RESUMEN
Although it is known that protein kinase A (PKA) in the nucleus regulates gene expression, the specificities of nuclear PKA signaling remain poorly understood. Here, we combined computational modeling and live-cell imaging of PKA-dependent phosphorylation in mouse brain slices to investigate how transient dopamine signals are translated into nuclear PKA activity in cortical pyramidal neurons and striatal medium spiny neurons. We observed that the nuclear PKA signal in striatal neurons featured an ultrasensitive responsiveness, associated with fast all-or-none responses, which is not consistent with the commonly accepted theory of a slow and passive diffusion of catalytic PKA in the nucleus. Our numerical model suggests that a positive feed-forward mechanism inhibiting nuclear phosphatase activity - possibly mediated by DARPP-32 (also known as PPP1R1B) - could be responsible for this non-linear pattern of nuclear PKA response, allowing for a better detection of the transient dopamine signals that are often associated with reward-mediated learning.
Asunto(s)
Núcleo Celular/enzimología , Cuerpo Estriado/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neuronas/enzimología , Animales , Núcleo Celular/genética , Cuerpo Estriado/citología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Dopamina/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/genética , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Fosforilación , Transducción de SeñalRESUMEN
The calcium-regulated phosphodiesterase 1 (PDE1) family is highly expressed in the brain, but its functional role in neurones is poorly understood. Using the selective PDE1 inhibitor Lu AF64196 and biosensors for cyclic nucleotides including a novel biosensor for cGMP, we analyzed the effect of PDE1 on cAMP and cGMP in individual neurones in brain slices from male newborn mice. Release of caged NMDA triggered a transient increase of intracellular calcium, which was associated with a decrease in cAMP and cGMP in medium spiny neurones in the striatum. Lu AF64196 alone did not increase neuronal cyclic nucleotide levels, but blocked the NMDA-induced reduction in cyclic nucleotides indicating that this was mediated by calcium-activated PDE1. Similar effects were observed in the prefrontal cortex and the hippocampus. Upon corelease of dopamine and NMDA, PDE1 was shown to down-regulate the D1-receptor mediated increase in cAMP. PDE1 inhibition increased long-term potentiation in rat ventral striatum, showing that PDE1 is implicated in the regulation of synaptic plasticity. Overall, our results show that PDE1 reduces cyclic nucleotide signaling in the context of glutamate and dopamine coincidence. This effect could have a therapeutic value for treating brain disorders related to dysfunctions in dopamine neuromodulation.
Asunto(s)
Cuerpo Estriado/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Nucleótidos Cíclicos/metabolismo , Animales , Dopamina/metabolismo , Ácido Glutámico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Ratas , Ratas WistarRESUMEN
KEY POINTS: Brief dopamine events are critical actors of reward-mediated learning in the striatum; the intracellular cAMP-protein kinase A (PKA) response of striatal medium spiny neurons to such events was studied dynamically using a combination of biosensor imaging in mouse brain slices and in silico simulations. Both D1 and D2 medium spiny neurons can sense brief dopamine transients in the sub-micromolar range. While dopamine transients profoundly change cAMP levels in both types of medium spiny neurons, the PKA-dependent phosphorylation level remains unaffected in D2 neurons. At the level of PKA-dependent phosphorylation, D2 unresponsiveness depends on protein phosphatase-1 (PP1) inhibition by DARPP-32. Simulations suggest that D2 medium spiny neurons could detect transient dips in dopamine level. ABSTRACT: The phasic release of dopamine in the striatum determines various aspects of reward and action selection, but the dynamics of the dopamine effect on intracellular signalling remains poorly understood. We used genetically encoded FRET biosensors in striatal brain slices to quantify the effect of transient dopamine on cAMP or PKA-dependent phosphorylation levels, and computational modelling to further explore the dynamics of this signalling pathway. Medium-sized spiny neurons (MSNs), which express either D1 or D2 dopamine receptors, responded to dopamine by an increase or a decrease in cAMP, respectively. Transient dopamine showed similar sub-micromolar efficacies on cAMP in both D1 and D2 MSNs, thus challenging the commonly accepted notion that dopamine efficacy is much higher on D2 than on D1 receptors. However, in D2 MSNs, the large decrease in cAMP level triggered by transient dopamine did not translate to a decrease in PKA-dependent phosphorylation level, owing to the efficient inhibition of protein phosphatase 1 by DARPP-32. Simulations further suggested that D2 MSNs can also operate in a 'tone-sensing' mode, allowing them to detect transient dips in basal dopamine. Overall, our results show that D2 MSNs may sense much more complex patterns of dopamine than previously thought.
