RESUMO
Ciliary neurotrophic factor (CNTF) was identified as a survival factor in various types of peripheral and central neurons, glia and non-neural cells. At present, there is no available data on the expression and localization of CNTF-receptors in cementoblasts as well as on the role of exogenous CNTF on this cell line. The purpose of this study was to determine if cementoblasts express CNTF-receptors and analyze the mechanism of its apoptotic regulation effects on cementoblasts. OCCM-30 cementoblasts were cultivated and stimulated kinetically using CNTF protein (NBP2-35168, Novus Biologicals). Quantified transcriptional (RT-qPCR) and translational (WB) products of CNTFRα, IL-6Rα (CD126), LIFR, p-GP130, GP130, p-ERK1/2, ERK1/2, Caspase-8, -9, -3 and cleaved-caspase-3 were evaluated. Immunofluorescence (IF) staining was applied to visualize the localization of the CNTF-receptors within cells. The apoptosis ratio was measured with an Annexin-V FITC/PI kit. The ERK1/2 antagonist (FR180204, Calbiochem) was added for further investigation by flow cytometry analysis. The CNTF-receptor complex (CNTFRα, LIFR, GP130) was functionally up-regulated in cementoblasts while cultivated with exogenous CNTF. CNTF significantly attenuated cell viability and proliferation for long-term stimulation. Flow cytometry analysis shows that CNTF enhanced the apoptosis after prolonged duration. However, after only a short-term period, CNTF halts the apoptosis of cementoblasts. Further studies revealed that CNTF activated phosphorylated GP130 and the anti-apoptotic molecule ERK1/2 signaling to participate in the regulation of the apoptosis ratio of cementoblasts. In conclusion, CNTF elicited the cellular functions through a notable induction of its receptor complex in cementoblasts. CNTF has an inhibitory effect on the cementoblast homeostasis. These data also elucidate a cellular mechanism for an exogenous CNTF-triggered apoptosis regulation in a mechanism of ERK1/2 and caspase signaling and provides insight into the complex cellular responses induced by CNTF in cementoblasts.
Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar , Fator Neurotrófico Ciliar , Apoptose , Caspases/metabolismo , Fator Neurotrófico Ciliar/metabolismo , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Receptor gp130 de Citocina/metabolismo , Cemento Dentário/metabolismo , Sistema de Sinalização das MAP Quinases , Receptor do Fator Neutrófico Ciliar/metabolismoRESUMO
Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine that signals through a receptor complex containing a specific subunit, CNTF receptor α (CNTFRα). The two molecules are constitutively expressed in key structures for human placental growth and differentiation. The possible role of CNTF in enhancing cell proliferation and/or invasion during placental development and remodelling was investigated using HTR-8/SVneo and BeWo cells, taken respectively as cytotrophoblast and syncytiotrophoblast models. In both cell lines, treatment with human recombinant (hr) CNTF activated JAK2/STAT3 signalling and inhibited the ERK pathway. Interestingly, in HTR-8/SVneo cells, 50 ng hrCNTF induced significant downregulation of matrix metalloprotease (MMP)-1 and significant upregulation of MMP-9. Moreover, pharmacological inhibition of JAK2/STAT3 signalling by AG490 and curcumin resulted in MMP-9 downregulation; it activated the ERK signalling pathway and upregulated MMP-1 expression. Collectively, these data suggest a role for CNTF signalling in extravillous cytotrophoblast invasion through the modulation of specific MMPs.
