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Amyotrophic lateral sclerosis (ALS) is a lethal and incurable neurodegenerative disease due to the loss of upper and lower motor neurons, which leads to muscle weakness, atrophy, and paralysis. Sigma-1 receptor (σ-1R) is a ligand-operated protein that exhibits pro-survival and anti-apoptotic properties. In addition, mutations in its codifying gene are linked to development of juvenile ALS pointing to an important role in ALS. Here, we investigated the disease-modifying effects of pridopidine, a σ-1R agonist, using a delayed onset SOD1 G93A mouse model of ALS. Mice were administered a continuous release of pridopidine (3.0 mg/kg/day) for 4 weeks starting before the appearance of any sign of muscle weakness. Mice were monitored weekly and several behavioural tests were used to evaluate muscle strength, motor coordination and gait patterns. Pridopidine-treated SOD1 G93A mice showed genotype-specific effects with the prevention of cachexia. In addition, these effects exhibited significant improvement of motor behaviour 5 weeks after treatment ended. However, the survival of the animals was not extended. In summary, these results show that pridopidine can modify the disease phenotype of ALS-associated cachexia and motor deficits in a SOD1 G93A mouse model.
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Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Esclerose Lateral Amiotrófica/genética , Animais , Caquexia , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Debilidade Muscular , Fenótipo , Piperidinas , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
It has been suggested that the favorable side-effect profiles of atypical antipsychotics (e.g. clozapine and amisulpride) are related to their â¼100-fold faster dissociation from dopamine D2 receptors (D2R) compared with typical antipsychotics (e.g. haloperidol and chlorpromazine). Fast dissociation would entail rapidly reversible antagonism; however, this has not been thoroughly studied using functional assays. We compared the reversibilities of D2R antagonism by 17 compounds using an electrophysiological method to measure dopamine-evoked potassium channel activation via D2R. Varying rates and amplitudes of D2R response recovery were observed following antagonist removal. Whereas recovery rates differed 15-fold between atypical drugs, recovery from clozapine and amisulpride antagonism was, unexpectedly, less than twofold faster than from chlorpromazine. The recovery amplitude correlated with calculated water solubility and lipid/water distribution coefficients, suggesting variable drug partitioning into cell membranes. Our data do not support the notion that the rate of reversibility of D2R antagonism is what distinguishes atypical from typical antipsychotics.
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Antipsicóticos/classificação , Antipsicóticos/farmacologia , Antagonistas dos Receptores de Dopamina D2 , Animais , Relação Dose-Resposta a Droga , Interações Hidrofóbicas e Hidrofílicas , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Oócitos , Canais de Potássio/metabolismo , Solubilidade , XenopusRESUMO
The human aging brain is characterized by changes in network efficiency that are currently best captured through longitudinal resting-state functional MRI (rs-fMRI). These studies however are challenging due to the long human lifespan. Here we show that the mouse animal model with a much shorter lifespan allows us to follow the functional network organization over most of the animal's adult lifetime. We used a longitudinal study of the functional connectivity of different brain regions with rs-fMRI under anesthesia. Our analysis uncovers network modules similar to those reported in younger mice and in humans (i.e., prefrontal/default mode network (DMN), somatomotor and somatosensory networks). Statistical analysis reveals different patterns of network reorganization during aging. Female mice showed a pattern akin to human aging, with de-differentiation of the connectome, mainly due to increases in connectivity of the prefrontal/DMN cortical networks to other modules. Our male cohorts revealed heterogenous aging patterns with only one group confirming the de- differentiation, while the majority showed an increase in connectivity of the somatomotor cortex to the Nucleus accumbens. In summary, in line with human work, our analysis in mice supports the concept of de-differentiation in the aging mammalian brain and reveals additional trajectories in aging mice networks.
