RESUMO
Ras/Raf/MEK/ERK (Ras-ERK) signaling has been implicated in the effects of drugs of abuse. Inhibitors of MEK1/2, the kinases upstream of ERK1/2, have been critical in defining the role of the Ras-ERK cascade in drug-dependent alterations in behavioral plasticity, but the Ras family of small GTPases has not been extensively examined in drug-related behaviors. We examined the role of Ras Guanine Nucleotide Releasing Factor 1 (RasGRF1) and 2 (RasGRF2), upstream regulators of the Ras-ERK signaling cascade, on cocaine self-administration (SA) in male mice. We first established a role for Ras-ERK signaling in cocaine SA, demonstrating that pERK1/2 is upregulated following SA in C57BL/6N mice in striatum. We then compared RasGRF1 and RasGRF2 KO mouse lines, demonstrating that cocaine SA in RasGRF2 KO mice was increased relative to WT controls, whereas RasGRF1 KO and WT mice did not differ. This effect in RasGRF2 mice is likely mediated by the Ras-ERK signaling pathway, as pERK1/2 upregulation following cocaine SA was absent in RasGRF2 KO mice. Interestingly, the lentiviral knockdown of RasGRF2 in the NAc had the opposite effect to that in RasGRF2 KO mice, reducing cocaine SA. We subsequently demonstrated that the MEK inhibitor PD325901 administered peripherally prior to cocaine SA increased cocaine intake, replicating the increase seen in RasGRF2 KO mice, whereas PD325901 administered into the NAc decreased cocaine intake, similar to the effect seen following lentiviral knockdown of RasGRF2. These data indicate a role for RasGRF2 in cocaine SA in mice that is ERK-dependent, and suggest a differential effect of global versus site-specific RasGRF2 inhibition.SIGNIFICANCE STATEMENT Exposure to drugs of abuse activates a variety of intracellular pathways, and following repeated exposure, persistent changes in these pathways contribute to drug dependence. Downstream components of the Ras-ERK signaling cascade are involved in the acute and chronic effects of drugs of abuse, but their upstream mediators have not been extensively characterized. Here we show, using a combination of molecular, pharmacological, and lentiviral techniques, that the guanine nucleotide exchange factor RasGRF2 mediates cocaine self-administration via an ERK-dependent mechanism, whereas RasGRF1 has no effect on responding for cocaine. These data indicate dissociative effects of mediators of Ras activity on cocaine reward and expand the understanding of the contribution of Ras-ERK signaling to drug-taking behavior.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Cocaína/farmacologia , Corpo Estriado/fisiopatologia , Sistema de Sinalização das MAP Quinases/fisiologia , Recompensa , Fatores ras de Troca de Nucleotídeo Guanina/fisiologia , Acetilação , Animais , Benzamidas/farmacologia , Cocaína/administração & dosagem , Condicionamento Operante , Corpo Estriado/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Histonas/metabolismo , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiopatologia , Especificidade de Órgãos , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Autoadministração , Fatores ras de Troca de Nucleotídeo Guanina/deficiência , Fatores ras de Troca de Nucleotídeo Guanina/genética , ras-GRF1/deficiência , ras-GRF1/genética , ras-GRF1/fisiologiaRESUMO
Dopamine dysregulation syndrome shares some core behavioral features with psychostimulant addiction, suggesting that dopamine replacement therapy can acquire psychostimulantlike properties in some patients with Parkinson disease (PD). We here report strong experimental evidence supporting this hypothesis in an α-synuclein rat model of PD. Although levodopa had no effect in controls, it acquired 2 prominent psychostimulantlike properties in Parkinsonian rats: (1) it produced intense reward on its own and in parallel (2) decreased interest in other nondrug reward. These 2 effects may combine to explain the addictive use of levodopa after loss of midbrain dopamine neurons in some PD patients.