Asunto(s)
Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Animales , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/fisiología , Ratones , Ratones Endogámicos C57BL , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 1/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismoRESUMEN
Reading requires the integration of several central cognitive subsystems, ranging from attention and oculomotor control to word identification and language comprehension. Reading saccades and fixations contain information that can be correlated with word properties. When reading a sentence, the brain must decide where to direct the next saccade according to what has been read up to the actual fixation. In this process, the retrieval memory brings information about the current word features and attributes into working memory. According to this information, the prefrontal cortex predicts and triggers the next saccade. The frequency and cloze predictability of the fixated word, the preceding words and the upcoming ones affect when and where the eyes will move next. In this paper we present a diagnostic technique for early stage cognitive impairment detection by analyzing eye movements during reading proverbs. We performed a case-control study involving 20 patients with probable Alzheimer's disease and 40 age-matched, healthy control patients. The measurements were analyzed using linear mixed-effects models, revealing that eye movement behavior while reading can provide valuable information about whether a person is cognitively impaired. To the best of our knowledge, this is the first study using word-based properties, proverbs and linear mixed-effect models for identifying cognitive abnormalities.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/fisiopatología , Atención/fisiología , Movimientos Oculares/fisiología , Lectura , Semántica , Anciano , Análisis de Varianza , Femenino , Fijación Ocular , Humanos , Modelos Lineales , Masculino , Persona de Mediana EdadRESUMEN
The cAMP/protein kinase A (PKA) signalling cascade is ubiquitous, and each step in this cascade involves enzymes that are expressed in multiple isoforms. We investigated the effects of this diversity on the integration of the pathway in the target cell by comparing prefrontal cortical neurones with striatal neurones which express a very specific set of signalling proteins. The prefrontal cortex and striatum both receive dopaminergic inputs and we analysed the dynamics of the cAMP/PKA signal triggered by dopamine D1 receptors in these two brain structures. Biosensor imaging in mouse brain slice preparations showed profound differences in the D1 response between pyramidal cortical neurones and striatal medium spiny neurones: the cAMP/PKA response was much stronger, faster and longer lasting in striatal neurones than in pyramidal cortical neurones. We identified three molecular determinants underlying these differences: different activities of phosphodiesterases, particularly those of type 4, which strongly damp the cAMP signal in the cortex but not in the striatum; stronger adenylyl cyclase activity in the striatum, generating responses with a faster onset than in the cortex; and DARPP-32, a phosphatase inhibitor which prolongs PKA action in the striatum. Striatal neurones were also highly responsive in terms of gene expression since a single sub-second dopamine stimulation is sufficient to trigger c-Fos expression in the striatum, but not in the cortex. Our data show how specific molecular elements of the cAMP/PKA signalling cascade selectively enable the principal striatal neurones to respond to brief dopamine stimuli, a critical process in incentive learning.
Asunto(s)
Cuerpo Estriado/fisiología , Dopamina/fisiología , Neuronas/fisiología , 1-Metil-3-Isobutilxantina/farmacología , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Animales , Técnicas Biosensibles , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Agonistas de Dopamina/farmacología , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibidores de Fosfodiesterasa/farmacología , Corteza Prefrontal/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismoRESUMEN
RATIONALE: We have shown recently that particulate (pGC) and soluble guanylyl (sGC) cyclases synthesize cGMP in different compartments in adult rat ventricular myocytes (ARVMs). OBJECTIVE: We hypothesized that cGMP-dependent protein kinase (PKG) exerts a feedback control on cGMP concentration contributing to its intracellular compartmentation. METHODS AND RESULTS: Global cGMP levels, cGMP-phosphodiesterase (PDE) and pGC enzymatic activities were determined in purified ARVMs. Subsarcolemmal cGMP signals were monitored in single cells by recording the cGMP-gated current (I(CNG)) in myocytes expressing the wild-type rat olfactory cyclic nucleotide-gated (CNG) channel. Whereas the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (100 µmol/L) produced little effect on I(CNG), the response