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Envelhecimento , Imageamento por Ressonância Magnética , Adulto , Masculino , Humanos , Feminino , Animais , Camundongos , Estudos Longitudinais , Encéfalo/diagnóstico por imagem , Córtex Cerebral , MamíferosRESUMO
Alzheimer's disease (AD) presents complex challenges due to its multifactorial nature, poorly understood etiology, and late detection. The mechanisms through which genetic, fixed and modifiable risk factors influence susceptibility to AD are under intense investigation, yet the impact of unique risk factors on brain networks is difficult to disentangle, and their interactions remain unclear. To model multiple risk factors including APOE genotype, age, sex, diet, and immunity we leveraged mice expressing the human APOE and NOS2 genes, conferring a reduced immune response compared to mouse Nos2. Employing graph analyses of brain connectomes derived from accelerated diffusion-weighted MRI, we assessed the global and local impact of risk factors in the absence of AD pathology. Aging and a high-fat diet impacted extensive networks comprising AD-vulnerable regions, including the temporal association cortex, amygdala, and the periaqueductal gray, involved in stress responses. Sex impacted networks including sexually dimorphic regions (thalamus, insula, hypothalamus) and key memory-processing areas (fimbria, septum). APOE genotypes modulated connectivity in memory, sensory, and motor regions, while diet and immunity both impacted the insula and hypothalamus. Notably, these risk factors converged on a circuit comprising 63 of 54,946 total connections (0.11% of the connectome), highlighting shared vulnerability amongst multiple AD risk factors in regions essential for sensory integration, emotional regulation, decision making, motor coordination, memory, homeostasis, and interoception. These network-based biomarkers hold translational value for distinguishing high-risk versus low-risk participants at preclinical AD stages, suggest circuits as potential therapeutic targets, and advance our understanding of network fingerprints associated with AD risk. Significance Statement: Current interventions for Alzheimer's disease (AD) do not provide a cure, and are delivered years after neuropathological onset. Addressing the impact of risk factors on brain networks holds promises for early detection, prevention, and revealing putative therapeutic targets at preclinical stages. We utilized six mouse models to investigate the impact of factors, including APOE genotype, age, sex, immunity, and diet, on brain networks. Large structural connectomes were derived from high resolution compressed sensing diffusion MRI. A highly parallelized graph classification identified subnetworks associated with unique risk factors, revealing their network fingerprints, and a common network composed of 63 connections with shared vulnerability to all risk factors. APOE genotype specific immune signatures support the design of interventions tailored to risk profiles.
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Introduction: Optical Projection Tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high resolution optical imaging techniques, ideally suited for ex vivo 3D whole mouse brain imaging. Although they exhibit high specificity for their targets, the anatomical detail provided by tissue autofluorescence remains limited. Methods: T1-weighted images were acquired from 19 BABB or DBE cleared brains to create an MR template using serial longitudinal registration. Afterwards, fluorescent OPT and LSFM images were coregistered/normalized to the MR template to create fusion images. Results: Volumetric calculations revealed a significant difference between BABB and DBE cleared brains, leading to develop two optimized templates, with associated tissue priors and brain atlas, for BABB (OCUM) and DBE (iOCUM). By creating fusion images, we identified virus infected brain regions, mapped dopamine transporter and translocator protein expression, and traced innervation from the eye along the optic tract to the thalamus and superior colliculus using cholera toxin B. Fusion images allowed for precise anatomical identification of fluorescent signal in the detailed anatomical context provided by MR. Discussion: The possibility to anatomically map fluorescent signals on magnetic resonance (MR) images, widely used in clinical and preclinical neuroscience, would greatly benefit applications of optical imaging of mouse brain. These specific MR templates for cleared brains enable a broad range of neuroscientific applications integrating 3D optical brain imaging.
