RESUMO
Extracellular molecular cues guide migrating growth cones along specific routes during development of axon tracts. Such processes rely on asymmetric elevation of cytosolic Ca(2+) concentrations across the growth cone that mediates its attractive or repulsive turning toward or away from the side with Ca(2+) elevation, respectively. Downstream of these Ca(2+) signals, localized activation of membrane trafficking steers the growth cone bidirectionally, with endocytosis driving repulsion and exocytosis causing attraction. However, it remains unclear how Ca(2+) can differentially regulate these opposite membrane-trafficking events. Here, we show that growth cone turning depends on localized imbalance between exocytosis and endocytosis and identify Ca(2+)-dependent signaling pathways mediating such imbalance. In embryonic chicken dorsal root ganglion neurons, repulsive Ca(2+) signals promote clathrin-mediated endocytosis through a 90 kDa splice variant of phosphatidylinositol-4-phosphate 5-kinase type-1γ (PIPKIγ90). In contrast, attractive Ca(2+) signals facilitate exocytosis but suppress endocytosis via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (Cdk5) that can inactivate PIPKIγ90. Blocking CaMKII or Cdk5 leads to balanced activation of both exocytosis and endocytosis that causes straight growth cone migration even in the presence of guidance signals, whereas experimentally perturbing the balance restores the growth cone's turning response. Remarkably, the direction of this resumed turning depends on relative activities of exocytosis and endocytosis, but not on the type of guidance signals. Our results suggest that navigating growth cones can be redirected by shifting the imbalance between exocytosis and endocytosis, highlighting the importance of membrane-trafficking imbalance for axon guidance and, possibly, for polarized cell migration in general.
Assuntos
Endocitose/fisiologia , Exocitose/fisiologia , Cones de Crescimento/fisiologia , Neurônios/citologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Células Cultivadas , Embrião de Galinha , Clatrina/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Gânglios Espinais/citologia , Cones de Crescimento/efeitos dos fármacos , Glicoproteína Associada a Mielina/farmacologia , Neurônios/efeitos dos fármacos , Organofosfonatos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fotólise , Piperazinas/farmacologia , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Negatively targeting the tumor suppressor and phosphoinositide phosphatase PTEN (phosphatase and tensin homologue) promotes axon regrowth after injury. How PTEN functions in axon guidance has remained unknown. Here we report the differential role of PTEN in chemotactic guidance of axonal growth cones. Down-regulating PTEN expression in Xenopus laevis spinal neurons selectively abolished growth cone chemorepulsion but permitted chemoattraction. These findings persisted during cAMP-dependent switching of turning behaviors. Live cell imaging using a GFP biosensor revealed rapid PTEN-dependent depression of phosphatidylinositol 3,4,5-trisphosphate levels in the growth cone induced by the repellent myelin-associated glycoprotein. Moreover, down-regulating PTEN expression blocked negative remodeling of ß1-integrin adhesions triggered by myelin-associated glycoprotein, yet permitted integrin clustering by a positive chemotropic treatment. Thus, PTEN negatively regulates growth cone phosphatidylinositol 3,4,5-trisphosphate levels and mediates chemorepulsion, whereas chemoattraction is PTEN-independent. Regenerative therapies targeting PTEN may therefore suppress growth cone repulsion to soluble cues while permitting attractive guidance, an essential feature for re-forming functional neural circuits.
Assuntos
Quimiotaxia , Cones de Crescimento/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Quimiotaxia/efeitos dos fármacos , Análise por Conglomerados , AMP Cíclico/farmacologia , Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Cones de Crescimento/efeitos dos fármacos , Integrina beta1/metabolismo , Glicoproteína Associada a Mielina/farmacologia , Fosfatos de Fosfatidilinositol/metabolismoRESUMO
Demyelinating diseases, such as multiple sclerosis, are characterized by the loss of the myelin sheath around neurons, owing to inflammation and gliosis in the central nervous system (CNS). Current treatments therefore target anti-inflammatory mechanisms to impede or slow disease progression. The identification of a means to enhance axon myelination would present new therapeutic approaches to inhibit and possibly reverse disease progression. Previously, LRR and Ig domain-containing, Nogo receptor-interacting protein (LINGO-1) has been identified as an in vitro and in vivo negative regulator of oligodendrocyte differentiation and myelination. Here we show that loss of LINGO-1 function by Lingo1 gene knockout or by treatment with an antibody antagonist of LINGO-1 function leads to functional recovery from experimental autoimmune encephalomyelitis. This is reflected biologically by improved axonal integrity, as confirmed by magnetic resonance diffusion tensor imaging, and by newly formed myelin sheaths, as determined by electron microscopy. Antagonism of LINGO-1 or its pathway is therefore a promising approach for the treatment of demyelinating diseases of the CNS.