Assuntos
Antiparkinsonianos/uso terapêutico , Neurônios Dopaminérgicos/patologia , Levodopa/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Substância Negra/patologia , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Antiparkinsonianos/farmacologia , Condicionamento Operante/efeitos dos fármacos , Condicionamento Operante/fisiologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Preferências Alimentares/efeitos dos fármacos , Humanos , Levodopa/farmacologia , Masculino , Mutação/genética , Doença de Parkinson/etiologia , Doença de Parkinson/genética , Ratos , Ratos Wistar , Recompensa , Sacarina/administração & dosagem , Edulcorantes/administração & dosagem , Paladar/efeitos dos fármacos , Transdução Genética , Tirosina 3-Mono-Oxigenase/metabolismo , Ubiquitina/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidadeRESUMO
Due to the increase in the rate of male and female infertility, assisted fertilization practices are currently adopted as valid support for couples unable to get pregnant. Analytical approaches for fertility hormone dosages are constantly being developed, following the technological progress of fertilization methods that have evolved for more than a century. Indeed, the analysis of fertility hormones in serum samples is a common clinical practice to check the fertility state, but absolute quantification of these hormones is a great challenge due to biological variability and low serum concentrations. Currently, ELISA (enzyme-linked immunosorbent assay) based methods are the most used analytical techniques to quantify hormones in blood in clinical settings. The current Article discusses the development of a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) to monitor multiple fertility hormones of a protein nature in a single chromatographic run, i.e., LH (luteinizing hormone), FSH (follicle-stimulating hormone), TSH (thyroid-stimulating hormone), AMH (anti-Müllerian hormone), adiponectin, ghrelin, leptin, glucagon, and obestatin. Particular attention has been paid to the AMH hormone, whose ELISA-based quantification is known to be controversial due to the poor reproducibility between the various kits used. For AMH, the internal standard method was used for the quantitative determination to compare mass spectrometry data to the ELISA assays performed by an accredited analysis laboratory on a cohort of samples from women aged between 18 and 60 years. The ability to monitor multiple transitions by LC-MRM/MS ensured both high specificity and high selectivity, which is necessary for the quantification of protein and steroid hormones, besides improvements in data reproducibility and reduced analysis times and costs.
RESUMO
In the present study we investigated whether the neuropeptide nociceptin/orphanin FQ (N/OFQ), previously implicated in the pathogenesis of Parkinson's disease, also affects L-DOPA-induced dyskinesia. In striatal slices of naive rodents, N/OFQ (0.1-1 µm) prevented the increase of ERK phosphorylation and the loss of depotentiation of synaptic plasticity induced by the D1 receptor agonist SKF38393 in spiny neurons. In vivo, exogenous N/OFQ (0.03-1 nmol, i.c.v.) or a synthetic N/OFQ receptor agonist given systemically (0.01-1 mg/Kg) attenuated dyskinesias expression in 6-hydroxydopamine hemilesioned rats primed with L-DOPA, without causing primary hypolocomotive effects. Conversely, N/OFQ receptor antagonists worsened dyskinesia expression. In vivo microdialysis revealed that N/OFQ prevented dyskinesias simultaneously with its neurochemical correlates such as the surge of nigral GABA and glutamate, and the reduction of thalamic GABA. Regional microinjections revealed that N/OFQ attenuated dyskinesias more potently and effectively when microinjected in striatum than substantia nigra (SN) reticulata, whereas N/OFQ receptor antagonists were ineffective in striatum but worsened dyskinesias when given in SN. Quantitative autoradiography showed an increase in N/OFQ receptor binding in striatum and a reduction in SN of both unprimed and dyskinetic 6-hydroxydopamine rats, consistent with opposite adaptive changes of N/OFQ transmission. Finally, the N/OFQ receptor synthetic agonist also reduced dyskinesia expression in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated dyskinetic macaques without affecting the global parkinsonian score. We conclude that N/OFQ receptor agonists may represent a novel strategy to counteract L-DOPA-induced dyskinesias. Their action is possibly mediated by upregulated striatal N/OFQ receptors opposing the D1 receptor-mediated overactivation of the striatonigral direct pathway.