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Agonist potency at some neurotransmitter receptors has been shown to be regulated by voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by inhibitory autoreceptors. Likewise, receptor deactivation rates upon agonist removal have been implicated in autoreceptor function. Using G protein-coupled potassium (GIRK) channel activation in Xenopus oocytes as readout of receptor activity, we have investigated the voltage sensitivities and signaling kinetics of the hH(3)(445) and hH(3)(365) isoforms of the human histamine H3 receptor, which functions as an inhibitory auto- and heteroreceptor in the nervous system. We have also investigated both the human and the mouse homologues of the related histamine H4 receptor, which is expressed mainly on hematopoietic cells. We found that the hH(3)(445) receptor is the most sensitive to voltage, whereas the hH(3)(365) and H(4) receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH(3)(445) and hH(3)(365) isoforms, with the hH(3)(365) isoform being five to six-fold slower than the hH(3)(445) receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH4 receptor. The differences in voltage sensitivities and deactivation kinetics between the hH(3)(445), hH(3)(365), and H4 receptors might be relevant to their respective physiological roles.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos H3/metabolismo , Receptores Histamínicos/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Eletrofisiológicos , Humanos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores Histamínicos H3/química , Receptores Histamínicos H4 , Alinhamento de Sequência , Xenopus laevisRESUMO
Striatal adenosine (A)2 -dopamine (D)2 receptor (R) heteromers exist with antagonistic interactions. We have studied these Rs and their interactions during cocaine self-administration and extinction using a 'yoked' protocol to understand the role of motivational mechanisms behind the adaptive observed. In the ventral striatum, a significant increase in the A2A R density was observed in rats that received 'yoked' cocaine during maintenance phase and following its extinction while this significant increase was only observed after extinction from cocaine self-administration. In the dorsal striatum, a significant increase in the affinity of A2A Rs was determined in the two groups of rats that received cocaine during maintenance. D2 -like Rs were significantly increased in the dorsal striatum of animals that received 'yoked' cocaine during maintenance. In the rat dorsal, but not the ventral, striatum significant reductions in the EC50 values for dopamine and increases in the guanosine5'-([γ]-thio)triphosphate (GTPγS) accumulation were observed following active and passive cocaine injections during maintenance. After 10-day extinction, a significant reduction of the Bmax value of GTPγS accumulation was demonstrated in the dorsal striatum of rats previously self-administered cocaine, while a significant reduction of the EC50 value for dopamine in the ventral striatum was found in the 'yoked' cocaine group. By comparing the cocaine self-administration group with the 'yoked' cocaine group, evidence for the existence of motivational mechanisms that guide adaptive changes in the A2A R and D2 R and in the D2 -Gi coupling differentially developed in the ventral and dorsal striatum during cocaine maintenance and its extinction has been demonstrated.
Assuntos
Cocaína/farmacologia , Corpo Estriado/efeitos dos fármacos , Inibidores da Captação de Dopamina/farmacologia , Extinção Psicológica/efeitos dos fármacos , Receptor A2A de Adenosina/efeitos dos fármacos , Receptores de Dopamina D2/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Animais , Cocaína/administração & dosagem , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Infusões Intravenosas , Masculino , Fenetilaminas/farmacologia , Ratos , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D2/metabolismo , Esquema de Reforço , AutoadministraçãoRESUMO
It is well known that cocaine blocks the dopamine transporter. This mechanism should lead to a general increase in dopaminergic neurotransmission, and yet dopamine D(1) receptors (D(1)Rs) play a more significant role in the behavioral effects of cocaine than the other dopamine receptor subtypes. Cocaine also binds to σ-1 receptors, the physiological role of which is largely unknown. In the present study, D(1)R and σ(1)R were found to heteromerize in transfected cells, where cocaine robustly potentiated D(1)R-mediated adenylyl cyclase activation, induced MAPK activation per se and counteracted MAPK activation induced by D(1)R stimulation in a dopamine transporter-independent and σ(1)R-dependent manner. Some of these effects were also demonstrated in murine striatal slices and were absent in σ(1)R KO mice, providing evidence for the existence of σ(1)R-D(1)R heteromers in the brain. Therefore, these results provide a molecular explanation for which D(1)R plays a more significant role in the behavioral effects of cocaine, through σ(1)R-D(1)R heteromerization, and provide a unique perspective toward understanding the molecular basis of cocaine addiction.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/metabolismo , Cocaína/toxicidade , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D1/metabolismo , Receptores sigma/efeitos dos fármacos , Receptores sigma/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células CHO , Linhagem Celular , Transtornos Relacionados ao Uso de Cocaína/etiologia , Cricetinae , Cricetulus , Dimerização , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Estrutura Quaternária de Proteína/efeitos dos fármacos , Receptores de Dopamina D1/química , Receptores de Dopamina D1/genética , Receptores sigma/química , Receptores sigma/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Receptor Sigma-1RESUMO
Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combine multimodal imaging (cm-nm scale) with single nuclei RNA-sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar-/-) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection is imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection is limited to grey matter of sensory systems in wildtype mice, but extends into white matter, meninges and choroid plexus in Ifnar-/- mice. Cells in wildtype display strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+ NK cells, whereas in Ifnar-/-, the absence of this response contributes to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos , Interferon Tipo I , Carrapatos , Camundongos , Animais , Interferon Tipo I/metabolismo , Neurônios/metabolismo , Camundongos Knockout , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Tropismo , Carrapatos/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Evidence indicates that dopamine (DA) mainly acts as a volume transmission (VT) transmitter through its release into the extracellular fluid where the D(1) -like and D(2) -like receptors are predominantly extrasynaptic. It was therefore of interest to compare the affinities of the two major families of DA receptors. [(3)H] raclopride /DA and [(3)H] SCH23390/DA competition assays compared the affinity of DA at D(2) -like and D(1) -like receptors in rat dorsal striatal membrane preparations as well as in membrane preparations from CHO cell lines stably transfected with human D(2L) and D(1) receptors. The IC(50) values of DA at D(2) -like receptors in dorsal striatal membranes and CHO cell membranes were markedly and significantly reduced compared with the IC(50) values of DA at D(1) -like receptors. These IC(50) values reflect differences in both the high and low affinity states. The K(iH) value for DA at [(3)H] raclopride-labeled D(2) -like receptors in dorsal striatum was 12 nM, and this can help explain PET findings that amphetamine-induced increases in DA release can produce an up to 50% decrease of [(11)C] raclopride binding in the dorsal striatum in vivo. These combined results give indications for the existence of striatal D(2) -like receptor-mediated DA VT at the local circuit level in vivo. The demonstration of a K(iH) value of 183 nM for DA at D(1) antagonist-labeled D(1) -like receptors instead gives a likely explanation for the failure of a reduction of D(1) -like receptor binding after amphetamine-induced DA release in PET studies using the D(1) -like antagonist radioligands [(11)C] SCH23390 and [(11)C] NNC. It seems difficult to evaluate the role of the extrasynaptic D(1) receptors in VT in vivo with the PET radioligands available for this receptor.
Assuntos
Antagonistas de Dopamina/metabolismo , Dopamina/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Benzazepinas/metabolismo , Células CHO , Corpo Estriado/diagnóstico por imagem , Cricetinae , Cricetulus , Antagonistas dos Receptores de Dopamina D2 , Neurônios Dopaminérgicos/metabolismo , Masculino , Tomografia por Emissão de Pósitrons , Racloprida/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D2/agonistasRESUMO
Motor learning induces plasticity in multiple brain regions involving the cerebellum as a crucial player. Synaptic plasticity in the excitatory collaterals to the cerebellar output, the deep cerebellar nuclei (DCN), have recently been shown to be an important part of motor learning. These synapses are composed of climbing fiber (CF) and mossy fiber synapses, with the former conveying unconditioned and the latter conditioned responses in classical conditioning paradigms. The CF synapse on to the cerebellar cortex and the DCN express vesicular transporter 2 (vGluT2), whereas mossy fibers express vGluT1 and /or vGluT2 in their terminals. However, the underlying regulatory mechanism of vGluT expression in the DCN remains unknown. Here we confirm the increase of vGluT2 in a specific part of the DCN during the acquisition of a skilled reaching task in mice. Furthermore, our findings show that this is due to an increase in co-expression of vGluT2 in vGluT1 presynapses instead of the formation of new vGluT2 synapses. Our data indicate that remodeling of synapses - in contrast to synaptogenesis - also plays an important role in motor learning and may explain the presence of both vGluT's in some mossy fiber synapses.
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Núcleos Cerebelares , Cerebelo , Aprendizagem , Proteína Vesicular 2 de Transporte de Glutamato , Animais , Córtex Cerebelar/metabolismo , Núcleos Cerebelares/metabolismo , Cerebelo/metabolismo , Camundongos , Sinapses/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
Sigma-1 receptor agonists have recently gained a great deal of interest due to their anti-amnesic, neuroprotective, and neurorestorative properties. Compounds such as PRE-084 or pridopidine (ACR16) are being studied as a potential treatment against cognitive decline associated with neurodegenerative disease, also to include Alzheimer's disease. Here, we performed in vitro experiments using primary neuronal cell cultures from rats to evaluate the abilities of ACR16 and PRE-084 to induce new synapses and spines formation, analyzing the expression of the possible genes and proteins involved. We additionally examined their neuroprotective properties against neuronal death mediated by oxidative stress and excitotoxicity. Both ACR16 and PRE-084 exhibited a concentration-dependent neuroprotective effect against NMDA- and H2O2-related toxicity, in addition to promoting the formation of new synapses and dendritic spines. However, only ACR16 generated dendritic spines involved in new synapse establishment, maintaining a more expanded activation of MAPK/ERK and PI3K/Akt signaling cascades. Consequently, ACR16 was also evaluated in vivo, and a dose of 1.5 mg/kg/day was administered intraperitoneally in APP/PS1 mice before performing the Morris water maze. ACR16 diminished the spatial learning and memory deficits observed in APP/PS1 transgenic mice via PI3K/Akt pathway activation. These data point to ACR16 as a pharmacological tool to prevent synapse loss and memory deficits associated with Alzheimer's disease, due to its neuroprotective properties against oxidative stress and excitotoxicity, as well as the promotion of new synapses and spines through a mechanism that involves AKT and ERK signaling pathways.