Assuntos
Axônios/fisiologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Proteínas de Membrana/antagonistas & inibidores , Bainha de Mielina/fisiologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Traumatismos da Medula Espinal/terapia , Animais , Axônios/diagnóstico por imagem , Axônios/ultraestrutura , Encefalomielite Autoimune Experimental/patologia , Injeções Espinhais , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Proteínas da Mielina , Bainha de Mielina/ultraestrutura , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Associada a Mielina/farmacologia , Glicoproteína Mielina-Oligodendrócito , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/fisiologia , Ratos , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Tomografia Computadorizada por Raios XRESUMO
The ability of IFN-ß to induce IL-10 production from innate immune cells is important for its anti-inflammatory properties and is believed to contribute to its therapeutic value in treating multiple sclerosis patients. In this study, we identified that IFN-ß stimulates IL-10 production by activating the JAK1- and PI3K-signaling pathways. JAK1 activity was required for IFN-ß to activate PI3K and Akt1 that resulted in repression of glycogen synthase kinase 3 (GSK3)-ß activity. IFN-ß-mediated suppression of GSK3-ß promoted IL-10, because IL-10 production by IFN-ß-stimulated dendritic cells (DC) expressing an active GSK3-ß knockin was severely reduced, whereas pharmacological or genetic inhibition of GSK3-ß augmented IL-10 production. IFN-ß increased the phosphorylated levels of CREB and STAT3 but only CREB levels were affected by PI3K. Also, a knockdown in CREB, but not STAT3, affected the capacity of IFN-ß to induce IL-10 from DC. IL-10 production by IFN-ß-stimulated DC was shown to suppress IFN-γ and IL-17 production by myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, and this IL-10-dependent anti-inflammatory effect was enhanced by directly targeting GSK3 in DC. These findings highlight how IFN-ß induces IL-10 production and the importance that IL-10 plays in its anti-inflammatory properties, as well as identify a therapeutic target that could be used to increase the IL-10-dependent anti-inflammatory properties of IFN-ß.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Interferon beta/fisiologia , Interleucina-10/biossíntese , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Células Dendríticas/enzimologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Epitopos de Linfócito T/imunologia , Técnicas de Introdução de Genes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Interleucina-10/fisiologia , Interleucina-17/antagonistas & inibidores , Interleucina-17/biossíntese , Líquido Intracelular/enzimologia , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Janus Quinase 1/metabolismo , Janus Quinase 1/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas da Mielina , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Associada a Mielina/farmacologia , Glicoproteína Mielina-Oligodendrócito , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Transdução de Sinais/imunologiaRESUMO
The neuropeptide vasoactive intestinal peptide (VIP) has been shown to inhibit macrophage proinflammatory actions, promote a positive Th2/Th1 balance, and stimulate regulatory T-cell production. The fact that this peptide is highly efficacious in animal models of inflammatory diseases such as collagen-induced arthritis and experimental autoimmune encephalomyelitis (EAE) suggests that the endogenous peptide might normally provide protection against such pathologies. We thus studied the response of VIP-deficient (i.e., VIP KO) mice to myelin oligodendrocyte protein-induced EAE. Surprisingly, VIP KO mice were almost completely resistant to EAE, with delayed onset and mild or absent clinical profile. Despite this, flow cytometric analyses and antigen-rechallenge experiments indicated that myelin oligodendrocyte protein-treated VIP KO mice exhibited robust Th1/Th17 cell inductions and antigen-specific proliferation and cytokine responses. Moreover, adoptive transfer of lymphocytes from immunized VIP KO mice to WT recipients resulted in full-blown EAE, supporting their encephalitogenic potential. In contrast, transfer of encephalitogenic WT cells to VIP KO hosts did not produce EAE, suggesting that loss of VIP specifically affected the effector phase of the disease. Histological analyses indicated that CD4 T cells entered the meningeal and perivascular areas of VIP-deficient mice, but that parenchymal infiltration was strongly impaired. Finally, VIP pretreatment of VIP KO mice before immunization was able to restore their sensitivity to EAE. These results indicate that VIP plays an unanticipated permissive and/or proinflammatory role in the propagation of the inflammatory response in the CNS, a finding with potential therapeutic relevance in autoimmune neuroinflammatory diseases such as multiple sclerosis.
