RESUMO
In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin degradation results in loss of axonal function and eventual axonal degeneration. Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to remyelination of denuded axons occurs regularly in early stages of MS but halts as the pathology transitions into progressive MS. Pharmacological potentiation of endogenous OPC maturation and remyelination is now recognized as a promising therapeutic approach for MS. In this study, we analyzed the effects of modulating the Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective inhibitors of ROCK, on the transformation of OPCs into mature, myelinating oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent and human origin, that ROCK inhibition in OPCs results in a significant generation of branches and cell processes in early differentiation stages, followed by accelerated production of myelin protein as an indication of advanced maturation. Furthermore, inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons and remyelination in rat cerebellar tissue explants previously demyelinated with lysolecithin. Our findings indicate that by direct inhibition of this signaling molecule, the OPC differentiation program is activated resulting in morphological and functional cell maturation, myelin formation, and regeneration. Altogether, we show evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the induction of remyelination in demyelinating pathologies.
Assuntos
Diferenciação Celular/fisiologia , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Gânglios Espinais/citologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/fisiologia , Oligodendroglia , Nervo Óptico/citologia , Ratos , Células-Tronco , Fatores de Tempo , Quinases Associadas a rho/metabolismoRESUMO
Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme within the amidase-signature family. It catalyzes the hydrolysis of several endogenous biologically active lipids, including anandamide (arachidonoyl ethanolamide), oleoyl ethanolamide, and palmitoyl ethanolamide. These endogenous FAAH substrates have been shown to be involved in a variety of physiological and pathological processes, including synaptic regulation, regulation of sleep and feeding, locomotor activity, pain and inflammation. Here we describe the biochemical and biological properties of a potent and selective FAAH inhibitor, 4-(3-phenyl-[1,2,4]thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide (JNJ-1661010). The time-dependence of apparent IC(50) values at rat and human recombinant FAAH, dialysis and mass spectrometry data indicate that the acyl piperazinyl fragment of JNJ-1661010 forms a covalent bond with the enzyme. This bond is slowly hydrolyzed, with release of the piperazinyl fragment and recovery of enzyme activity. The lack of inhibition observed in a rat liver esterase assay suggests that JNJ-1661010 is not a general esterase inhibitor. JNJ-1661010 is >100-fold preferentially selective for FAAH-1 when compared to FAAH-2. JNJ-1661010 dose-dependently increases arachidonoyl ethanolamide, oleoyl ethanolamide, and palmitoyl ethanolamide in the rat brain. The compound attenuates tactile allodynia in the rat mild thermal injury model of acute tissue damage and in the rat spinal nerve ligation (Chung) model of neuropathic pain. JNJ-1661010 also diminishes thermal hyperalgesia in the inflammatory rat carrageenan paw model. These data suggest that FAAH inhibitors with modes of action similar to JNJ-1661010 may be useful clinically as broad-spectrum analgesics.
Assuntos
Amidoidrolases/antagonistas & inibidores , Analgésicos/farmacologia , Encéfalo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Dor/prevenção & controle , Piperazinas/farmacologia , Tiadiazóis/farmacologia , Amidas , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Encéfalo/enzimologia , Carragenina , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endocanabinoides , Etanolaminas , Temperatura Alta , Humanos , Hidrólise , Isoenzimas , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuralgia/etiologia , Neuralgia/prevenção & controle , Ácidos Oleicos/metabolismo , Dor/etiologia , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Ácidos Palmíticos/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Proteínas Recombinantes/antagonistas & inibidoresRESUMO
A series of thiadiazolopiperazinyl aryl urea fatty acid amide hydrolase (FAAH) inhibitors is described. The molecules were found to inhibit the enzyme by acting as mechanism-based substrates, forming a covalent bond with Ser241. SAR and PK properties are presented.
Assuntos
Amidoidrolases/antagonistas & inibidores , Piperazinas/farmacologia , Tiadiazóis/farmacologia , Ureia/análogos & derivados , Ureia/farmacologia , Amidoidrolases/deficiência , Amidoidrolases/genética , Animais , Camundongos , Camundongos Knockout , Piperazinas/química , Piperazinas/farmacocinética , Tiadiazóis/química , Tiadiazóis/farmacocinética , Ureia/química , Ureia/farmacocinéticaRESUMO
The enzyme Fatty Acid Amide Hydrolase (FAAH) is a key regulator of the endogenous levels of a family of biologically active lipid mediators, the fatty acid amides. These include anandamide, oleoyl ethanolamide and palmitoyl ethanolamide, and their effects are mediated by a variety of downstream targets including cannabinoid receptors and peroxisome proliferator-activated receptors (PPARs). Activation of both of these may have anti-inflammatory and neuroprotective effects. Levels of all three mediators are low in normal nervous tissue, but substantially elevated in mice lacking FAAH as a result of genetic deletion. There is a long anecdotal history of cannabis use by patients suffering from multiple sclerosis, and preclinical studies have indicated beneficial effects of cannabinoid receptor stimulation on both long-term outcome and acute muscle spasm in rodent models of multiple sclerosis (experimental autoimmune encephalitis; EAE). Thus far no report has appeared on the effect of inhibition of FAAH on the progression of EAE. Using a chronic mouse EAE model, we present data indicating that mice lacking FAAH experience an initial inflammatory phase of EAE similar in severity to wild type controls, but exhibited a more substantial clinical remission compared to wild type mice.