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Doença de Alzheimer , Doenças Neurodegenerativas , Fármacos Neuroprotetores , Camundongos , Animais , Ratos , Doença de Alzheimer/complicações , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Fosfatidilinositol 3-Quinases/farmacologia , Fosfatidilinositol 3-Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/uso terapêutico , N-Metilaspartato/farmacologia , N-Metilaspartato/uso terapêutico , Transtornos da Memória/metabolismo , Camundongos Transgênicos , Modelos Animais de Doenças , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Aprendizagem em LabirintoRESUMO
To study the aging human brain requires significant resources and time. Thus, mice models of aging can provide insight into changes in brain biological functions at a fraction of the time when compared to humans. This study aims to explore changes in dopamine D1 and D2 receptor availability and of gray matter density in striatum during aging in mice and to evaluate whether longitudinal imaging in mice may serve as a model for normal brain aging to complement cross-sectional research in humans. Mice underwent repeated structural magnetic resonance imaging (sMRI), and [11C]Raclopride and [11C]SCH23390 positron emission tomography (PET) was performed on a subset of aging mice. PET and sMRI data were analyzed by binding potential (BP ND ), voxel- and tensor-based morphometry (VBM and TBM, respectively). Longitudinal PET revealed a significant reduction in striatal BP ND for D2 receptors over time, whereas no significant change was found for D1 receptors. sMRI indicated a significant increase in modulated gray matter density (mGMD) over time in striatum, with limited clusters showing decreased mGMD. Mouse [11C]Raclopride data is compatible with previous reports in human cross-sectional studies, suggesting that a natural loss of dopaminergic D2 receptors in striatum can be assessed in mice, reflecting estimates from humans. No changes in D1 were found, which may be attributed to altered [11C]SCH23390 kinetics in anesthetized mice, suggesting that this tracer is not yet able to replicate human findings. sMRI revealed a significant increase in mGMD. Although contrary to expectations, this increase in modulated GM density may be attributed to an age-related increase in non-neuronal cells.
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From observations in rodents, it has been suggested that the cellular basis of learning-dependent changes, detected using structural MRI, may be increased dendritic spine density, alterations in astrocyte volume, and adaptations within intracortical myelin. Myelin plasticity is crucial for neurological function, and active myelination is required for learning and memory. However, the dynamics of myelin plasticity and how it relates to morphometric-based measurements of structural plasticity remains unknown. We used a motor skill learning paradigm in male mice to evaluate experience-dependent brain plasticity by voxel-based morphometry (VBM) in longitudinal MRI, combined with a cross-sectional immunohistochemical investigation. Whole-brain VBM revealed nonlinear decreases in gray matter volume (GMV) juxtaposed to nonlinear increases in white matter volume (WMV) within GM that were best modeled by an asymptotic time course. Using an atlas-based cortical mask, we found nonlinear changes with learning in primary and secondary motor areas and in somatosensory cortex. Analysis of cross-sectional myelin immunoreactivity in forelimb somatosensory cortex confirmed an increase in myelin immunoreactivity followed by a return towards baseline levels. Further investigations using quantitative confocal microscopy confirmed these changes specifically to the length density of myelinated axons. The absence of significant histological changes in cortical thickness suggests that nonlinear morphometric changes are likely due to changes in intracortical myelin for which morphometric WMV in somatosensory cortex significantly correlated with myelin immunoreactivity. Together, these observations indicate a nonlinear increase of intracortical myelin during learning and support the hypothesis that myelin is a component of structural changes observed by VBM during learning.