Assuntos
Movimento Celular/imunologia , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/imunologia , Linfócitos T/imunologia , Peptídeo Intestinal Vasoativo/deficiência , Peptídeo Intestinal Vasoativo/imunologia , Animais , Doenças Autoimunes/etiologia , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/etiologia , Inflamação/etiologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Glicoproteína Associada a Mielina/farmacologia , Células Th1 , Células Th17RESUMO
Decreased expression of prosurvival and progrowth-stimulatory pathways, in addition to an environment that inhibits neuronal growth, contribute to the limited regenerative capacity in the central nervous system following injury or neurodegeneration. Membrane/lipid rafts, plasmalemmal microdomains enriched in cholesterol, sphingolipids, and the protein caveolin (Cav) are essential for synaptic development/stabilization and neuronal signaling. Cav-1 concentrates glutamate and neurotrophin receptors and prosurvival kinases and regulates cAMP formation. Here, we show that primary neurons that express a synapsin-driven Cav-1 vector (SynCav1) have increased raft formation, neurotransmitter and neurotrophin receptor expression, NMDA- and BDNF-mediated prosurvival kinase activation, agonist-stimulated cAMP formation, and dendritic growth. Moreover, expression of SynCav1 in Cav-1 KO neurons restores NMDA- and BDNF-mediated signaling and enhances dendritic growth. The enhanced dendritic growth occurred even in the presence of inhibitory cytokines (TNFα, IL-1ß) and myelin-associated glycoproteins (MAG, Nogo). Targeting of Cav-1 to neurons thus enhances prosurvival and progrowth signaling and may be a novel means to repair the injured and neurodegenerative brain.
Assuntos
Caveolina 1/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colesterol/metabolismo , Citocinas/farmacologia , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Associada a Mielina/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sinapsinas/metabolismoRESUMO
The second messenger cyclic adenosine monophosphate (cAMP) plays a pivotal role in axonal growth and guidance, but its downstream mechanisms remain elusive. In this study, we report that type II protein kinase A (PKA) is highly enriched in growth cone filopodia, and this spatial localization enables the coupling of cAMP signaling to its specific effectors to regulate guidance responses. Disrupting the localization of PKA to filopodia impairs cAMP-mediated growth cone attraction and prevents the switching of repulsive responses to attraction by elevated cAMP. Our data further show that PKA targets protein phosphatase-1 (PP1) through the phosphorylation of a regulatory protein inhibitor-1 (I-1) to promote growth cone attraction. Finally, we find that I-1 and PP1 mediate growth cone repulsion induced by myelin-associated glycoprotein. These findings demonstrate that the spatial localization of type II PKA to growth cone filopodia plays an important role in the regulation of growth cone motility and guidance by cAMP.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Cones de Crescimento/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/enzimologia , Animais , Cálcio/metabolismo , Proteína Quinase Tipo II Dependente de AMP Cíclico , Ativação Enzimática/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Glicoproteína Associada a Mielina/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1 , Transporte Proteico/efeitos dos fármacos , Proteínas/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , XenopusRESUMO
In the adult mammalian CNS, the growth inhibitors oligodendrocyte-myelin glycoprotein (OMgp) and the reticulon RTN4 (Nogo) are broadly expressed in oligodendrocytes and neurons. Nogo and OMgp complex with the neuronal cell surface receptors Nogo receptor-1 (NgR1) and paired Ig-like receptor-B (PirB) to regulate neuronal morphology. In the healthy CNS, NgR1 regulates dendritic spine shape and attenuates activity-driven synaptic plasticity at Schaffer collateral-CA1 synapses. Here, we examine whether Nogo and OMgp influence functional synaptic plasticity, the efficacy by which synaptic transmission occurs. In acute hippocampal slices of adult mice, Nogo-66 and OMgp suppress NMDA receptor-dependent long-term potentiation (LTP) when locally