Assuntos
Amidoidrolases/deficiência , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/fisiopatologia , Animais , Complexo CD3/metabolismo , Doença Crônica , Doenças Desmielinizantes/etiologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Glicoproteínas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , Medula Espinal/patologia , Linfócitos T/patologia , Linfócitos T/fisiologia , Fatores de TempoRESUMO
Anandamide and other fatty acid amides are metabolised by the enzyme fatty acid amide hydrolase (FAAH), which thereby regulates their endogenous levels. Here we demonstrate that cultured rat cortical microglia express FAAH at low levels. The potent FAAH inhibitor URB597 reduced the LPS stimulated microglial expression of cyclo-oxygenase 2 and inducible nitric oxide, with concomitant attenuation of the release of PGE2 and NO. Additional of supplemental exogenous anandamide did not increase the magnitude of attenuation of mediator release. The effect of URB597 on LPS stimulated PGE2 release was not blocked by selective CB1 or CB2 receptor antagonists.
Assuntos
Amidoidrolases/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Amidoidrolases/fisiologia , Animais , Benzamidas/farmacologia , Carbamatos/farmacologia , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Microglia/enzimologia , Microglia/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
FTY720 is a prodrug for FTY-phosphate, an agonist at four of the five known receptors for sphingosine-1-phosphate (S1P). We show that administration of either FTY720 or FTY-P to SJL mice with established relapsing-remitting experimental autoimmune encephalitis (EAE) results in a rapid and sustained improvement in their clinical status, and a reversal of changes in expression of mRNAs encoding some myelin proteins and inflammatory mediators. EAE produced by adoptively transferring lymph node cells from immunized mice to naïve hosts is similarly ameliorated by FTY-P. Treatment with FTY-P is accompanied by a dose-responsive peripheral lymphopoenia.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Imunossupressores/uso terapêutico , Propilenoglicóis/uso terapêutico , Receptores Acoplados a Proteínas G/agonistas , Animais , Antineoplásicos/uso terapêutico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Cloridrato de Fingolimode , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunossupressores/farmacologia , Interferon gama/genética , Interferon gama/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/fisiologia , Linfopenia/tratamento farmacológico , Linfopenia/etiologia , Camundongos , Camundongos Endogâmicos , Mitoxantrona/uso terapêutico , Dados de Sequência Molecular , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Proteína Proteolipídica de Mielina , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Fragmentos de Peptídeos , Propilenoglicóis/farmacologia , RNA Mensageiro/biossíntese , Receptores de Lisofosfolipídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Esfingosina/análogos & derivados , Fatores de TempoRESUMO
Reactive gliosis is an aspect of neural plasticity and growth factor (GF) stimulation of astrocytes in vitro is widely regarded as a model system to study astrocyte plasticity. Astrocytes express receptors for several ligands including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), agonists for the G-protein-coupled lysophospholipid receptors (lpRs). Activation of lpRs by LPA or S1P leads to multiple pharmacological effects including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK), release of arachidonic acid, and induces mitogenesis. Treatment of astrocytes in vitro with a growth factor cocktail (containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF] and insulin) led to a marked attenuation of lpR-induced PI hydrolysis. In contrast, under identical conditions, GF treatment led to marked potentiation of PI hydrolysis downstream of activation of another abundantly expressed G-protein coupled receptor, mGluR5. Quantitative gene expression analysis of GF-treated or control astrocytes by TaqMan RT-PCR indicated that GF treatment did not change gene expression of lpa1 and s1p1, but increased gene expression of s1p5 which is expressed at very low levels in basal conditions. These results suggest that GF differentially affected PLC activation downstream of mGluR5 versus lpR activation and that the changes in mRNA levels of lpRs do not account for marked attenuation of agonist-induced phosphoinositide turnover.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Substâncias de Crescimento/farmacologia , Fosfatidilinositóis/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Fosfatos de Inositol/metabolismo , Insulina/farmacologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5 , Receptores de Lisofosfolipídeos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
We used a simple commercial magnetic immunobead method for the preparation of acutely isolated microglial cells from postnatal days 1-3 rat brain. With the exception of a 15 min enzyme incubation, all stages are carried out at 4 degrees C, minimizing the opportunity for changes in gene expression during the isolation to be reflected in changes in accumulated mRNA. The composition of the isolated cells was compared with that of microglial cultures prepared by conventional tissue culture methods, and the purity of microglia was comparable between the two preparations. RT-PCR analysis of several genes related to inflammatory products indicated that the acutely prepared cells were in a less activated condition than the conventionally tissue cultured cells. We examined the pattern of expression of receptors for lysophosphatidic acid (lpa) and sphingosine-1-phosphate (S1P) using quantitative real-time PCR (TaqMan PCR) techniques. mRNA for LPA1, S1P1, S1P2, S1P3 and S1P5 was detected in these preparations, but the levels of the different receptor mRNAs varied according to the state of activation of the cells. mRNA for LPA3 was only detected significantly in cultured cell after lipopolysaccharide (LPS) stimulation, being almost absent in cultured microglia and undetectable in the acutely isolated preparations. The levels of mRNA of LPA1 and S1P receptors was reduced by overnight exposure to S1P, while the same treatment significantly up-regulated the level of LPA3 mRNA.