYN-induced bioenergetic deficit, the autonomous spiking of dopaminergic neurons slowed or stopped. PFFs also up-regulated lysosomal gene expression and increased lysosomal abundance, leading to the formation of Lewy-like inclusions. Similar changes were observed in PPN cholinergic neurons following aSYN PFF exposure. Conclusions: Taken together, our findings suggest that disruption of mitochondrial function, and the subsequent bioenergetic deficit, is a proximal step in the cascade of events induced by aSYN pathology leading to dysfunction and degeneration of neurons at-risk in PD.
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Recombinant adeno-associated viruses (rAAV) are extensively used in both research and clinical applications. Despite significant advances, there is a lack of short promoters able to drive the expression of virus delivered genes in specific classes of neurons. We designed an efficient rAAV vector suitable for the rAAV-mediated gene expression in cortical interneurons, mainly in the parvalbumin expressing cells. The vector includes a short parvalbumin promoter and a specialized poly(A) sequence. The degree of conservation of the parvalbumin gene adjoining non-coding regions was used in both the promoter design and the selection of the poly(A) sequence. The specificity was established by co-localizing the fluorescence of the virus delivered eGFP and the antibody for a neuronal marker. rAAV particles were injected in the visual cortex area V1/V2 of adult rats (2-4 months old). Neurons expressing the virus delivered eGFP were mainly positive for interneuronal markers: 66.5 ± 2.8% for parvalbumin, 14.6 ± 2.4% for somatostatin, 7.1 ± 1.2% for vasoactive intestinal peptide, 2.8 ± 0.6% for cholecystokinin. Meanwhile, only 2.1 ± 0.5% were positive for CaMKII, a marker for principal cells in the cortex. The efficiency of the construct was verified by optogenetic experiments: the expression of the virus delivered ChR2 channels was sufficient to evoke by blue light laser high frequency bursts of action potentials in putative fast spiking neurons. We conclude that our promoter allows highly specific expression of the rAAV delivered cDNAs in cortical interneurons with a strong preference for the parvalbumin positive cells.
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Parvalbúminas , Péptido Intestinal Vasoactivo , Animales , Ratas , Parvalbúminas/genética , Péptido Intestinal Vasoactivo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Interneuronas/metabolismo , Dependovirus/genética , Somatostatina/metabolismo , Colecistoquinina/metabolismoRESUMEN
How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca2+) entry through Cav1 channels triggered Ca2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca2+-dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.
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Calcio , Neuronas Dopaminérgicas , Adenosina Trifosfato/metabolismo , Ácido Aspártico , Calcio/metabolismo , Neuronas Dopaminérgicas/metabolismo , Malatos/metabolismo , Malatos/farmacología , Mitocondrias/metabolismo , Oxidantes , Sustancia Negra/metabolismoRESUMEN
Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.
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Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Axones/patología , Muerte Celular , Dendritas/metabolismo , Dendritas/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Levodopa/farmacología , Levodopa/uso terapéutico , Masculino , Ratones , Destreza Motora/efectos de los fármacos , NADH Deshidrogenasa/deficiencia , NADH Deshidrogenasa/genética , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/fisiopatología , Fenotipo , Sustancia Negra/citología , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismoRESUMEN
Local control of protein translation is a fundamental process for the regulation of synaptic plasticity. It has been demonstrated that the local protein synthesis occurring in axons and dendrites can be shaped by numerous mechanisms, including miRNA-mediated regulation. However, several aspects underlying this regulatory process have not been elucidated yet. Here, we analyze the differential miRNA profile in cell bodies and neurites of primary hippocampal neurons and find an enrichment of the precursor and mature forms of miR-218 in the neuritic projections. We show that miR-218 abundance is regulated during hippocampal development and by chronic silencing or activation of neuronal network. Overexpression and knockdown of miR-218 demonstrated that miR-218 targets the mRNA encoding the GluA2 subunit of AMPA receptors and modulates its expression. At the functional level, miR-218 overexpression increases glutamatergic synaptic transmission at both single neuron and network levels. Our data demonstrate that miR-218 may play a key role in the regulation of AMPA-mediated excitatory transmission and in the homeostatic regulation of synaptic plasticity.