Assuntos
Antidiscinéticos , Antiparkinsonianos/efeitos adversos , Discinesia Induzida por Medicamentos/tratamento farmacológico , Levodopa/efeitos adversos , Peptídeos Opioides/agonistas , Animais , Autorradiografia , Comportamento Animal/efeitos dos fármacos , Fenômenos Eletrofisiológicos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Macaca , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microdiálise , Microinjeções , Peptídeos Opioides/antagonistas & inibidores , Peptídeos Opioides/genética , Oxidopamina/toxicidade , Equilíbrio Postural/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Ácido gama-Aminobutírico/metabolismo , NociceptinaRESUMO
Aberrant membrane localization of dopamine D(1) receptor (D1R) is associated with L-DOPA-induced dyskinesia (LID), a major complication of L-DOPA treatment in Parkinson's disease (PD). Since the proteasome plays a central role in modulating neuronal response through regulation of neurotransmitter receptor intraneuronal fate, we hypothesized that the ubiquitine-proteasome proteolytic pathway could be impaired in LID. Those LIDs are actually associated with a striatum-specific decrease in proteasome catalytic activity and accumulation of polyubiquitinated proteins in experimental rodent and monkey parkinsonism. We then demonstrated that such decreased proteasome catalytic activity (1) results from D1R activation and (2) feed-back the D1R abnormal trafficking, i.e., its exaggerated cell surface abundance. We further showed that the genetic invalidation of the E3 ubiquitin-protein ligase parkin PD gene leads to exaggerated abnormal involuntary movements compared with wild-type mice. We thus established in an unprecedented series of experimental models that impairment of the ubiquitine-proteasome system at specific nodes (E3 ligase parkin, polyubiquitination, proteasome catalytic activity) leads to the same phenomenon, i.e., aberrant behavioral response to dopamine replacement therapy in PD, highlighting the intimate interplay between dopamine receptor and proteasome activity in a nondegenerative context.
Assuntos
Discinesia Induzida por Medicamentos/metabolismo , Levodopa/toxicidade , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Receptores de Dopamina D1/agonistas , Animais , Modelos Animais de Doenças , Agonistas de Dopamina/toxicidade , Discinesia Induzida por Medicamentos/fisiopatologia , Feminino , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Transtornos Parkinsonianos/enzimologia , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/fisiologiaRESUMO
L-dopa-induced dyskinesia (LID) is a common debilitating complication of dopamine replacement therapy in Parkinson's disease. Recent evidence suggests that LID may be linked causally to a hyperactivation of the Ras-ERK signaling cascade in the basal ganglia. We set out to determine whether specific targeting of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a brain-specific activator of the Ras-ERK pathway, may provide a therapy for LID. On the rodent abnormal involuntary movements scale, Ras-GRF1-deficient mice were significantly resistant to the development of dyskinesia during chronic L-dopa treatment. Furthermore, in a nonhuman primate model of LID, lentiviral vectors expressing dominant negative forms of Ras-GRF1 caused a dramatic reversion of dyskinesia severity leaving intact the therapeutic effect of L-dopa. These data reveal the central role of Ras-GRF1 in governing striatal adaptations to dopamine replacement therapy and validate a viable treatment for LID based on intracellular signaling modulation.
Assuntos
Corpo Estriado/efeitos dos fármacos , Corpo Estriado/fisiologia , Discinesia Induzida por Medicamentos/fisiopatologia , Levodopa/farmacologia , Transdução de Sinais/fisiologia , ras-GRF1/metabolismo , Animais , Corpo Estriado/citologia , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , ras-GRF1/genéticaRESUMO
BACKGROUND AND PURPOSE: Regulator of G-protein signalling 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gαi/o and Gαq subunits, suppressing GPCR signalling. Here, we investigate whether RGS4 modulates nociceptin/orphanin FQ (N/OFQ) opioid (NOP) receptor signalling and if this modulation has relevance for l-Dopa-induced dyskinesia. EXPERIMENTAL APPROACH: HEK293T cells transfected with NOP, NOP/RGS4 or NOP/RGS19 were challenged with N/OFQ and the small-molecule NOP agonist AT-403, using D1-stimulated cAMP levels as a readout. Primary rat striatal neurons and adult mouse striatal slices were challenged with either N/OFQ or AT-403 in the presence of the experimental RGS4 chemical probe, CCG-203920, and D1-stimulated cAMP or phosphorylated extracellular signal regulated kinase 1/2 (pERK) responses were monitored. In vivo, CCG-203920 was co-administered with AT-403 and l-Dopa to 6-hydroxydopamine hemilesioned rats, and dyskinetic movements, striatal biochemical correlates of dyskinesia (pERK and pGluR1 levels) and striatal RGS4 levels were measured. KEY RESULTS: RGS4 expression reduced NOFQ and AT-403 potency and efficacy in HEK293T cells. CCG-203920 increased N/OFQ potency in primary rat striatal neurons and potentiated AT-403 response in mouse striatal slices. CCG-203920 enhanced AT-403-mediated inhibition of dyskinesia and its biochemical correlates, without compromising its motor-improving effects. Unilateral dopamine depletion caused bilateral reduction of RGS4 levels, which was reversed by l-Dopa. l-Dopa acutely up-regulated RGS4 in the lesioned striatum. CONCLUSIONS AND IMPLICATIONS: RGS4 physiologically inhibits NOP receptor signalling. CCG-203920 enhanced NOP responses and improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of striatal RGS4 up-regulation occurring during dyskinesia expression. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.