Assuntos
Fator Neurotrófico Ciliar , Curcumina , Fator Neurotrófico Ciliar/metabolismo , Fator Neurotrófico Ciliar/farmacologia , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Metaloproteinase 1 da Matriz , Metaloproteinase 9 da Matriz , Placenta/metabolismo , Placentação , Gravidez , Receptor do Fator Neutrófico Ciliar/metabolismoRESUMO
PURPOSE: The purpose of this study was to examine the expression of glial-derived neurotrophic factor (GDNF), the GDNF receptors GFRα1 and GFRα2, ciliary neurotrophic factor (CNTF), and the CNTF receptor CNTFRα in normal and glaucomatous human tissue. METHODS: Human retinas were collected from 8 donors that had been clinically diagnosed and treated for glaucoma, and also from 9 healthy control donors. Immunohistochemical analysis for each trophic factor and receptor was performed. The percent of each retinal section labeled with each antibody was quantified for the total retinal thickness, and separately for the retinal ganglion cell (RGC) complex + retinal nerve fiber layer (RNFL). The expression of each protein was correlated with measures of the subject's ocular histories. RESULTS: The percentage area immunopositive for GFRα2 was significantly decreased in the total retinal thickness containing all retinal layers and in the combined RGC complex + RNFL in glaucomatous eyes in both the peripapillary region and more peripheral retinal locations. We also observed a decrease in GFRα1 expression in the peripapillary RGC Complex + RNFL in glaucoma patients compared to healthy control patients. We also observed a relationship between GDNF and its receptors with several outcomes obtained from the medical record. No differences in CNTF or CNTFR labeling were observed. CONCLUSION: Decreases in GDNF receptor expression in glaucomatous tissue may limit the potential for neuroprotective therapy by supplementation with GDNF.
Assuntos
Glaucoma , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Retina , Fator Neurotrófico Ciliar/metabolismo , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Glaucoma/diagnóstico , Glaucoma/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Retina/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
Reactive astrocytes are implicated in traumatic spinal cord injury (TSCI). Interestingly, naïve astrocytes can easily transform into neurotoxic reactive astrocytes (A1s) with inflammatory stimulation. Previous studies demonstrated that microRNA(miR)-21a-5p was up-regulated in spinal cord tissue after TSCI; however, it is not clear whether this affected reactive astrocyte polarization. Here, we aim to detect the effects of miR-21a-5p on the induction of A1 formation and the underlying mechanisms. Our study found that the expression of miR-21a-5p was significantly increased while that of Cntfr α was decreased, since naïve astrocytes transformed into A1s 3 days post-TSCI; the binding site between miR-21a-5p and Cntfr α was further confirmed in astrocytes. After treatment with CNTF, the levels of A1 markers decreased while that of A2 increased. The expression of A1 markers significantly decreased with the downregulation of miR-21a-5p, while Cntfr α siRNA treatment caused the opposite both in vitro and in vivo. To summarize, miR-21a-5p/Cntfr α promotes A1 induction and might enhance the inflammatory process of TSCI; furthermore, we identified, for the first time, the effect and potential mechanism by which CNTF inhibits naïve astrocytes transformation into A1s. Collectively, our findings demonstrate that targeting miR-21a-5p represents a prospective therapy for promoting the recovery of TSCI.
Assuntos
Astrócitos/metabolismo , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Fator Neurotrófico Ciliar/metabolismo , MicroRNAs/metabolismo , Fator de Transcrição STAT3/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Astrócitos/citologia , Modelos Animais de Doenças , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Traumatismos da Medula Espinal/patologia , Regulação para CimaRESUMO
Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.
Assuntos
Quimiocina CCL5/metabolismo , Fator Neurotrófico Ciliar/genética , Regeneração Nervosa , Nervo Óptico/metabolismo , Animais , Sistemas CRISPR-Cas , Quimiocina CCL5/genética , Fator Neurotrófico Ciliar/metabolismo , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/genética , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Edição de Genes , Terapia Genética , Vetores Genéticos/genética , Regeneração Nervosa/genética , Traumatismos do Nervo Óptico/etiologia , Traumatismos do Nervo Óptico/terapia , Células Ganglionares da Retina/metabolismoRESUMO
Ciliary neurotrophic factor (CNTF) is a member of interleukin-6 type cytokine family. The CNTF receptor complex is a heterodimer including gp130 and CNTF receptor α (CNTFRα) proteins triggering the activation of multiple intracellular signaling pathways including AKT/PI3K, MAPK/ERK and Jak/STAT pathways. At present no data are available on the localization of CNTF and CNTFRα in prostate as well as on the role of CNTF in this organ. In this study we have analyzed the localization of CNTF and CNTFRα by immunohistochemistry and we have used PWR-1E cell line as a model for normal glandular cell to investigate the role of this cytokine. Our results show that CNTF and CNTFRa are expressed in the staminal compart of the prostate and that CNTF selectively inhibits ERK pathway. In conclusion, we suggest that CNTF could be considered as key molecule to maintenance epithelium homeostasis via pERK downregulation by an autocrine mechanism. Further CNTF studies in prostate cancer could be useful to verify the potential role of this cytokine in carcinogenesis.
Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Fator Neurotrófico Ciliar/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Próstata/metabolismo , Linhagem Celular , Humanos , Imuno-Histoquímica , Masculino , Próstata/citologiaAssuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mitocôndrias/metabolismo , Envelhecimento , Aminoácidos , Apoptose , Biomarcadores , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , DNA Mitocondrial/genética , Metabolismo Energético , Humanos , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Isoformas de Proteínas , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismoRESUMO
Interleukin-6 (IL-6) family cytokines signal through multimeric receptor complexes, providing unique opportunities to create novel ligand-based therapeutics. The cardiotrophin-like cytokine factor 1 (CLCF1) ligand has been shown to play a role in cancer, osteoporosis, and atherosclerosis. Once bound to ciliary neurotrophic factor receptor (CNTFR), CLCF1 mediates interactions to coreceptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). By increasing CNTFR-mediated binding to these coreceptors we generated a receptor superagonist which surpassed the potency of natural CNTFR ligands in neuronal signaling. Through additional mutations, we generated a receptor antagonist with increased binding to CNTFR but lack of binding to the coreceptors that inhibited tumor progression in murine xenograft models of nonsmall cell lung cancer. These studies further validate the CLCF1-CNTFR signaling axis as a therapeutic target and highlight an approach of engineering cytokine activity through a small number of mutations.
Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/agonistas , Citocinas/metabolismo , Engenharia de Proteínas/métodos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/antagonistas & inibidores , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Receptor gp130 de Citocina/metabolismo , Citocinas/química , Citocinas/genética , Humanos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Ligantes , Neurônios/metabolismo , Ligação Proteica , Ratos , Transdução de SinaisRESUMO
Oligodendrocyte development is tightly controlled by extrinsic signals; however, mechanisms that modulate cellular responses to these factors remain unclear. Six-transmembrane glycerophosphodiester phosphodiesterases (GDEs) are emerging as central regulators of cellular differentiation via their ability to shed glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. We show here that GDE3 controls the pace of oligodendrocyte generation by negatively regulating oligodendrocyte precursor cell (OPC) proliferation. GDE3 inhibits OPC proliferation by stimulating ciliary neurotrophic factor (CNTF)-mediated signaling through release of CNTFRα, the ligand-binding component of the CNTF-receptor multiprotein complex, which can function as a soluble factor to activate CNTF signaling. GDE3 releases soluble CNTFRα by GPI-anchor cleavage from the plasma membrane and from extracellular vesicles (EVs) after co-recruitment of CNTFRα in EVs. These studies uncover new physiological roles for GDE3 in gliogenesis and identify GDE3 as a key regulator of CNTF-dependent regulation of OPC proliferation through release of CNTFRα.
Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Células Precursoras de Oligodendrócitos/citologia , Células Precursoras de Oligodendrócitos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Animais , Membrana Celular/metabolismo , Proliferação de Células , Fator Neurotrófico Ciliar/metabolismo , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestrutura , Deleção de Genes , Células HEK293 , Humanos , Camundongos , Transdução de Sinais , Solubilidade , Medula Espinal/embriologia , Medula Espinal/metabolismoRESUMO
Systemic ciliary neurotrophic factor (CNTF) administration protects motor neurons from denervating diseases and lesions but produces non-neuromuscular side effects. Therefore, CNTF related therapeutics will need to specifically target motor neuron protective receptor mechanisms. Expression of the essential ligand binding subunit of the CNTF receptor, CNTF receptor α (CNTFRα), is induced in skeletal muscle by denervating lesion and in human denervating diseases. We show here, with muscle-specific in vivo genetic disruption, that muscle CNTFRα makes an essential/non-redundant contribution to maintaining choline acetyltransferase levels in denervated motor neurons following nerve crush, suggesting the muscle CNTFRα induction is an endogenous denervation-induced neuroprotective response that could be enhanced to treat nerve lesion and denervating diseases. Notably, unlike motor neuron gene expression, skeletal muscle gene expression can be specifically targeted with human gene therapy vectors already approved for market.