increased 2-fold in the presence of the PKG inhibitors KT5823 (50 nmol/L) or DT-2 (2 µmol/L). The effect of KT5823 was abolished in the presence of the nonselective cyclic nucleotide PDE inhibitor 3-isobutyl-1-methylxantine (IBMX) (100 µmol/L) or the selective cGMP-PDE5 inhibitor sildenafil (100 nmol/L). PKG inhibition also potentiated the effect of SNAP on global cGMP levels and fully blocked the increase in cGMP-PDE5 activity. In contrast, PKG inhibition decreased by ≈50% the I(CNG) response to ANP (10 and 100 nmol/L), even in the presence of IBMX. Conversely, PKG activation increased the I(CNG) response to ANP and amplified the stimulatory effect of ANP on pGC activity. CONCLUSIONS: PKG activation in adult cardiomyocytes limits the accumulation of cGMP induced by NO donors via PDE5 stimulation but increases that induced by natriuretic peptides. These findings support the paradigm that cGMP is not uniformly distributed in the cytosol and identifies PKG as a key component in this process.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Miocitos Cardíacos/enzimología , Sistemas de Mensajero Secundario , Animales , Factor Natriurético Atrial/metabolismo , Células Cultivadas , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Citosol/enzimología , Activación Enzimática , Retroalimentación Fisiológica , Guanilato Ciclasa/metabolismo , Masculino , Potenciales de la Membrana , Miocitos Cardíacos/efectos de los fármacos , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Técnicas de Placa-Clamp , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Radioinmunoensayo , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/metabolismo , Sarcolema/enzimología , Sistemas de Mensajero Secundario/efectos de los fármacos , Guanilil Ciclasa Soluble , Factores de Tiempo , TransfecciónRESUMEN
We investigated the role of phosphodiesterases (PDEs) in the integration of cAMP signals and protein kinase A (PKA) activity following beta-adrenergic stimulation, by carrying out real-time imaging of male mouse pyramidal cortical neurons expressing biosensors to monitor cAMP levels (Epac1-camps and Epac2-camps300) or PKA activity (AKAR2). In the soma, isoproterenol (ISO) increased the PKA signal to approximately half the maximal response obtained with forskolin, with a characteristic beta(1) pharmacology and an EC(50) of 4.5 nm. This response was related to free cAMP levels in the submicromolar range. The specific type 4 PDE (PDE4) inhibitor rolipram had a very small effect alone, but strongly potentiated the PKA response to ISO. Blockers of other PDEs had no effect. PDE4 thus acts as a brake in the propagation of the beta(1)-adrenergic signal from the membrane to the bulk somatic cytosol. The results for a submembrane domain were markedly different, whether recorded with a PKA-sensitive potassium current related to the slow AHP or by two-photon imaging of small distal dendrites. The responses to ISO were stronger than in the bulk cytosol. This is consistent with the cAMP/PKA signal being strong at the membrane, as shown by electrophysiology, and favored in cellular domains with a high surface area to volume ratio, in which this signal was detected by imaging. Rolipram alone also produced a strong cAMP/PKA signal, revealing tonic cAMP production. PDE4 thus appears as a crucial integrator with different physiological implications in different subcellular domains.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Lóbulo Parietal/enzimología , Células Piramidales/enzimología , Adenilil Ciclasas/metabolismo , Agonistas de Receptores Adrenérgicos beta 1 , Agonistas Adrenérgicos beta/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/metabolismo , Fármacos del Sistema Nervioso Central/farmacología , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dendritas/efectos de los fármacos , Dendritas/enzimología , Dendritas/metabolismo , Técnicas In Vitro , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Lóbulo Parietal/efectos de los fármacos , Lóbulo Parietal/metabolismo , Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/farmacología , Potasio/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Rolipram/farmacologíaRESUMEN
Intracellular signaling with cyclic nucleotides are ubiquitous signaling pathways, yet the dynamics of these signals profoundly differ in different cell types. Biosensor imaging experiments, by providing direct measurements in intact cellular environment, reveal which receptors are activated by neuromodulators and how the coincidence of different neuromodulators is integrated at various levels in the signaling cascade. Phosphodiesterases appear as one important determinant of cross-talk between different signaling pathways. Finally, analysis of signal dynamics reveal that striatal medium-sized spiny neuron obey a different logic than other brain regions such as cortex, probably in relation with the function of this brain region which efficiently detects transient dopamine.