Assuntos
Discinesia Induzida por Medicamentos , Levodopa , Camundongos , Ratos , Humanos , Animais , Levodopa/farmacologia , Analgésicos Opioides , Células HEK293 , Transdução de Sinais , Discinesia Induzida por Medicamentos/tratamento farmacológico , Receptores Opioides/metabolismo , NociceptinaRESUMO
Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneration and cognitive decline. Here, we show that selective genetic potentiation of neuronal ERK signaling prevents cell death in vitro and in vivo in the mouse brain, while attenuation of ERK signaling does the opposite. This neuroprotective effect mediated by an enhanced nuclear ERK activity can also be induced by the novel cell penetrating peptide RB5. In vitro administration of RB5 disrupts the preferential interaction of ERK1 MAP kinase with importinα1/KPNA2 over ERK2, facilitates ERK1/2 nuclear translocation, and enhances global ERK activity. Importantly, RB5 treatment in vivo promotes neuroprotection in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) disease, and enhances ERK signaling in a human cellular model of HD. Additionally, RB5-mediated potentiation of ERK nuclear signaling facilitates synaptic plasticity, enhances cognition in healthy rodents, and rescues cognitive impairments in AD and HD models. The reported molecular mechanism shared across multiple neurodegenerative disorders reveals a potential new therapeutic target approach based on the modulation of KPNA2-ERK1/2 interactions.
Assuntos
Sistema de Sinalização das MAP Quinases , Neuroproteção , Animais , Humanos , Camundongos , alfa Carioferinas/farmacologia , Cognição , Fosforilação , Transdução de SinaisRESUMO
Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice, thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely, a peripheral source of enzyme, established by transplanting ARSA-overexpressing hepatocytes from transgenic donors, failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified, enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall, our data provide a strong rationale for implementing HSPC gene therapy in MLD patients.
Assuntos
Terapia Genética/efeitos adversos , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/terapia , Animais , Comportamento Animal , Diferenciação Celular , Cerebrosídeo Sulfatase/deficiência , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/metabolismo , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Leucodistrofia Metacromática/metabolismo , Leucodistrofia Metacromática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Microglia/patologia , Neurofisiologia/estatística & dados numéricos , Sulfoglicoesfingolipídeos/metabolismoRESUMO
Extracellular signal-regulated kinases (ERK1 and 2) are synaptic signaling components necessary for several forms of learning. In mice lacking ERK1, we observe a dramatic enhancement of striatum-dependent long-term memory, which correlates with a facilitation of long-term potentiation in the nucleus accumbens. At the cellular level, we find that ablation of ERK1 results in a stimulus-dependent increase of ERK2 signaling, likely due to its enhanced interaction with the upstream kinase MEK. Consistently, such activity change is responsible for the hypersensitivity of ERK1 mutant mice to the rewarding properties of morphine. Our results reveal an unexpected complexity of ERK-dependent signaling in the brain and a critical regulatory role for ERK1 in the long-term adaptive changes underlying striatum-dependent behavioral plasticity and drug addiction.