Assuntos
Colina O-Acetiltransferase/metabolismo , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Neurônios Motores/metabolismo , Músculo Esquelético/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Músculo Esquelético/inervação , Compressão NervosaRESUMO
Within the supraoptic nucleus (SON) of a 35-day-old rat, we previously demonstrated a collateral sprouting response that reinnervates the partially denervated neural lobe (NL) after unilateral lesion of the hypothalamo-neurohypophysial tract. Others have shown a decreased propensity for axonal sprouting in an aged brain; therefore, to see if the SON exhibits a decreased propensity for axonal sprouting as the animal ages, we performed a unilateral lesion in the 125-day-old rat SON. Ultrastructural analysis of axon profiles in the NL of the 125-day-old rat demonstrated an absence of axonal sprouting following injury. We previously demonstrated that ciliary neurotrophic factor (CNTF) promotes process outgrowth from injured magnocellular neuron axons in vitro. Thus, we hypothesized that the lack of axonal sprouting in the 125-day-old rat SON may be due to a reduction in CNTF or the CNTF receptor components. To this point, we found that as the rat ages there is significantly less CNTF receptor alpha (CNTFRα) protein in the uninjured, 125-day-old rat compared to the uninjured, 35-day-old rat. We also observed that protein levels of CNTF and the CNTF receptor components were increased in the SON and NL following injury in the 35-day-old rat, but there was no difference in the protein levels in the 125-day-old rat. Altogether, the results presented herein demonstrate that the plasticity within the SON is highly dependent on the age of the rat, and that a decrease in CNTFRα protein levels in the 125-day-old rat may contribute to the loss of axonal sprouting following axotomy.
Assuntos
Envelhecimento/metabolismo , Axônios/metabolismo , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Núcleo Supraóptico/metabolismo , Animais , Axônios/química , Axotomia/métodos , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/análise , Masculino , Vias Neurais/química , Vias Neurais/metabolismo , Ratos , Ratos Sprague-Dawley , Núcleo Supraóptico/químicaRESUMO
Expression of the ciliary neurotrophic factor (CNTF) receptor essential ligand binding subunit, CNTF receptor α (CNTFRα), is induced in motor neurons and skeletal muscle following peripheral nerve lesion. We previously found muscle CNTFRα promotes motor neuron axon regeneration post-lesion. Both nerve lesion and CNTF administration activate motor neuron signal transducer and activator of transcription 3 (STAT3), a transcription factor implicated in axon growth, suggesting CNTF receptors may contribute to the lesion-induced STAT3 activation. However, many receptor types signal through STAT3, and if CNTF receptors contribute, motor neuron receptors seemed most likely to regulate motor neuron STAT3. To determine the role played by muscle CNTFRα, we used in vivo, muscle-specific CNTFRα depletion in mice and report here that this selectively impairs the second phase, sustained motor neuron STAT3 activation post-lesion. Thus, muscle CNTFRα makes an essential contribution to motor neuron STAT3 activation during axon regeneration and may thereby promote axon regeneration through such signaling. We also report CNTFRα quantitative PCR suggesting involvement of many denervated muscle types, as well as muscle damaged at the lesion site. The present data add to the evidence suggesting that enhancing muscle CNTFRα expression may promote motor neuron regeneration in trauma and disease.
Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Neurônios Motores/metabolismo , Músculo Esquelético/metabolismo , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Camundongos , Camundongos KnockoutRESUMO
Micro-696 (miR-696) has been previously known as an exercise related miRNA, which has a profound role in fatty acid oxidation and mitochondrial biogenesis of skeletal muscle. However, its role in skeletal myoblast proliferation and differentiation is still unclear. In this study, we found that miR-696 expressed highly in skeletal muscle and reduced during C2C12 myoblasts differentiation. MiR-696 overexpression repressed C2C12 myoblast proliferation and myofiber formation, while knockdown of endogenous miR-696 expression showed opposite results. During myogenesis, we observed an inversed expression pattern between miR-696 and CNTFRα in vitro, and demonstrated that miR-696 could specifically target CNTFRα and repress the expression of CNTFRα. Additionally, we further found that knockdown of CNTFRα suppressed the proliferation and differentiation of C2C12 cells. Taking all things together, we propose a novel insight that miR-696 down-regulates C2C12 cell myogenesis by inhibiting CNTFRα expression.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , MicroRNAs/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/genética , Camundongos , MicroRNAs/genética , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Mioblastos/citologia , Mioblastos/metabolismoRESUMO
Human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC CMs) may be used in regenerative medicine for individualized tissue transplants in the future. For application in patients, the generated CMs have to be highly pure and well characterized. In order to overcome the prevalent scarcity of CM-specific markers, we quantitatively assessed cell-surface-exposed sialo-glycoproteins and N-glycans of hiPSCs, CM progenitors, and CMs. Applying a combination of metabolic labeling and specific sialo-glycoprotein capture, we could highly enrich and quantify membrane proteins during cardiomyogenic differentiation. Among them we identified a number of novel, putative biomarkers for hiPSC CMs. Analysis of the N-glycome by capillary gel electrophoresis revealed three novel structures comprising ß1,3-linked galactose, α2,6-linked sialic acid and complex fucosylation; these were highly specific for hiPSCs. Bisecting GlcNAc structures strongly increased during differentiation, and we propose that they are characteristic of early, immature CMs.
Assuntos
Membrana Celular/química , Glicômica/métodos , Células-Tronco Pluripotentes Induzidas/química , Miócitos Cardíacos/química , Polissacarídeos/química , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Sequência de Carboidratos , Diferenciação Celular , Membrana Celular/metabolismo , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/genética , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Fucose/química , Fucose/metabolismo , Galactose/química , Galactose/metabolismo , Gastrinas/genética , Gastrinas/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Laminina/genética , Laminina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Polissacarídeos/metabolismo , Receptor EphA7/genética , Receptor EphA7/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Coloração e Rotulagem/métodosRESUMO
Numerous neurotrophic factors promote the survival of developing motor neurons but their combinatorial actions remain poorly understood; to address this, we here screened 66 combinations of 12 neurotrophic factors on pure, highly viable, and standardized embryonic mouse motor neurons isolated by a unique FACS technique. We demonstrate potent, strictly additive, survival effects of hepatocyte growth factor (HGF), ciliary neurotrophic factor (CNTF), and Artemin through specific activation of their receptor complexes in distinct subsets of lumbar motor neurons: HGF supports hindlimb motor neurons through c-Met; CNTF supports subsets of axial motor neurons through CNTFRα; and Artemin acts as the first survival factor for parasympathetic preganglionic motor neurons through GFRα3/Syndecan-3 activation. These data show that neurotrophic factors can selectively promote the survival of distinct classes of embryonic motor neurons. Similar studies on postnatal motor neurons may provide a conceptual framework for the combined therapeutic use of neurotrophic factors in degenerative motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy, and spinobulbar muscular atrophy.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Fator Neurotrófico Ciliar/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Sobrevivência Celular , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/genética , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Feminino , Citometria de Fluxo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Sindecana-3/genética , Sindecana-3/metabolismoRESUMO