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Substância Cinzenta , Córtex Motor , Masculino , Animais , Camundongos , Substância Cinzenta/patologia , Estudos Transversais , Roedores , Imageamento por Ressonância Magnética , Córtex Motor/diagnóstico por imagem , Córtex Motor/patologiaRESUMO
The Ca(2+)-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D(2) receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D(2) receptor, within an Arg-rich epitope that is also involved in the binding to G(i/o) proteins and to the adenosine A(2A) receptor, with the formation of A(2A)-D(2) receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A(2A) receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A(2A)-D(2) receptor oligomerization. BRET competition experiments indicated that, in the A(2A)-D(2) receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A(2A) receptor. Furthermore, Ca(2+) was found to induce conformational changes in the CaM-A(2A)-D(2) receptor oligomer and to selectively modulate A(2A) and D(2) receptor-mediated MAPK signaling in the A(2A)-D(2) receptor heteromer. These results may have implications for basal ganglia disorders, since A(2A)-D(2) receptor heteromers are being considered as a target for anti-parkinsonian agents.
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Calmodulina/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D2/metabolismo , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/química , Calmodulina/genética , Linhagem Celular , Dopamina/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Dados de Sequência Molecular , Complexos Multiproteicos , Multimerização Proteica , Estrutura Terciária de Proteína , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/genética , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Adenosine A(2A) receptor (A(2A)R) antagonists are being investigated as promising treatment strategy for Parkinson's disease (PD). To test whether A(2A)R antagonists are beneficial in early PD stages we used MitoPark mice, a genetic model with gradual degeneration of DA cells. Daily treatment of young MitoPark mice for eight weeks with the A(2A)R antagonist MSX-3 prevented the reduction of spontaneous locomotor activity observed in saline or L-DOPA treated animals. Chronic A(2A)R antagonist treatment neither induced desensitization of receptors nor accumulation of the drug in brain tissue. Despite beneficial effects on behavior, which are not improved upon addition of a low dose of L-DOPA, the characteristic decline of dopamine levels was not changed. Our results indicate that effective dosing with A(2A)R antagonists should be tested as monotherapy in early PD, and serves to remind us that positive behavioral effects of such treatment need not be reflected in rescue of striatal dopamine levels.
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Antagonistas do Receptor A2 de Adenosina/farmacologia , Dopamina/metabolismo , Atividade Motora/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Xantinas/farmacologia , Antagonistas do Receptor A2 de Adenosina/efeitos adversos , Animais , Modelos Animais de Doenças , Dopaminérgicos/farmacologia , Levodopa/farmacologia , Camundongos , Camundongos Endogâmicos , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Fatores de Tempo , Resultado do Tratamento , Xantinas/efeitos adversosRESUMO
In view of the co-distribution of dopamine D(2L)R and 5-hydroxytryptamine 5-HT(2A) receptors (D(2L)R and 5-HT(2A)R, respectively) within inter alia regions of the dorsal and ventral striatum and their role as a target of antipsychotic drugs; in this study we assessed the potential existence of D(2L)R-5-HT(2A)R heteromers in living cells and the functional consequences of this interaction. Thus, by means of a proximity-based bioluminescence resonance energy transfer (BRET) approach we demonstrated that the D(2L)R and the 5-HT(2A)R form stable and specific heteromers when expressed in HEK293T mammalian cells. Furthermore, when the D(2L)R-5-HT(2A)R heteromeric signaling was analyzed we found that the 5-HT(2A)R-mediated phospholipase C (PLC) activation was synergistically enhanced by the concomitant activation of the D(2L)R as shown in a NFAT-luciferase reporter gene assay and a specific and significant rise of the intracellular calcium levels were observed when both receptors were simultaneously activated. Conversely, when the D(2L)R-mediated adenylyl cyclase (AC) inhibition was assayed we showed that costimulation of D(2L)R and 5-HT(2A)R within the heteromer led to inhibition of the D(2L)R functioning, thus suggesting the existence of a 5-HT(2A)R-mediated D(2L)R trans-inhibition phenomenon. Finally, a bioinformatics study reveals that the triplet amino acid homologies LLT (Leu-Leu-Thr) and AIS (Ala-Ile-Ser) in TM1 and TM3, respectively of the D2R-5-HT(2A)R may be involved in the receptor interface. Overall, the presence of the D(2L)R-5-HT(2A)R heteromer in discrete brain regions is postulated based on the existence of D(2L)R-5-HT(2A) receptor-receptor interactions in living cells and their codistribution inter alia in striatal regions. Possible novel therapeutic strategies for treatment of schizophrenia should be explored by targeting this heteromer.