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MicroARNs/metabolismo , Neuritas/metabolismo , Biosíntesis de Proteínas , Subunidades de Proteína/metabolismo , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Animales , Secuencia de Bases , Cuerpo Celular/metabolismo , Potenciales Postsinápticos Excitadores , Hipocampo/metabolismo , Ratones Endogámicos C57BL , MicroARNs/genética , Red Nerviosa/metabolismoRESUMEN
Key mitochondrial functions such as ATP production, Ca2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔµH+) across the inner membrane. Although several drugs can modulate ΔµH+, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψm) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca2+ dynamics, and respiratory metabolism. By directly modulating Δψm, the mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic ß-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.
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Señalización del Calcio/fisiología , Channelrhodopsins/metabolismo , Células Secretoras de Insulina/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Optogenética , Animales , Células HEK293 , Células HeLa , Humanos , Células Secretoras de Insulina/citología , Consumo de Oxígeno/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
In the superior colliculus, visual stimuli can induce gamma frequency oscillations of neuronal activity. It has been shown that in cats, these oscillations are synchronized over distances of greater than 300 µm that may contribute toward visual information processing. We investigated the spatial properties of such oscillations in a rodent because the availability of molecular tools could enable future studies on the role of these oscillations in visual information processing. Using extracellular electrode array recordings in anesthetized rats, we found that visual stimuli-induced gamma and eta frequency (30-115 Hz) oscillations of the local field potential that were synchronized over distances of â¼ 600 µm. Multiple-unit events were phase locked to the local field potential signal and showed prominent oscillations during OFF responses. The rate of lower than 5 ms cross-electrode coincidences was in line with the response-corrected predictions for each electrode. These data suggest that the synchronized superior colliculus neuronal activity is largely network driven, whereas common synaptic inputs play a minor role.
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Sincronización de Fase en Electroencefalografía/fisiología , Ritmo Gamma/fisiología , Red Nerviosa/fisiología , Colículos Superiores/fisiología , Percepción Visual/fisiología , Animales , Masculino , Ratas , Ratas WistarRESUMEN
The superior colliculus in mammals or the optic tectum in amphibians is a major visual information processing center responsible for generation of orientating responses such as saccades in monkeys or prey catching avoidance behavior in frogs. The conserved structure function of the superior colliculus the optic tectum across distant species such as frogs, birds monkeys permits to draw rather general conclusions after studying a single species. We chose the frog optic tectum because we are able to perform whole-cell voltage-clamp recordings fluorescence imaging of tectal neurons while they respond to a visual stimulus. In the optic tectum of amphibians most visual information is processed by pear-shaped neurons possessing long dendritic branches, which receive the majority of synapses originating from the retinal ganglion cells. Since the first step of the retinal input integration is performed on these dendrites, it is important to know whether this integration is enhanced by active dendritic properties. We demonstrate that rapid calcium transients coinciding with the visual stimulus evoked action potentials in the somatic recordings can be readily detected up to the fine branches of these dendrites. These transients were blocked by calcium channel blockers nifedipine CdCl2 indicating that calcium entered dendrites via voltage-activated L-type calcium channels. The high speed of calcium transient propagation, >300 µm in <10 ms, is consistent with the notion that action potentials, actively propagating along dendrites, open voltage-gated L-type calcium channels causing rapid calcium concentration transients in the dendrites. We conclude that such activation by somatic action potentials of the dendritic voltage gated calcium channels in the close vicinity to the synapses formed by axons of the retinal ganglion cells may facilitate visual information processing in the principal neurons of the frog optic tectum.
