RESUMEN
BACKGROUND & AIMS: Interactions between C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC) is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC's anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis. METHODS: Monocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC's antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH) and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses. RESULTS: CVC significantly reduced monocyte/macrophage recruitment in vivo at doses ≥20 mg/kg/day (p < 0.05). At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05), and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls). CVC treatment had no notable effect on body or liver/kidney weight. CONCLUSIONS: CVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC's antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475).
Asunto(s)
Antagonistas de los Receptores CCR5/uso terapéutico , Imidazoles/uso terapéutico , Enfermedades Renales/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Animales , Antagonistas de los Receptores CCR5/administración & dosificación , Antagonistas de los Receptores CCR5/farmacología , Quimiotaxis , Fibrosis , Imidazoles/administración & dosificación , Imidazoles/farmacología , Enfermedades Renales/patología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/patología , Ratas , Ratas Sprague-Dawley , Receptores CCR2/antagonistas & inhibidores , SulfóxidosRESUMEN
Behavioral responsiveness to initial cocaine use varies among individuals and may contribute to differential vulnerability to cocaine addiction. Rats also exhibit individual differences in cocaine's effects and can be classified as low or high cocaine responders (LCRs or HCRs, respectively), based on their initial cocaine-induced locomotor activity (10 mg/kg, i.p.). Here, we used the extinction/reinstatement model to address whether or not LCRs and HCRs differ in (i) extinction/reinstatement of cocaine self-administration behavior and (ii) levels of metabotropic glutamate receptors (mGluRs) following these behaviors. During the earliest acquisition sessions, LCRs exhibited significantly greater cocaine intake (0.8 mg/kg/infusion) and cocaine-paired lever responding than HCRs, but intake and lever responding converged by the end of the cocaine self-administration portion of the study. LCRs and HCRs did not differ in cocaine seeking during the first extinction session and extinguished cocaine seeking similarly. HCRs exhibited greater reinstatement than LCRs to lower (2.5 and 5 mg/kg), but not higher (10 mg/kg), i.p. priming doses of cocaine. The effect of drug-paired cues on reinstatement following extinction was complex, with HCRs and LCRs showing the greater effect of cue depending on the order in which cue- and drug-primed tests were given. Western blot analysis revealed that mGluR5 heteromers were significantly higher in the dorsal striatum of HCRs than LCRs following reinstatement testing. Although our previous findings with the LCR/HCR model have uniformly supported the idea that lower initial cocaine-induced activation predicts more ready development of cocaine addiction-like behaviors, here, we show a more complex relationship with cocaine reinstatement.
Asunto(s)
Trastornos Relacionados con Cocaína/patología , Cocaína/farmacología , Cuerpo Estriado/metabolismo , Inhibidores de Captación de Dopamina/farmacología , Actividad Motora/efectos de los fármacos , Receptor del Glutamato Metabotropico 5/metabolismo , Refuerzo en Psicología , Análisis de Varianza , Animales , Trastornos Relacionados con Cocaína/metabolismo , Condicionamiento Operante/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Extinción Psicológica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5/genética , AutoadministraciónRESUMEN
Individual differences are a hallmark of drug addiction. Here, we describe a rat model based on differential initial responsiveness to low dose cocaine. Despite similar brain cocaine levels, individual outbred Sprague-Dawley rats exhibit markedly different magnitudes of acute cocaine-induced locomotor activity and, thereby, can be classified as low or high cocaine responders (LCRs or HCRs). LCRs and HCRs differ in drug-induced, but not novelty-associated, hyperactivity. LCRs have higher basal numbers of striatal dopamine transporters (DATs) than HCRs and exhibit marginal cocaine inhibition of in vivo DAT activity and cocaine-induced increases in extracellular DA. Importantly, lower initial cocaine response predicts greater locomotor sensitization, conditioned place preference and greater motivation to self-administer cocaine following low dose acquisition. Further, outbred Long-Evans rats classified as LCRs, versus HCRs, are more sensitive to cocaine's discriminative stimulus effects. Overall, results to date with the LCR/HCR model underscore the contribution of striatal DATs to individual differences in initial cocaine responsiveness and the value of assessing the influence of initial drug response on subsequent expression of addiction-like behaviors.
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Trastornos Relacionados con Cocaína/metabolismo , Cocaína/farmacología , Cuerpo Estriado/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Actividad Motora/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , RatasRESUMEN
The plasma membrane dopamine (DA) transporter (DAT) is essential for reuptake of extracellular DA. DAT function in heterologous cells is regulated by subcellular targeting, endocytosis, and intracellular trafficking, but the mechanisms regulating neuronal DAT remain poorly understood. Hence, we generated a knock-in mouse expressing a hemagglutinin (HA)-epitope-tagged DAT to study endogenous transporter trafficking. Introduction of the HA tag into the second extracellular loop of mouse DAT did not perturb its expression level, distribution pattern, or substrate uptake kinetics. Live-cell fluorescence microscopy imaging using fluorescently labeled HA-specific antibody and a quantitative HA-antibody endocytosis assay demonstrated that in axons HA-DAT was primarily located in the plasma membrane and internalized mostly in growth cones and varicosities, where synaptic vesicle markers were also concentrated. Formation of varicosities was frequently preceded or accompanied by highly dynamic filopodia-like membrane protrusions. Remarkably, HA-DAT often concentrated at the tips of these filopodia. This pool of HA-DATs exhibited low lateral membrane mobility. Thus, DAT-containing filopodia may be involved in synaptogenesis in developing DA neurons. Treatment of neurons with amphetamine increased mobility of filopodial HA-DAT and accelerated HA-DAT endocytosis in axons, suggesting that chronic amphetamine may interfere with DA synapse development. Interestingly, phorbol esters did not accelerate endocytosis of axonal DAT.