applied to Schaffer collateral-CA1 synapses. Neither Nogo-66 nor OMgp influences basal synaptic transmission or paired-pulse facilitation, a form of short-term synaptic plasticity. PirB(-/-) and NgR1(-/-) single mutants and NgR1(-/-);PirB(-/-) double mutants show normal LTP, indistinguishable from wild-type controls. In juvenile mice, LTD in NgR1(-/-), but not PirB(-/-), slices is absent. Mechanistic studies revealed that Nogo-66 and OMgp suppress LTP in an NgR1-dependent manner. OMgp inhibits LTP in part through PirB but independently of p75. This suggests that NgR1 and PirB participate in ligand-dependent inhibition of synaptic plasticity. Loss of NgR1 leads to increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling intermediates known to regulate neuronal growth and synaptic function. In primary cortical neurons, BDNF elicited phosphorylation of AKT and p70S6 kinase is attenuated in the presence of myelin inhibitors. Collectively, we provide evidence that mechanisms of neuronal growth inhibition and inhibition of synaptic strength are related. Thus, myelin inhibitors and their receptors may coordinate structural and functional neuronal plasticity in CNS health and disease.
Assuntos
Regulação para Baixo/fisiologia , Proteínas da Mielina/fisiologia , Glicoproteína Associada a Mielina/fisiologia , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Linhagem Celular , Regulação para Baixo/genética , Proteínas Ligadas por GPI , Humanos , Potenciação de Longa Duração/genética , Potenciação de Longa Duração/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Glicoproteína Associada a Mielina/genética , Glicoproteína Associada a Mielina/farmacologia , Glicoproteína Mielina-Oligodendrócito , Inibição Neural/genética , Plasticidade Neuronal/genética , Proteínas Nogo , Receptor Nogo 1 , Ratos , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/metabolismo , Receptores Imunológicos/fisiologiaRESUMO
An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1's protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood-brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.
Assuntos
Arginase/genética , AMP Cíclico/metabolismo , Isoflavonas/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Regiões Promotoras Genéticas/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Arginase/metabolismo , Células CHO , Células Cultivadas , Cerebelo/citologia , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Proteína GAP-43/metabolismo , Gânglios Espinais/citologia , Ensaios de Triagem em Larga Escala/métodos , Hipocampo/citologia , Masculino , Glicoproteína Associada a Mielina/farmacologia , Regeneração Nervosa/fisiologia , Neurônios/citologia , Doenças do Nervo Óptico/tratamento farmacológico , Doenças do Nervo Óptico/patologia , Estresse Oxidativo/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
NogoA, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein are CNS myelin molecules that bind to the neuronal Nogo-66 receptor (NgR) and inhibit axon growth. The NgR antagonist, soluble NgR1-Fc protein (sNgR-Fc), facilitates axon regeneration by neutralizing the inhibitory effects of myelin proteins in experimental models of CNS injury. Here we aim to investigate the effect of sNgR-Fc on the proliferation of neural progenitor cells (NPCs). The hippocampus cells of embryonic rats were isolated and cultured in vitro. The expression of nestin, ßIII-Tubulin, GFAP and Nogo-A on these cells was observed using immunocytochemistry. In order to investigate the effect on proliferation of NPCs, sNgR-Fc, MAG-Fc chimera and Notch1 blocker were added respectively. The total cell number for the proliferated NPCs was counted. BrdU was applied and the rate of proliferating cells was examined. The level of Notch1 was analyzed using Western blotting. We identified that NogoA is expressed in NPCs. sNgR-Fc significantly enhanced the proliferation of NPCs in vitro as indicated by BrdU labeling and total cell count. This proliferation effect was abolished by the administration of MAG suggesting specificity. In addition, we demonstrate that sNgR-Fc is a potent activator for Notch1 and Notch1 antagonist reversed the effect of sNgR-Fc on NPC proliferation. Our results suggest that sNgR-Fc may modulate Nogo activity to induce NPC proliferation via the Notch pathway.