Asunto(s)
AMP Cíclico , Nucleótidos Cíclicos , Cuerpo Estriado , Neuronas , Transducción de SeñalRESUMEN
BACKGROUND AND PURPOSE: Dopamine in the striatum plays a crucial role in reward processes and action selection. Dopamine signals are transduced by D1 and D2 dopamine receptors which trigger mirror effects through the cAMP/PKA signalling cascade in D1 and D2 medium-sized spiny neurons (MSNs). Phosphodiesterases (PDEs), which determine the profile of cAMP signals, are highly expressed in MSNs, but their respective roles in dopamine signal integration remain poorly understood. EXPERIMENTAL APPROACH: We used genetically encoded FRET biosensors to monitor at the single cell level the functional contribution of PDE2A, PDE4 and PDE10A in the changes of the cAMP/PKA response to transient and continuous dopamine in mouse striatal brain slices. KEY RESULTS: We found that PDE2A, PDE4 and PDE10A operate on the moderate to high cAMP levels elicited by D1 or A2A receptor stimulation. In contrast, only PDE10A is able to reduce cAMP down to baseline in both type of neurones, leading to the dephosphorylation of PKA substrates. CONCLUSION AND IMPLICATIONS: In both MSN types, PDE10A inhibition blunts the responsiveness to dopamine, whereas PDE2A or PDE4 inhibition reinforces dopamine action.
Asunto(s)
Cuerpo Estriado , Dopamina , Hidrolasas Diéster Fosfóricas , Animales , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Receptores de Dopamina D1/metabolismoRESUMEN
The opposing action of dopamine and acetylcholine has long been known to play an important role in basal ganglia physiology. However, the quantitative analysis of dopamine and acetylcholine signal interaction has been difficult to perform in the native context because the striatum comprises mainly two subtypes of medium-sized spiny neurons (MSNs) on which these neuromodulators exert different actions. We used biosensor imaging in live brain slices of dorsomedial striatum to monitor changes in intracellular cAMP at the level of individual MSNs. We observed that the muscarinic agonist oxotremorine decreases cAMP selectively in the MSN subpopulation that also expresses D1 dopamine receptors, an action mediated by the M4 muscarinic receptor. This receptor has a high efficacy on cAMP signaling and can shut down the positive cAMP response induced by dopamine, at acetylcholine concentrations which are consistent with physiological levels. This supports our prediction based on theoretical modeling that acetylcholine could exert a tonic inhibition on striatal cAMP signaling, thus supporting the possibility that a pause in acetylcholine release is required for phasic dopamine to transduce a cAMP signal in D1 MSNs. In vivo experiments with acetylcholinesterase inhibitors donepezil and tacrine, as well as with the positive allosteric modulators of M4 receptor VU0152100 and VU0010010 show that this effect is sufficient to reverse the increased locomotor activity of DAT-knockout mice. This suggests that M4 receptors could be a novel therapeutic target to treat hyperactivity disorders.
Asunto(s)
Acetilcolina/farmacología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , AMP Cíclico/metabolismo , Dopamina/farmacología , Receptor Muscarínico M4/agonistas , Receptores de Dopamina D1/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Agonistas Muscarínicos , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Oxotremorina/farmacologíaRESUMEN
A current challenge in cellular signaling is to decipher the complex intracellular spatiotemporal organization that any given cell type has developed to discriminate among different external stimuli acting via a common signaling pathway. This obviously applies to cAMP and cGMP signaling in the heart, where these cyclic nucleotides determine the regulation of cardiac function by many hormones and neuromediators. Recent studies have identified cyclic nucleotide phosphodiesterases as key actors in limiting the spread of cAMP and cGMP, and in shaping and organizing intracellular signaling microdomains. With this new role, phosphodiesterases have been promoted from the rank of a housekeeping attendant to that of an executive officer.