Assuntos
Encéfalo/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Receptores de Dopamina D2/metabolismo , Cálcio/metabolismo , Linhagem Celular , Genes Reporter , Humanos , Luciferases/genética , Multimerização Proteica , Receptor 5-HT2A de Serotonina/genética , Receptores de Dopamina D2/genética , TransfecçãoRESUMO
Evidence exists that the adenosine receptor A(2A)R and the dopamine receptor D(2)R form constitutive heteromers in living cells. Mass spectrometry and pull-down data showed that an arginine-rich domain of the D(2)R third intracellular loop binds via electrostatic interactions to a specific motif of the A(2A)R C-terminal tail. It has been indicated that the phosphorylated serine 374 might represent an important residue in this motif. In the present study, it was found that a point mutation of serine 374 to alanine reduced the A(2A)R ability to interact with D(2)R. Also, this point mutation abolished the A(2A)R-mediated inhibition of both the D(2)R high affinity agonist binding and signaling. These results point to a key role of serine 374 in the A(2A)R-D(2)R interface. All together these results indicate that by targeting A(2A)R serine 374 it will be possible to allosterically modulate A(2A)R-D(2)R function, thus representing a new approach for therapeutically modulate D(2)R function.
Assuntos
Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D2/metabolismo , Serina/metabolismo , Regulação Alostérica , Linhagem Celular , Humanos , Mutação Puntual , Conformação Proteica , Multimerização Proteica , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/genética , Receptores de Dopamina D2/química , Serina/genéticaRESUMO
A single serine point mutation (S374A) in the adenosine A(2A) receptor (A(2A)R) C-terminal tail reduces A(2A)R-D(2)R heteromerization and prevents its allosteric modulation of the dopamine D(2) receptor (D(2)R). By means of site directed mutagenesis of the A(2A)R and synthetic transmembrane (TM) α-helix peptides of the D(2)R we further explored the role of electrostatic interactions and TM helix interactions of the A(2A)R-D(2)R heteromer interface. We found evidence that the TM domains IV and V of the D(2)R play a major role in the A(2A)R-D(2)R heteromer interface since the incubation with peptides corresponding to these domains significantly reduced the ability of A(2A)R and D(2)R to heteromerize. In addition, the incubation with TM-IV or TM-V blocked the allosteric modulation normally found in A(2A)R-D(2)R heteromers. The mutation of two negatively charged aspartates in the A(2A)R C-terminal tail (D401A/D402A) in combination with the S374A mutation drastically reduced the physical A(2A)R-D(2)R interaction and lost the ability of antagonistic allosteric modulation over the A(2A)R-D(2)R interface, suggesting further evidence for the existence of an electrostatic interaction between the C-terminal tail of A(2A)R and the intracellular loop 3 (IL3) of D(2)R. On the other hand, molecular dynamic model and bioinformatic analysis propose that specific AAR, AQE, and VLS protriplets as an important motive in the A(2A)R-D(2L)R heteromer interface together with D(2L)R TM segments IV/V interacting with A(2A)R TM-IV/V or TM-I/VII.
Assuntos
Receptor A2A de Adenosina/química , Receptores de Dopamina D2/química , Regulação Alostérica , Sequência de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Linhagem Celular , Humanos , Dados de Sequência Molecular , Mutação , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor A2A de Adenosina/genética , Receptores de Dopamina D2/genética , Serina/química , Serina/genéticaRESUMO
Previous biochemical, cardiovascular and behavioral work has given evidence for the existence of antagonistic galanin receptor-5-HT1A receptor interactions in the brain. In this study we investigated the existence of GalR1-5-HT1A receptor heteromers and their functional characteristics. In mammalian cells transfected with GFP2-tagged 5-HT1A receptor and YFP-tagged GalR1 receptor, a proximity-based fluorescence resonance energy transfer technique was used and it has been demonstrated that GalR1-5-HT1A receptors heteromerize. Furthermore, signaling by either the mitogen-activated protein kinase (MAPK) or adenylyl cyclase (AC) pathways by these heteromers indicates a trans-inhibition phenomenon through their interacting interface via allosteric mechanisms that block the development of an excessive activation of G(i/o) and an exaggerated inhibition of AC or stimulation of MAPK activity. The presence of these heteromers in the discrete brain regions is postulated based on the existence of GalR-5-HT1A receptor-receptor interactions previously described in the brain and gives rise to explore possible novel therapeutic strategies for treatment of depression by targeting the GalR1-5-HT1A heteromers.