Assuntos
Hipocampo/citologia , Proteínas da Mielina/metabolismo , Glicoproteína Associada a Mielina/farmacologia , Receptor Notch1/fisiologia , Proteínas Recombinantes de Fusão/farmacologia , Células-Tronco/fisiologia , Animais , Proliferação de Células/efeitos dos fármacos , Proteínas da Mielina/biossíntese , Glicoproteína Mielina-Oligodendrócito , Proteínas Nogo , Ratos , Células-Tronco/efeitos dos fármacosRESUMO
Genes controlling immunopathologic diseases of differing etiopathology may also influence susceptibility to autoimmune disease. B10.D1-H2(q)/SgJ mice with a 2538 G-->A missense mutation in the tyrosine kinase-2 gene (Tyk2) are susceptible to Toxoplasma gondii yet resistant to autoimmune arthritis, unlike the wild-type B10.Q/Ai substrain. To understand whether Tyk2 is also important in a second autoimmune model, experimental allergic encephalomyelitis (EAE) was induced in B10.D1-H2(q)/SgJ (Tyk2(A)) and B10.Q/Ai (Tyk2(G)) mice with the myelin oligodendrocyte glycoprotein peptide 79-96. B10.D1-H2(q)/SgJ mice were resistant to EAE whereas B10.Q/Ai mice were susceptible, and a single copy of the Tyk2(G) allele conferred EAE susceptibility in F(1) hybrids. Furthermore, EAE resistance in B10.D1-H2(q)/SgJ mice was overridden when pertussis toxin (PTX) was used to mimic the effects of environmental factors derived from infectious agents. Numerous cytokines and chemokines were increased when PTX was included in the immunization protocol. However, only RANTES, IL-6, and IFN-gamma increased significantly with both genetic compensation and PTX treatment. These data indicate that Tyk2 is a shared autoimmune disease susceptibility gene whose genetic contribution to disease susceptibility can be modified by environmental factors. Single nucleotide polymorphisms like the one that distinguishes Tyk2 alleles are of considerable significance given the potential role of gene-by-environment interactions in autoimmune disease susceptibility.
Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único/genética , TYK2 Quinase/genética , TYK2 Quinase/metabolismo , Alelos , Animais , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Camundongos , Proteínas da Mielina , Glicoproteína Associada a Mielina/farmacologia , Glicoproteína Mielina-Oligodendrócito , Toxina Pertussis/farmacologiaRESUMO
One of the major barriers to successful axon regeneration in the adult CNS is the presence of inhibitory molecules that originate from the myelin sheath and glial scar. So far, only a small number of pharmacological compounds have exhibited functional activity against CNS inhibitors in promoting axon regeneration after injury. To search for novel compounds that enhance neurite outgrowth in vitro, we initiated a screen of a collection of natural products. We identified four compounds with the potential to promote growth over a myelin substrate. Of these, Amphotericin B (AmB) was shown to enhance neurite outgrowth and antagonize activities of major myelin associated inhibitors and glial-scar-derived chondroitin sulfate proteoglycans. AmB was found to activate Akt and thereby suppress the activity of glycogen synthase kinase 3 beta. Also, a cell permeable peptide that inhibits Akt activity was shown to block the effect of AmB in promoting axonal growth, while another peptide that increases Akt activity stimulated axonal growth in the presence of the myelin associated inhibitors. Our results suggest that AmB can promote neurite outgrowth over a wide range of inhibitory substrates via a mechanism that involves activation of Akt.