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Axones , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/fisiología , Dopamina/metabolismo , Endocitosis , Epítopos/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , NeurogénesisRESUMEN
Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinson's disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.
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Reprogramación Celular , Neuronas Dopaminérgicas/citología , Fibroblastos/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proliferación Celular , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/trasplante , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Enfermedad de Parkinson/terapia , Ratas , Ratas Sprague-Dawley , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The plasma membrane dopamine transporter (DAT) takes extracellular dopamine back up into dopaminergic neurons. Although the number of DATs at the cell surface is regulated by endocytosis and recycling, the molecular mechanisms that control this endocytic trafficking of DAT are not defined. To map the sequence motifs that are involved in constitutive DAT endocytosis, mutagenesis of human DAT tagged with yellow fluorescent protein (YFP) and an extracellular HA epitope was performed. Removal of the entire N terminus of DAT resulted in accumulation of the resulting DAT mutant (YFP-HA-DeltaN-DAT) in early and recycling endosomes in HeLa and PAE cells, and in primary rat mesencephalic-striatal neuronal cocultures. This endosomal accumulation was due to rapid constitutive internalization of YFP-HA-DeltaN-DAT by the clathrin-dependent pathway. Small deletions and multialanine substitutions in the N terminus revealed two molecular determinants within the membrane proximal residues 60-65 that are important for preventing rapid internalization of DAT. First, mutations of Arg60 or Trp63, leading to disruption of the "outward facing" DAT conformation, correlated with an increased pool of mobile DATs in the plasma membrane and accelerated constitutive internalization of the DAT mutants. Second, mutation of Lys65 also correlated with elevated endocytosis. While none of these mutations alone recapitulated the marked endocytic phenotype of YFP-HA-DeltaN-DAT, simultaneous elimination of both the outward conformation of DAT and Lys65 resulted in DAT mutants that were rapidly internalized. Thus, our studies reveal a new link between DAT endocytosis and conformation-dependent uptake activity that represents a novel mode for regulating DAT function.
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Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Endocitosis/fisiología , Proteínas de la Membrana/metabolismo , Animales , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/fisiología , Femenino , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Mutación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Embarazo , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-DawleyRESUMEN
The dopamine transporter (DAT) substrates dopamine, d-amphetamine (AMPH), and methamphetamine are known to rapidly and transiently reduce DAT activity and/or surface expression in dorsal striatum and heterologous expression systems. We sought to determine if similar substrate-induced regulation of DATs occurs in rat nucleus accumbens. In dorsal striatum synaptosomes, brief (15-min) in vitro substrate pre-exposure markedly decreased maximal [(3)H]dopamine uptake velocity whereas identical substrate pre-exposure in nucleus accumbens synaptosomes produced a smaller, non-significant reduction. However, 45 min after systemic AMPH administration, maximal ex vivo [(3)H]dopamine uptake velocity was significantly reduced in both brain regions. Protein kinase C inhibition blocked AMPH's down-regulation of DAT activity. DAT synaptosomal surface expression was not modified following either the brief in vitro or in vivo AMPH pre-exposure but was reduced after a longer (1-h) in vitro pre-exposure in both brain regions. Together, our findings suggest that relatively brief substrate exposure results in greater down-regulation of DAT activity in dorsal striatum than in nucleus accumbens. Moreover, exposure to AMPH appears to regulate striatal DATs in a biphasic manner, with an initial protein kinase C-dependent decrease in DAT-mediated uptake velocity and then, with longer exposure, a reduction in DAT surface expression.