Assuntos
Corpo Estriado/enzimologia , Potenciação de Longa Duração/genética , Memória/fisiologia , Proteínas Quinases Ativadas por Mitógeno/deficiência , Núcleo Accumbens/enzimologia , Terminações Pré-Sinápticas/enzimologia , Transmissão Sináptica/genética , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/enzimologia , Animais , Aprendizagem da Esquiva/fisiologia , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Transtornos Cognitivos/enzimologia , Transtornos Cognitivos/genética , Transtornos Cognitivos/patologia , Corpo Estriado/anormalidades , Corpo Estriado/citologia , Feminino , Hipocampo/citologia , Hipocampo/enzimologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/genética , Morfina/farmacologia , Atividade Motora/genética , Mutação/genética , Rede Nervosa/anormalidades , Rede Nervosa/citologia , Rede Nervosa/enzimologia , Malformações do Sistema Nervoso/enzimologia , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Núcleo Accumbens/anormalidades , Núcleo Accumbens/citologia , Terminações Pré-Sinápticas/ultraestrutura , Regulação para Cima/genéticaRESUMO
Increasing evidence supports a close relationship between Ras-ERK1/2 activation in the striatum and L-DOPA-induced dyskinesia (LID). ERK1/2 activation by L-DOPA takes place through the crosstalk between D1R/AC/PKA/DARPP-32 pathway and NMDA/Ras pathway. Compelling genetic and pharmacological evidence indicates that Ras-ERK1/2 inhibition prevents LID onset and may even revert already established dyskinetic symptoms. However, it is currently unclear whether exacerbation of Ras-ERK1/2 activity in the striatum may further aggravate dyskinesia in experimental animal models. Here we took advantage of two genetic models in which Ras-ERK1/2 signaling is hyperactivated, the Nf1+/- mice, in which the Ras inhibitor neurofibromin is reduced, and the Ras-GRF1 overexpressing (Ras-GRF1 OE) transgenic mice in which a specific neuronal activator of Ras is enhanced. Nf1+/- and Ras-GRF1 OE mice were unilaterally lesioned with 6-OHDA and treated with an escalating L-DOPA dosing regimen. In addition, a subset of Nf1+/- hemi-parkinsonian animals was also co-treated with the Ras inhibitor lovastatin. Our results revealed that Nf1+/- and Ras-GRF1 OE mice displayed similar dyskinetic symptoms to their wild-type counterparts. This observation was confirmed by the lack of differences between mutant and wild-type mice in striatal molecular changes associated to LID (i.e., FosB, and pERK1/2 expression). Interestingly, attenuation of Ras activity with lovastatin does not weaken dyskinetic symptoms in Nf1+/- mice. Altogether, these data suggest that ERK1/2-signaling activation in dyskinetic animals is maximal and does not require further genetic enhancement in the upstream Ras pathway. However, our data also demonstrate that such a genetic enhancement may reduce the efficacy of anti-dyskinetic drugs like lovastatin.
Assuntos
Discinesia Induzida por Medicamentos/tratamento farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Levodopa/toxicidade , Lovastatina/farmacologia , Neurofibromina 1/fisiologia , Proteínas ras/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Dopaminérgicos/toxicidade , Discinesia Induzida por Medicamentos/metabolismo , Discinesia Induzida por Medicamentos/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Transdução de Sinais , Proteínas ras/genéticaRESUMO
BACKGROUND AND PURPOSE: We previously showed that nociceptin/orphanin FQ opioid peptide (NOP) receptor agonists attenuate the expression of levodopa-induced dyskinesia in animal models of Parkinson's disease. We now investigate the efficacy of two novel, potent and chemically distinct NOP receptor agonists, AT-390 and AT-403, to improve Parkinsonian disabilities and attenuate dyskinesia development and expression. EXPERIMENTAL APPROACH: Binding affinity and functional efficacy of AT-390 and AT-403 at the opioid receptors were determined in radioligand displacement assays and in GTPγS binding assays respectively, conducted in CHO cells. Their anti-Parkinsonian activity was evaluated in 6-hydroxydopamine hemi-lesioned rats whereas the anti-dyskinetic properties were assessed in 6-hydroxydopamine hemi-lesioned rats chronically treated with levodopa. The ability of AT-403 to inhibit the D1 receptor-induced phosphorylation of striatal ERK was investigated. KEY RESULTS: AT-390 and AT-403 selectively improved akinesia at low doses and disrupted global motor activity at higher doses. AT-403 palliated dyskinesia expression without causing sedation in a narrow therapeutic window, whereas AT-390 delayed the appearance of abnormal involuntary movements and increased their duration at doses causing sedation. AT-403 did not prevent the priming to levodopa, although it significantly inhibited dyskinesia on the first day of administration. AT-403 reduced the ERK phosphorylation induced by SKF38393 in vitro and by levodopa in vivo. CONCLUSIONS AND IMPLICATIONS: NOP receptor stimulation can provide significant albeit mild anti-dyskinetic effect at doses not causing sedation. The therapeutic window, however, varies across compounds. AT-403 could be a potent and selective tool to investigate the role of NOP receptors in vivo.