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Axônios/efeitos dos fármacos , Produtos Biológicos/farmacologia , Neurônios/efeitos dos fármacos , Proteína Oncogênica v-akt/metabolismo , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/antagonistas & inibidores , Proteoglicanas de Sulfatos de Condroitina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Glicoproteína Associada a Mielina/antagonistas & inibidores , Glicoproteína Associada a Mielina/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Análise de Componente Principal , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Monocyte chemoattractant protein (MCP)-1 plays a critical role in innate immunity by directing the migration of monocytes into inflammatory sites. Recent data indicated a function for this chemokine in adaptive immunity as a regulator of T cell commitment to T helper cell type 2 (Th2) effector function. Studies in a Th1-dependent animal model, experimental autoimmune encephalomyelitis (EAE), showed that MCP-1 was highly expressed in the central nervous system (CNS) of affected rodents, and MCP-1 antibodies could block relapses of the disease. Mice deficient for the major MCP-1 receptor, CC chemokine receptor (CCR)2, did not develop EAE after active immunization but generated effector cells that could transfer the disease to naive wild-type recipients. We analyzed EAE in mice deficient for MCP-1 to define the relevant ligand for CCR2, which responds to murine MCP-1, MCP-2, MCP-3, and MCP-5. We found that C57BL/6 MCP-1-null mice were markedly resistant to EAE after active immunization, with drastically impaired recruitment of macrophages to the CNS, yet able to generate effector T cells that transferred severe disease to naive wild-type recipients. By contrast, adoptive transfer of primed T cells from wild-type mice into naive MCP-1-null recipients did not mediate clinical EAE. On the SJL background, disruption of the MCP-1 gene produced a milder EAE phenotype with diminished relapses that mimicked previous findings using anti-MCP-1 antibodies. There was no compensatory upregulation of MCP-2, MCP-3, or MCP-5 in MCP-1-null mice with EAE. These results indicated that MCP-1 is the major CCR2 ligand in mice with EAE, and provided an opportunity to define the role of MCP-1 in EAE. Compared with wild-type littermates, MCP-1-/- mice exhibited reduced expression of interferon gamma in draining lymph node and CNS and increased antigen-specific immunoglobulin G1 antibody production. Taken together, these data demonstrate that MCP-1 is crucial for Th1 immune responses in EAE induction and that macrophage recruitment to the inflamed CNS target organ is required for primed T cells to execute a Th1 effector program in EAE.
Assuntos
Quimiocina CCL2/imunologia , Citocinas , Encefalomielite Autoimune Experimental/imunologia , Macrófagos/imunologia , Células Th1/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD11/genética , Complexo CD3/genética , Antígenos CD4/genética , Antígenos CD8/genética , Divisão Celular , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Quimiocina CCL2/genética , Quimiocina CCL4 , Quimiocina CCL7 , Quimiocina CCL8 , Quimiocina CXCL10 , Quimiocinas CXC/biossíntese , Expressão Gênica , Imunidade Inata , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-10/biossíntese , Interleucina-10/genética , Interleucina-4/biossíntese , Interleucina-4/genética , Proteínas Inflamatórias de Macrófagos/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Quimioatraentes de Monócitos/genética , Proteínas da Mielina , Proteína Proteolipídica de Mielina/farmacologia , Glicoproteína Associada a Mielina/farmacologia , Glicoproteína Mielina-Oligodendrócito , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Regulação para CimaRESUMO
BACKGROUND: Multiple sclerosis (MS) is the endpoint of a complex and still poorly understood process which results in inflammation, demyelination and axonal and neuronal degeneration. Since the first description of MS, psychological stress has been suggested to be one of the trigger factors in the onset and/or relapse of symptoms. However, data from animal models of MS, such as experimental autoimmune encephalomyelitis (EAE) are inconsistent and the effect of stress on EAE onset and severity depends on duration and time of application of the stress protocol and the underlying mechanisms. METHODS: Dark Agouti rats were inoculated with MOG/CFA to induce EAE, and an immobilisation stress protocol with two different durations (12 and 21 days, starting at the moment of MOG-inoculation) was