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Cuerpo Estriado/metabolismo , Dopaminérgicos/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Núcleo Accumbens/metabolismo , Anfetamina/farmacología , Animales , Biotinilación/métodos , Cuerpo Estriado/ultraestructura , Dopamina/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Masculino , Maleimidas/farmacología , Metanfetamina/farmacología , Núcleo Accumbens/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Factores de TiempoRESUMEN
Sensitization to psychostimulants results in a behavioral response of a greater magnitude than that produced by a given single dose. Previously, we have shown that sensitization to the D(2)/D(3) dopamine receptor agonist quinpirole produces alterations in quinpirole-stimulated local cerebral glucose utilization (LCGU) in ventral striatal and limbic cortical regions. To determine whether basal neuronal activity is altered in the sensitized animal, this study examined the effects of a sensitizing course of quinpirole on basal neuronal activity using the [(14)C]-2-deoxyglucose (2-DG) method in rats with verified sensitization. Adult, male Long-Evans rats (n = 7 or 10/group) were subjected to 10 injections of quinpirole (0.5 mg/kg, s.c.) or saline administered every 3rd day. Sensitization was verified on the basis of locomotor activity. The 2-DG procedure was performed in freely moving rats 3 days after the last quinpirole injection. LCGU was determined by quantitative autoradiography. No alterations in basal LCGU were detected in quinpirole-sensitized rats compared to those treated with saline. The present finding suggests that either the basal activity of very discrete populations of neurons is affected by sensitization to quinpirole that are not likely to be detected by the 2-DG method, or that the neurobiological changes that result in the sensitized behavioral response affect only stimulated, but not basal, neuronal activity.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Agonistas de Dopamina/farmacología , Glucosa/metabolismo , Quinpirol/farmacología , Receptores de Dopamina D2/agonistas , Animales , Autorradiografía , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Estimulantes del Sistema Nervioso Central/farmacología , Desoxiglucosa/metabolismo , Dopamina/metabolismo , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Long-Evans , Receptores de Dopamina D2/metabolismo , Sensibilidad y EspecificidadRESUMEN
Sensitization refers to augmented behavioral responses produced by repeated, intermittent injections of dopaminergic psychostimulants. The locomotor manifestations observed after a sensitizing course of quinpirole, a D(2)/D(3) dopamine agonist, can be modified by the MAO(A) inhibitor clorgyline, by a mechanism apparently unrelated to its actions on MAO(A). Alterations in regional neuronal activity produced by quinpirole in quinpirole-sensitized rats with or without clorgyline pretreatment were assessed based on LCGU using the [(14)C]-2-deoxyglucose (2-DG) method. Adult, male Long-Evans rats (180-200 g, n=9-10/group) were subjected to an injection of either clorgyline (1.0 mg/kg, s.c.) or saline 90 min prior to an injection of quinpirole (0.5 mg/kg, s.c.) or saline, 1 set of injections administered every 3rd day for 10 sets. The 2-DG procedure was initiated 60 min after an 11th set of injections in freely moving rats. LCGU was determined by quantitative autoradiography. LCGU was decreased in a number of limbic (nucleus accumbens and ventral pallidum) and cortical (medial/ventral orbital and infralimbic) regions and in the raphe magnus nucleus in quinpirole-sensitized rats (P<0.05 vs. saline-saline). Quinpirole-sensitized rats pretreated with clorgyline had similar alterations in LCGU, but LCGU was higher in the locus coeruleus compared to quinpirole alone (P<0.05), was not decreased in the raphe magnus nucleus, and was decreased in the piriform cortex and septum. This implicates altered activity of the noradrenergic, serotonergic, olfactory, and limbic systems in the modified behavioral response to quinpirole with clorgyline pretreatment.
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Conducta Animal/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Clorgilina/farmacología , Agonistas de Dopamina/farmacología , Glucosa/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Quinpirol/farmacología , Animales , Antimetabolitos , Mapeo Encefálico , Radioisótopos de Carbono , Corteza Cerebral/metabolismo , Desoxiglucosa , Esquema de Medicación , Masculino , Actividad Motora/efectos de los fármacos , Ratas , Ratas Long-EvansRESUMEN
Dopaminergic psychostimulants produce behavioral responses of greater magnitude with repeated, intermittent administration, than a single, acute dose, a phenomenon known as "sensitization." Most studies of sensitization have focused on the "motive circuit"; however, some additional anterior cortical regions also appear to be affected. In this study, alterations in regional neuronal activity in anterior cortical brain areas produced by quinpirole, a D(2)/D(3) agonist, were assessed on the basis of local cerebral glucose utilization (LCGU) using the [(14)C]-2-deoxyglucose (2-DG) method. Adult, male Long-Evans rats (180-200 g, n = 7-9/group) were subjected to ten injections of quinpirole (0.5 mg/kg, s.c.) administered every third day; controls and drug-naive rats received saline. Locomotor activity was quantitated after injections one and ten to confirm sensitization. The 2-DG procedure was initiated 60 min after an 11th injection in freely moving rats. LCGU was determined in 11 anterior cortical brain regions by quantitative autoradiography. In drug-naive rats, quinpirole decreased LCGU in the cingulate cortex-area 3 (-16%) and infralimbic cortex (-16%). In sensitized rats, quinpirole decreased LCGU in the cingulate cortex-area 1 (-19%), frontal cortex-area 3 (-19%), lateral orbital cortex (-18%), medial/ventral orbital cortex (-17%), and parietal cortex (-17%) as well as in the cingulate cortex-area 3 (-19%) and infralimbic cortex (-20%); (all P < 0.05 v. control). This suggests that decreased neuronal activity in the cingulate cortex-area 1, frontal cortex-area 3, lateral orbital cortex, medial/ventral orbital cortex, and parietal cortex, in addition to altered activity in the motive circuit, may underlie the augmented behavioral response to quinpirole in sensitized animals.