PURPOSE: To demonstrate that vasoactive intestinal peptide (VIP), a corneal endothelial (CE) cell autocrine factor, maintains the integrity of corneal endothelium in human donor corneoscleral explants precut for endothelial keratoplasty. METHODS: Twelve paired human donor corneoscleral explants used as control versus VIP-treated explants (10 nM, 30 minutes, 37°C) were shipped (4°C) to the Lions Eye Institute for Transplantation and Research for precutting (Moria CBM-ALTK Keratome), shipped back to the laboratory, and cultured in ciliary neurotrophic factor (CNTF, 0.83 nM, 37°C, 24 hours). Trephined endothelial discs (8-8.5 mm) were analyzed for differentiation markers (N-cadherin, CNTF receptor α subunit [CNTFRα], and connexin 43) by Western blot after a quarter of the discs from 4 paired explants were cut away and stained with alizarin red S for microscopic damage analysis. Two additional paired explants (6 days in culture) were stained for panoramic view of central CE damage. RESULTS: VIP treatment increased N-cadherin and CNTFRα levels (mean ± SEM) to 1.38 ± 0.11-fold (P = 0.003) and 1.46 ± 0.22-fold (P = 0.03) of paired controls, respectively, whereas CE cell CNTF responsiveness in upregulation of connexin 43 increased to 2.02 ± 0.5 (mean ± SEM)-fold of the controls (P = 0.04). CE damage decreased from (mean ± SEM) 10.0% ± 1.2% to 1.6% ± 0.3% (P < 0.0001) and 9.1% ± 1.1% to 2.4% ± 1.0% (P = 0.0006). After 6 days in culture, the damage in whole CE discs decreased from 20.0% (control) to 5.5% (VIP treated). CONCLUSIONS: VIP treatment before precut enhanced the preservation of corneal endothelium.
Assuntos
Sobrevivência Celular/fisiologia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Endotélio Corneano/citologia , Endotélio Corneano/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Peptídeo Intestinal Vasoativo/uso terapêutico , Adulto , Idoso , Antígenos CD/metabolismo , Western Blotting , Caderinas/metabolismo , Fator Neurotrófico Ciliar/farmacologia , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Conexina 43/metabolismo , Endotélio Corneano/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Preservação de Órgãos , Soluções para Preservação de Órgãos , Doadores de Tecidos , Coleta de Tecidos e ÓrgãosRESUMO
The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored CNTFRα to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-ß (gp130/LIFRß) for signaling. Both CLC and CNTFRα are necessary for signaling but so far CLF-1 has only been known as a putative facilitator of CLC secretion. However, it has recently been shown that CLF-1 contains three binding sites: one for CLC; one for CNTFRα (that may promote assembly of the trimeric complex); and one for the endocytic receptor sorLA. The latter site provides high affinity binding of CLF-1, CLC:CLF-1, as well as the trimeric (CLC:CLF-1:CNTFRα) complex to sorLA, and in sorLA-expressing cells the soluble ligands CLF-1 and CLC:CLF-1 are rapidly taken up and internalized. In cells co-expressing CNTFRα and sorLA, CNTFRα first binds CLC:CLF-1 to form a membrane-associated trimeric complex, but it also connects to sorLA via the free sorLA-binding site in CLF-1. As a result, CNTFRα, which has no capacity for endocytosis on its own, is tugged along and internalized by the sorLA-mediated endocytosis of CLC:CLF-1. The present protocol describes the experimental procedures used to demonstrate i) the sorLA-mediated and CLC:CLF-1-dependent downregulation of surface-membrane CNTFRα expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFRα; and iii) the lowered cellular response to CLC:CLF-1-stimulation upon sorLA-mediated downregulation of CNTFRα.
Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Citocinas/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores de Citocinas/metabolismo , Sítios de Ligação , Western Blotting , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/genética , Citocinas/química , Regulação para Baixo , Endocitose , Células HEK293 , Humanos , Imuno-Histoquímica , Proteínas Relacionadas a Receptor de LDL/genética , Ligantes , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/enzimologia , Proteínas de Membrana Transportadoras/genética , Fosforilação , Receptores de Citocinas/química , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Exogenous ciliary neurotrophic factor (CNTF) administration promotes the survival of motor neurons in a wide range of models. It also increases the expression of the critical neurotransmitter enzyme choline acetyltransferase (ChAT) by in vitro motor neurons, likely independent of its effects on their survival. We have used the adult mouse facial nerve crush model and adult-onset conditional disruption of the CNTF receptor α (CNTFRα) gene to directly examine the in vivo roles played by endogenous CNTF receptors in adult motor neuron survival and ChAT maintenance, independent of developmental functions. We have previously shown that adult activation of the CreER gene construct in floxed CNTFRα mice depletes this essential receptor subunit in a large subset of motor neurons (and all skeletal muscle, as shown in this study) but has no effect on the survival of intact or lesioned motor neurons, indicating that these adult CNTF receptors play no essential survival role in this model, in contrast to their essential role during embryonic development. Here we show that this same CNTFRα depletion does not affect ChAT labeling in nonlesioned motor neurons, but it significantly increases the loss of ChAT following nerve crush. The data suggest that, although neither motor neuron nor muscle CNTF receptors play a significant, nonredundant role in the maintenance of ChAT in intact adult motor neurons, the receptors become essential for ChAT maintenance when the motor neurons are challenged by nerve crush. Therefore, the data suggest that the receptors act as a critical component of an endogenous neuroprotective mechanism. J. Comp. Neurol. 525:1206-1215, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Colina O-Acetiltransferase/biossíntese , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Traumatismos do Nervo Facial/metabolismo , Neurônios Motores/enzimologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Compressão Nervosa , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Cardiotrophin-like cytokine:cytokine-like factor-1 (CLC:CLF-1) is a heterodimeric neurotropic cytokine that plays a crucial role during neuronal development. Mice lacking CLC:CLF-1 die soon after birth due to a suckling defect and show reduced numbers of motor neurons. Humans carrying mutations in CLC:CLF-1 develop similar disorders, known as Sohar-Crisponi or cold-induced sweating syndrome, and have a high risk of early death. It is well known that CLC binds the ciliary neurotrophic factor receptor α (CNTFRα) and is a prerequisite for signaling through the gp130/leukemia inhibitory factor receptor ß (LIFRß) heterodimer, whereas CLF-1 serves to promote the cellular release of CLC. However, the precise role of CLF-1 is unclear. Here, we report that CLF-1, based on its binding site for CLC and on two additional and independent sites for CNTFRα and sorLA, is a key player in CLC and CNTFRα signaling and turnover. The site for CNTFRα enables CLF-1 to promote CLC:CNTFRα complex formation and signaling. The second site establishes a link between the endocytic receptor sorLA and the tripartite CLC:CLF-1:CNTFRα complex and allows sorLA to downregulate the CNTFRα pool in stimulated cells. Finally, sorLA may bind and concentrate the tripartite soluble CLC:CLF-1:CNTFRα complex on cell membranes and thus facilitate its signaling through gp130/LIFRß.
Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Citocinas/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores de Citocinas/metabolismo , Transdução de Sinais , Células HEK293 , Humanos , Ligação ProteicaRESUMO
Currently there is no neuroprotective or neurorestorative therapy for Parkinson's disease. Here we report that transient receptor potential vanilloid 1 (TRPV1) on astrocytes mediates endogenous production of ciliary neurotrophic factor (CNTF), which prevents the active degeneration of dopamine neurons and leads to behavioural recovery through CNTF receptor alpha (CNTFRα) on nigral dopamine neurons in both the MPP(+)-lesioned or adeno-associated virus α-synuclein rat models of Parkinson's disease. Western blot and immunohistochemical analysis of human post-mortem substantia nigra from Parkinson's disease suggests that this endogenous neuroprotective system (TRPV1 and CNTF on astrocytes, and CNTFRα on dopamine neurons) might have relevance to human Parkinson's disease. Our results suggest that activation of astrocytic TRPV1 activates endogenous neuroprotective machinery in vivo and that it is a novel therapeutic target for the treatment of Parkinson's disease.