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Potenciales de Acción , Calcio/metabolismo , Dendritas/metabolismo , Estimulación Luminosa , Ranidae/fisiología , Colículos Superiores/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Dendritas/efectos de los fármacos , Nimodipina/farmacología , Técnicas de Placa-Clamp , Colículos Superiores/efectos de los fármacosRESUMEN
Synapsin I (SynI) is a synaptic vesicle (SV) phosphoprotein playing multiple roles in synaptic transmission and plasticity by differentially affecting crucial steps of SV trafficking in excitatory and inhibitory synapses. SynI knockout (KO) mice are epileptic, and nonsense and missense mutations in the human SYN1 gene have a causal role in idiopathic epilepsy and autism. To get insights into the mechanisms of epileptogenesis linked to SYN1 mutations, we analyzed the effects of the recently identified Q555X mutation on neurotransmitter release dynamics and short-term plasticity (STP) in excitatory and inhibitory synapses. We used patch-clamp electrophysiology coupled to electron microscopy and multi-electrode arrays to dissect synaptic transmission of primary SynI KO hippocampal neurons in which the human wild-type and mutant SynI were expressed by lentiviral transduction. A parallel decrease in the SV readily releasable pool in inhibitory synapses and in the release probability in excitatory synapses caused a marked reduction in the evoked synchronous release. This effect was accompanied by an increase in asynchronous release that was much more intense in excitatory synapses and associated with an increased total charge transfer. Q555X-hSynI induced larger facilitation and post-tetanic potentiation in excitatory synapses and stronger depression after long trains in inhibitory synapses. These changes were associated with higher network excitability and firing/bursting activity. Our data indicate that imbalances in STP and release dynamics of inhibitory and excitatory synapses trigger network hyperexcitability potentially leading to epilepsy/autism manifestations.
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Epilepsia/genética , Epilepsia/metabolismo , Plasticidad Neuronal/genética , Sinapsis/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Animales , Femenino , Expresión Génica , Hipocampo/metabolismo , Humanos , Espacio Intracelular/metabolismo , Ratones , Ratones Noqueados , Neuronas/metabolismo , Técnicas de Placa-Clamp , Fenotipo , Multimerización de Proteína , Transporte de Proteínas , Sinapsinas/química , Potenciales Sinápticos , Vesículas Sinápticas/metabolismoRESUMEN
Endocannabinoids (eCBs) regulate neuronal activity in the dorso-lateral striatum (DLS), a brain region that is involved in habitual behaviors. How synaptic eCB signaling contributes to habitual behaviors under physiological and pathological conditions remains unclear. Using a mouse model of cannabinoid tolerance, we found that persistent activation of the eCB pathway impaired eCB-mediated long-term depression (LTD) and synaptic depotentiation in the DLS. The loss of eCB LTD, occurring preferentially at cortical connections to striatopallidal neurons, was associated with a shift in behavioral control from goal-directed action to habitual responding. eCB LTD and behavioral alterations were rescued by in vivo modulation of small-conductance calcium activated potassium channel (SK channel) activity in the DLS, which potentiates eCB signaling. Our results reveal a direct relationship between drug tolerance and changes in control of instrumental performance by establishing a central role for eCB LTD in habit expression. In addition, SK channels emerge as molecular targets to fine tune the eCB pathway under pathological conditions.
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Cannabinoides/administración & dosificación , Cuerpo Estriado/efectos de los fármacos , Tolerancia a Medicamentos/fisiología , Hábitos , Depresión Sináptica a Largo Plazo/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Animales , Apamina/farmacología , Benzamidas/farmacología , Biofisica , Cannabinoides/agonistas , Cannabinoides/antagonistas & inhibidores , Carbamatos/farmacología , Condicionamiento Operante/efectos de los fármacos , Cuerpo Estriado/citología , Ciclohexanoles/farmacocinética , Relación Dosis-Respuesta a Droga , Dronabinol/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Ácido Ocadaico/farmacología , Técnicas de Placa-Clamp , Piperidinas/farmacología , Unión Proteica/efectos de los fármacos , Pirazoles/farmacología , Rimonabant , Bloqueadores de los Canales de Sodio/farmacología , Tritio/farmacocinéticaRESUMEN
The RE-1-specific silencing transcription factor (REST or NRSF) is a transcription repressor that orchestrates differentiation and also operates in differentiated neurons and neurosecretory cells (neural cells). Its role in proliferation has been investigated so far only in rapidly growing tumors, with conflicting results: suppression in non-neural tumors, stimulation in medulloblastomas. Working with two clones of chromaffin-neuronal PC12 cells, which express different levels of REST, and using genetic complementation and knockdown approaches, we show that REST also promotes proliferation in differentiated neural cells. Mechanistically, this occurs by a signaling pathway involving REST, the GTPase-activating protein tuberin (TSC2) and the transcription co-factor ß-catenin. In PC12 cells, raised expression of REST correlates with reduced TSC2 levels, nuclear accumulation and co-transcriptional activation of ß-catenin, and increased expression of its target oncogenes Myc and Ccnd1, which might account for the proliferation advantage and the distinct morphology. Rest transcription is also increased, unveiling the existence of a self-sustaining, feed-forward REST-TSC2-ß-catenin signaling loop that is also operative in another neural cell model, NT2/D1 cells. Transfection of REST, knockdown of TSC2 or forced expression of active ß-catenin recapitulated the biochemical, functional and morphological properties of the high-expressing REST clone in wild-type PC12 cells. Upregulation of REST promoted proliferation and phenotypic changes, thus hindering neurosecretion. The new REST-TSC2-ß-catenin signaling paradigm might have an important role in various aspects of neural cell physiology and pathology, including the regulation of proliferation and neurosecretion.