Asunto(s)
Miocardio/metabolismo , Nucleótidos Cíclicos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Transducción de Señal , Animales , Humanos , Distribución TisularRESUMEN
BACKGROUND: Cyclic guanosine monophosphate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides, such as atrial or brain natriuretic peptide, which activate the soluble and particulate forms of guanylyl cyclase, respectively. However, natriuretic peptides and NO donors exert different effects on cardiac and vascular smooth muscle function. We therefore tested whether these differences are due to an intracellular compartmentation of cGMP and evaluated the role of phosphodiesterase (PDE) subtypes in this process. METHODS AND RESULTS: Subsarcolemmal cGMP signals were monitored in adult rat cardiomyocytes by expression of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit and recording of the associated cGMP-gated current (ICNG). Atrial natriuretic peptide (10 nmol/L) or brain natriuretic peptide (10 nmol/L) induced a clear activation of ICNG, whereas NO donors (S-nitroso-N-acetyl-penicillamine, diethylamine NONOate, 3-morpholinosydnonimine, and spermine NO, all at 100 micromol/L) had little effect. The ICNG current was strongly potentiated by nonselective PDE inhibition with isobutyl methylxanthine (100 micromol/L) and by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (10 micromol/L) and Bay 60-7550 (50 nmol/L). Surprisingly, sildenafil, a PDE5 inhibitor, produced a dose-dependent increase of I(CNG) activated by NO donors but had no effect (at 100 nmol/L) on the current elicited by atrial natriuretic peptide. CONCLUSIONS: These results indicate that in rat cardiomyocytes (1) the particulate cGMP pool is readily accessible at the plasma membrane, whereas the soluble pool is not; and (2) PDE5 controls the soluble but not the particulate pool, whereas the latter is under the exclusive control of PDE2. Differential spatiotemporal distributions of cGMP may therefore contribute to the specific effects of natriuretic peptides and NO donors on cardiac function.
Asunto(s)
Compartimento Celular , GMP Cíclico/metabolismo , Canales Iónicos/fisiología , Miocitos Cardíacos/química , Sistemas de Mensajero Secundario , 1-Metil-3-Isobutilxantina/farmacología , 3',5'-GMP Cíclico Fosfodiesterasas/fisiología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Factor Natriurético Atrial/farmacología , Transporte Biológico , Membrana Celular/metabolismo , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Ventrículos Cardíacos/citología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/análisis , Canales Iónicos/antagonistas & inhibidores , Isoenzimas/antagonistas & inhibidores , Isoenzimas/fisiología , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/ultraestructura , Péptido Natriurético Encefálico/farmacología , Donantes de Óxido Nítrico/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/fisiología , Piperazinas/farmacología , Purinas , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/fisiología , Sarcolema/metabolismo , Citrato de Sildenafil , Solubilidad , SulfonasRESUMEN
Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.
RESUMEN
Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP-dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, pathfinding, efficacy of synaptic transmission, regulation of excitability, or long term changes. Genetically encoded optical biosensors for cAMP or PKA are considerably improving our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progress made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the sub-cellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus, and axon. Combining this imaging approach with pharmacology or genetic models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly emerge as a forefront tool to decipher the subtle mechanics of intracellular signaling. This will certainly help us to understand the mechanism of action of current drugs and foster the development of novel molecules for neuropsychiatric diseases.
Asunto(s)
Técnicas Biosensibles , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Transducción de Señal , Animales , Encéfalo/citología , Encéfalo/metabolismo , Química Encefálica , RatonesRESUMEN
The NO-cGMP signaling plays an important role in the regulation of striatal function although the mechanisms of action of cGMP specifically in medium spiny neurons (MSNs) remain unclear. Using genetically encoded fluorescent biosensors, including a novel Epac-based sensor (EPAC-S(H150)) with increased sensitivity for cAMP, we analyze the cGMP response to NO and whether it affected cAMP/PKA signaling in MSNs. The Cygnet2 sensor for cGMP reported large responses to NO donors in both striatonigral and striatopallidal MSNs, this cGMP signal was controlled partially by PDE2. At the level of cAMP brief forskolin stimulations produced transient cAMP signals which differed between D1 and D2 MSNs. NO inhibited these cAMP transients through cGMP-dependent PDE2 activation, an effect that was translated and magnified downstream of cAMP, at the level of PKA. PDE2 thus appears as a critical effector of NO which modulates the post-synaptic response of MSNs to dopaminergic transmission.
RESUMEN
ß-Adrenergic receptors (ß-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca²âº-induced Ca²âº release via phosphorylation of L-type Ca²âº channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of ß-AR-dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that ß-AR stimulation of the L-type Ca²âº current (ICa,L) is increased only in Pde4b-/- mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under ß-AR stimulation, Ca²âº transients, cell contraction, and spontaneous Ca²âº release events were increased in Pde4b-/- and Pde4d-/- myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b-/- mice but not in WT mice. These results identify PDE4B in the CaV1.2 complex as a critical regulator of ICa,L during ß-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca²âº-induced Ca²âº release in cardiomyocytes.