applied in order to analyse the effect of stress on disease onset and neuroinflammation. RESULTS: Twelve days of stress exposure increased EAE clinical score in Dark Agouti rats. In addition, these animals presented higher levels of MMP-9 and proinflammatory PGE2 in spinal cord. In contrast, animals chronically exposed to stress (21 days) showed a significantly lower incidence of EAE clinical signs and reduced myelin loss, leukocyte infiltration and accumulation of inflammatory/oxidative mediators in spinal cord. Interestingly, chronically stressed animals showed a parallel increase in levels of the anti-inflammatory prostaglandin 15d-PGJ2, the main endogenous agonist of PPARγ. CONCLUSIONS: Our results demonstrate that, depending on duration, stress exposure elicits opposite effects on PGE2/15d-PGJ2 ratios in spinal cord of EAE-induced Dark Agouti rats. Further studies are needed to elucidate if these changes in prostaglandin balance are sufficient to mediate the differences in clinical score and inflammation here reported, and to establish the potential utility of pharmacological intervention in MS directed toward anti-inflammatory pathways.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Glicoproteína Associada a Mielina/farmacologia , Medula Espinal/patologia , Estresse Fisiológico , Análise de Variância , Animais , Western Blotting , Corticosterona/sangue , Dinoprostona/metabolismo , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Peroxidação de Lipídeos , Masculino , Proteínas da Mielina , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Peroxidase/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/metabolismo , Radioimunoensaio , Ratos , Restrição Física , Medula Espinal/imunologia , Medula Espinal/metabolismoRESUMO
IL-12Rß2(-/-) mice, which are unresponsive to IL-12, develop severe experimental autoimmune encephalomyelitis (EAE). The mechanisms for enhanced autoimmunity are incompletely understood. We report that in IL-12Rß2(-/-) mice, thymocytes undergo markedly accelerated maturation. This occurs at the transition from a double positive (DP) to a single positive (SP) phenotype, resulting in higher numbers of CD4 and CD8 SP cells, and to a lesser extent at the transition from double negative (DN) to DP cells. Accelerated maturation is observed in mice injected with anti-CD3 to mimic pre-T-cell receptor stimulation, and also in mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide to induce EAE.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Animais , Complexo CD3/imunologia , Células Cultivadas , Suscetibilidade a Doenças , Feminino , Interleucina-12/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Proteínas da Mielina , Glicoproteína Associada a Mielina/farmacologia , Glicoproteína Mielina-Oligodendrócito , Timo/imunologiaRESUMO
The p75 neurotrophin receptor (p75NTR) collaborates with the Nogo receptor (NgR) and LINGO-1 to activate RhoA in response to myelin-based growth inhibitors such as myelin-associated glycoprotein (MAG). In this issue of Neuron, Domeniconi et al., in a surprising turn, show that MAG induces intramembrane proteolysis (RIP) of p75NTR and find that p75NTR cleavage is required for MAG-induced RhoA activation and growth inhibition.
Assuntos
Glicoproteína Associada a Mielina/farmacologia , Neuritos/efeitos dos fármacos , Neurônios/citologia , Animais , Modelos Biológicos , Neuritos/metabolismo , Estrutura Terciária de Proteína/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The three known inhibitors of axonal regeneration present in myelin--MAG, Nogo, and OMgp--all interact with the same receptor complex to effect inhibition via protein kinase C (PKC)-dependent activation of the small GTPase Rho. The transducing component of this receptor complex is the p75 neurotrophin receptor. Here we show that MAG binding to cerebellar neurons induces alpha- and then gamma-secretase proteolytic cleavage of p75, in a protein kinase C-dependent manner, and that this cleavage is necessary for both activation of Rho and inhibition of neurite outgrowth.