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Retroalimentación Fisiológica , Neuronas/metabolismo , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismo , beta Catenina/metabolismo , Animales , Diferenciación Celular/genética , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neuronas/patología , Neurosecreción/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/genética , Ratas , Proteínas Represoras/genética , Transducción de Señal/genética , Transgenes/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , beta Catenina/genéticaRESUMEN
Parkinson's disease is a common neurodegenerative disorder characterized by a profound motor disability that is traceable to the emergence of synchronous, rhythmic spiking in neurons of the external segment of the globus pallidus (GPe). The origins of this pathophysiology are poorly defined for the generation of pacemaking. After the induction of a parkinsonian state in mice, there was a progressive decline in autonomous GPe pacemaking, which normally serves to desynchronize activity. The loss was attributable to the downregulation of an ion channel that is essential in pacemaking, the hyperpolarization and cyclic nucleotide-gated (HCN) channel. Viral delivery of HCN2 subunits restored pacemaking and reduced burst spiking in GPe neurons. However, the motor disability induced by dopamine (DA) depletion was not reversed, suggesting that the loss of pacemaking was a consequence, rather than a cause, of key network pathophysiology, a conclusion that is consistent with the ability of L-type channel antagonists to attenuate silencing after DA depletion.
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Canalopatías/fisiopatología , Globo Pálido/fisiopatología , Canales Iónicos/fisiología , Neuronas/fisiología , Enfermedad de Parkinson/fisiopatología , Animales , Calcio/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Dopamina/metabolismo , Regulación hacia Abajo , Vectores Genéticos/administración & dosificación , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales Iónicos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microinyecciones , Neuronas/metabolismo , Oxidopamina , Canales de Potasio , Sustancia Negra/metabolismoRESUMEN
The activity patterns of subthalamic nucleus (STN) neurons are intimately linked to motor function and dysfunction and arise through the complex interaction of intrinsic properties and inhibitory and excitatory synaptic inputs. In many neurons, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play key roles in intrinsic excitability and synaptic integration both under normal conditions and in disease states. However, in STN neurons, which strongly express HCN channels, their roles remain relatively obscure. To address this deficit, complementary molecular and cellular electrophysiological, imaging, and computational approaches were applied to the rat STN. Molecular profiling demonstrated that individual STN neurons express mRNA encoding several HCN subunits, with HCN2 and 3 being the most abundant. Light and electron microscopic analysis showed that HCN2 subunits are strongly expressed and distributed throughout the somatodendritic plasma membrane. Voltage-, current-, and dynamic-clamp analysis, two-photon Ca(2+) imaging, and computational modeling revealed that HCN channels are activated by GABA(A) receptor-mediated inputs and thus limit synaptic hyperpolarization and deinactivation of low-voltage-activated Ca(2+) channels. Although HCN channels also limited the temporal summation of EPSPs, generated through two-photon uncaging of glutamate, this action was largely shunted by GABAergic inhibition that was necessary for HCN channel activation. Together the data demonstrate that HCN channels in STN neurons selectively counteract GABA(A) receptor-mediated inhibition arising from the globus pallidus and thus promote single-spike activity rather than rebound burst firing.