Assuntos
Acetamidas/farmacologia , Antiparkinsonianos/farmacologia , Discinesia Induzida por Medicamentos/tratamento farmacológico , Piperidinas/farmacologia , Receptores Opioides/agonistas , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/antagonistas & inibidores , Acetamidas/uso terapêutico , Animais , Antiparkinsonianos/uso terapêutico , Corpo Estriado/metabolismo , Cricetinae , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Levodopa/antagonistas & inibidores , Masculino , Oxidopamina , Fosforilação/efeitos dos fármacos , Piperidinas/uso terapêutico , Ensaio Radioligante , Ratos , Receptor de NociceptinaRESUMO
The striatum has a well documented role in procedural learning and memory. However, the synaptic and molecular mechanisms of acquisition and storage of this form of memory remain poorly understood. We examined procedural memory and plasticity in transgenic mice reversibly expressing a dominant-negative cAMP response element-binding protein (CREB) mutant in the dorsal striatum. In these transgenic mice, corticostriatal long-term potentiation and depression are abolished, indicating that CREB function is essential for bidirectional long-term synaptic plasticity in this structure. Importantly, CREB-deficient animals show reversible alterations in several forms of striatum-dependent memory, including footshock avoidance learning and "response" learning in the cross maze. These findings implicate transcriptional regulation by CREB family transcription factors in striatum-dependent information processing and provide the first clear correlation between procedural learning and memory and synaptic plasticity at the corticostriatal synapse.
Assuntos
Corpo Estriado/citologia , Corpo Estriado/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/deficiência , Memória/fisiologia , Plasticidade Neuronal/genética , Sinapses/fisiologia , Animais , Aprendizagem da Esquiva/fisiologia , Comportamento Animal , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica/métodos , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Potenciação de Longa Duração/efeitos da radiação , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/genética , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp/métodos , Sinapses/metabolismoRESUMO
This work describes the first successful oligodendrocyte-based cell therapy for presymptomatic arylsulfatase A (ARSA) null neonate mice, a murine model for human metachromatic leukodystrophy (MLD). We found that oligodendrocyte progenitors (OLPs) engrafted and survived into adulthood when transplanted in the neonatal MLD brain. Transplanted cells integrated nondisruptively, did not produce tumors, and survived as proteolipid protein- and MBP-positive postmitotic myelinating oligodendrocytes (OLs) intermingled with endogenous MLD OLs within the adult MLD white matter. Transplanted MLD mice had reduced sulfatide accumulation in the CNS, increased brain ARSA activity, and full prevention of the electrophysiological and motor deficits that characterize untreated MLD mice. Our results provide direct evidence that healthy OLPs can tolerate the neurotoxic accumulation of sulfatides that evolves during the postnatal development of the MLD brain and contribute to OL cell replacement to limit the accumulation of sulfatides and the evolution of CNS defects in this lysosomal storage disease mouse model.