Assuntos
Glicoproteína Associada a Mielina/farmacologia , Inibição Neural/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de Fator de Crescimento Neural/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Animais Recém-Nascidos , Ácido Aspártico Endopeptidases/farmacologia , Western Blotting/métodos , Células Cultivadas , Cerebelo/citologia , Cricetinae , Cricetulus , Interações Medicamentosas , Endopeptidases , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Membrana/metabolismo , Neuritos/fisiologia , Neuroblastoma , Neurônios/fisiologia , Oligopeptídeos/farmacologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Transfecção/métodos , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Myelin-associated glycoprotein (MAG) is a well-characterized axon growth inhibitor in the adult vertebrate nervous system. Several signals that play roles in inhibiting axon growth have been identified. Here, we report that soluble MAG induces activation of Rap1 in postnatal cerebellar granule neurons (CGNs) and dorsal root ganglion (DRG) neurons. The p75 receptor associates with activated Rap1 and is internalized in response to MAG. After MAG is applied to the distal axons of the sciatic nerves, the activated Rap1, internalized p75 receptor, and MAG are retrogradely trafficked via axons to the cell bodies of the DRG neurons. Rap1 activity is required for survival of the DRG neurons as well as CGNs when treated with MAG. The transport of the signaling complex containing the p75 receptor and Rap1 may play a role in the effect of MAG.
Assuntos
Glicoproteína Associada a Mielina/fisiologia , Neurônios/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Células Cultivadas , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Associada a Mielina/farmacologia , Neurônios/efeitos dos fármacos , Ratos , Proteínas RecombinantesRESUMO
Elucidating the mechanisms that regulate the survival and outgrowth of corticospinal tract (CST) neurons and other CNS tracts will be a key component in developing novel approaches for the treatment of central nervous system (CNS) disorders, including stroke, spinal cord injury (SCI), and motor neuron disease (MND). However, the in vivo complexities of these diseases make a systematic evaluation of potential therapeutics that directly affect corticospinal regeneration or survival very challenging. Here, we use Thy1.2 transgenic mice expressing yellow fluorescent protein (YFP) in postnatal day 8 (P8) corticospinal neurons, as a source of CST neurons that have already established synapses in the spinal cord, to assess factors that influence neurite outgrowth and survival of axotomized CST neurons. After culture, YFP-positive corticospinal neurons represent an enriched neuronal population over other glia and interneurons, survive, and extend processes over time. YFP-positive CST neurons also continue to express the corticospinal markers CTIP2 and Otx1. CST neurons display different degrees of axon extension, dendritic branch length and elaboration, and neurite elongation in response to neurotrophin-3 and ciliary neurotrophic factor, and an inhibitory outgrowth response when cultured on myelin-associated glycoprotein. Some CST neurons are lost with extended culture, which provides a baseline from which we can also assess factors that enhance CST neuron survival. This assay thus allows us to assess independent aspects of CST axonal and dendritic outgrowth kinetics, which allows for the rapid and sensitive investigation of new therapies to address corticospinal neuron outgrowth in the context of CNS injury and neurodegenerative disorders.
Assuntos
Diferenciação Celular/fisiologia , Glicoproteína Associada a Mielina/farmacologia , Fatores de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Tratos Piramidais/citologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células CHO , Morte Celular , Células Cultivadas , Cricetinae , Cricetulus , Genótipo , Técnicas In Vitro , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Neuritos/efeitos dos fármacos , Neurônios/citologia , Antígenos Thy-1/genética , Fatores de TempoRESUMO
Calcium arising through release from intracellular stores and from influx across the plasma membrane is essential for signalling by specific guidance cues and by factors that inhibit axon regeneration. The mediators of calcium influx in these cases are largely unknown. Transient receptor potential channels (TRPCs) belong to a superfamily of Ca2+-permeable, receptor-operated channels that have important roles in sensing and responding to changes in the local environment. Here we report that XTRPC1, a Xenopus homolog of mammalian TRPC1, is required for proper growth cone turning responses of Xenopus spinal neurons to microscopic gradients of netrin-1, brain-derived neurotrophic factor and myelin-associated glycoprotein, but not to semaphorin 3A. Furthermore, XTRPC1 is required for midline guidance of axons of commissural interneurons in the developing Xenopus spinal cord. Thus, members of the TRPC family may serve as a key mediator for the Ca2+ influx that regulates axon guidance during development and inhibits axon regeneration in adulthood.