Assuntos
Transplante de Tecido Encefálico/métodos , Leucodistrofia Metacromática/terapia , Oligodendroglia/transplante , Transplante de Células-Tronco/métodos , Animais , Animais Recém-Nascidos , Transplante de Tecido Encefálico/tendências , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/metabolismo , Modelos Animais de Doenças , Sobrevivência de Enxerto/fisiologia , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/metabolismo , Camundongos , Camundongos Knockout , Proteína Básica da Mielina/metabolismo , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Oligodendroglia/metabolismo , Transplante de Células-Tronco/tendências , Sulfoglicoesfingolipídeos/metabolismo , Resultado do TratamentoRESUMO
Gene-based delivery can establish a sustained supply of therapeutic proteins within the nervous system. For diseases characterized by extensive CNS and peripheral nervous system (PNS) involvement, widespread distribution of the exogenous gene may be required, a challenge to in vivo gene transfer strategies. Here, using lentiviral vectors (LVs), we efficiently transduced hematopoietic stem cells (HSCs) ex vivo and evaluated the potential of their progeny to target therapeutic genes to the CNS and PNS of transplanted mice and correct a neurodegenerative disorder, metachromatic leukodystrophy (MLD). We proved extensive repopulation of CNS microglia and PNS endoneurial macrophages by transgene-expressing cells. Intriguingly, recruitment of these HSC-derived cells was faster and more robust in MLD mice. By transplanting HSCs transduced with the arylsulfatase A gene, we fully reconstituted enzyme activity in the hematopoietic system of MLD mice and prevented the development of motor conduction impairment, learning and coordination deficits, and neuropathological abnormalities typical of the disease. Remarkably, ex vivo gene therapy had a significantly higher therapeutic impact than WT HSC transplantation, indicating a critical role for enzyme overexpression in the HSC progeny. These results indicate that transplantation of LV-transduced autologous HSCs represents a potentially efficacious therapeutic strategy for MLD and possibly other neurodegenerative disorders.
Assuntos
Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Leucodistrofia Metacromática/terapia , Animais , Diferenciação Celular , Movimento Celular , Modelos Animais de Doenças , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora , Sistema Nervoso/citologiaRESUMO
BACKGROUND: Dysregulation of Ras-extracellular signal-related kinase (ERK) signaling gives rise to RASopathies, a class of neurodevelopmental syndromes associated with intellectual disability. Recently, much attention has been directed at models bearing mild forms of RASopathies whose behavioral impairments can be attenuated by inhibiting the Ras-ERK cascade in the adult. Little is known about the brain mechanisms in severe forms of these disorders. METHODS: We performed an extensive characterization of a new brain-specific model of severe forms of RASopathies, the KRAS12V mutant mouse. RESULTS: The KRAS12V mutation results in a severe form of intellectual disability, which parallels mental deficits found in patients bearing mutations in this gene. KRAS12V mice show a severe impairment of both short- and long-term memory in a number of behavioral tasks. At the cellular level, an upregulation of ERK signaling during early phases of postnatal development, but not in the adult state, results in a selective enhancement of synaptogenesis in gamma-aminobutyric acidergic interneurons. The enhancement of ERK activity in interneurons at this critical postnatal time leads to a permanent increase in the inhibitory tone throughout the brain, manifesting in reduced synaptic transmission and long-term plasticity in the hippocampus. In the adult, the behavioral and electrophysiological phenotypes in KRAS12V mice can be temporarily reverted by inhibiting gamma-aminobutyric acid signaling but not by a Ras-ERK blockade. Importantly, the synaptogenesis phenotype can be rescued by a treatment at the developmental stage with Ras-ERK inhibitors. CONCLUSIONS: These data demonstrate a novel mechanism underlying inhibitory synaptogenesis and provide new insights in understanding mental dysfunctions associated to RASopathies.
Assuntos
Encéfalo/fisiologia , Neurônios GABAérgicos/fisiologia , Deficiência Intelectual/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sinapses/fisiologia , Animais , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Modelos Animais de Doenças , Neurônios GABAérgicos/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiologia , Potenciais Pós-Sinápticos Inibidores , Potenciação de Longa Duração , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores de GABA/metabolismo , Comportamento Social , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
The ability of cocaine to produce lasting neural adaptations in mesocorticolimbic brain regions is thought to promote drug seeking and facilitate addiction in humans. The Ras-controlled Raf-MEK-ERK protein kinase signaling cascade has been implicated in the behavioral and neurobiological actions of cocaine in animals. However, these pharmacological studies have not been able to determine the specific role of the two predominant isoforms of ERK (ERK1 and ERK2) in these processes. We report here that deletion of the ERK1 isoform, which leads to increased ERK2 stimulus-dependent signaling, facilitates the development of cocaine-induced psychomotor sensitization and the acquisition of a cocaine conditioned place preference. Conversely, pharmacological blockade of ERK signaling attenuates the development of psychomotor sensitization to cocaine. Finally, cocaine-evoked gene expression in mesocorticolimbic brain regions is potentiated in ERK1-deficient mice. Thus, alterations in ERK signaling influence both the neurobiological impact of cocaine and its ability to produce enduring forms of drug experience-dependent behavioral plasticity. Our results suggest that enhanced ERK2 signaling following repeated drug exposure may facilitate the development of forms of cocaine-induced plasticity that contribute to addiction.
Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Transtornos Relacionados ao Uso de Cocaína/genética , Cocaína/efeitos adversos , Genes Precoces/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/genética , Animais , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Transtornos Relacionados ao Uso de Cocaína/enzimologia , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/enzimologia , Corpo Estriado/fisiopatologia , Modelos Animais de Doenças , Inibidores da Captação de Dopamina/efeitos adversos , Encefalinas/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes Precoces/genética , Sistema Límbico/efeitos dos fármacos , Sistema Límbico/enzimologia , Sistema Límbico/fisiopatologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/enzimologia , Área Tegmentar Ventral/fisiopatologiaRESUMO
Ras-ERK signalling in the brain plays a central role in drug addiction. However, to date, no clinically relevant inhibitor of this cascade has been tested in experimental models of addiction, a necessary step toward clinical trials. We designed two new cell-penetrating peptides - RB1 and RB3 - that penetrate the brain and, in the micromolar range, inhibit phosphorylation of ERK, histone H3 and S6 ribosomal protein in striatal slices. Furthermore, a screening of small therapeutics currently in clinical trials for cancer therapy revealed PD325901 as a brain-penetrating drug that blocks ERK signalling in the nanomolar range. All three compounds have an inhibitory effect on cocaine-induced ERK activation and reward in mice. In particular, PD325901 persistently blocks cocaine-induced place preference and accelerates extinction following cocaine self-administration. Thus, clinically relevant, systemically administered drugs that attenuate Ras-ERK signalling in the brain may be valuable tools for the treatment of cocaine addiction.
Assuntos
Comportamento Animal/efeitos dos fármacos , Cocaína/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , Proteínas ras/antagonistas & inibidores , Animais , Benzamidas/metabolismo , Peptídeos Penetradores de Células/metabolismo , Corpo Estriado/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/metabolismo , CamundongosRESUMO
OBJECTIVE: Recent findings have shown that pharmacogenetic manipulations of the Ras-ERK pathway provide a therapeutic means to tackle l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID). First, we investigated whether a prolonged l-DOPA treatment differentially affected ERK signaling in medium spiny neurons of the direct pathway (dMSNs) and in cholinergic aspiny interneurons (ChIs) and assessed the role of Ras-GRF1 in both subpopulations. Second, using viral-assisted technology, we probed Ras-GRF1 and Ras-GRF2 as potential targets in this pathway. We investigated how selective blockade of striatal Ras-GRF1 or Ras-GRF2 expression impacted on LID (induction, maintenance, and reversion) and its neurochemical correlates. METHODS: We used both Ras-GRF1 knockout mice and lentiviral vectors (LVs) delivering short-hairpin RNA sequences (shRNAs) to obtain striatum-specific gene knockdown of Ras-GRF1 and Ras-GRF2. The consequences of these genetic manipulations were evaluated in the 6-hydroxydopamine mouse model of Parkinson's disease. Escalating doses of l-DOPA were administered and then behavioral analysis with immunohistochemical assays and in vivo microdialysis were performed. RESULTS: Ras-GRF1 was found essential in controlling ERK signaling in dMSNs, but its ablation did not prevent ERK activation in ChIs. Moreover, striatal injection of LV-shRNA/Ras-GRF1 attenuated dyskinesia development and ERK-dependent signaling, whereas LV-shRNA/Ras-GRF2 was without effect, ruling out the involvement of Ras-GRF2 in LID expression. Accordingly, Ras-GRF1 but not Ras-GRF2 striatal gene-knockdown reduced l-DOPA-induced GABA and glutamate release in the substantia nigra pars reticulata, a neurochemical correlate of dyskinesia. Finally, inactivation of Ras-GRF1 provided a prolonged anti-dyskinetic effect for up to 7 weeks and significantly attenuated symptoms in animals with established LID. INTERPRETATION: Our results suggest that Ras-GRF1 is a promising target for LID therapy based on Ras-ERK signaling inhibition in the striatum.