RESUMO
It is suggested that activation of receptor for advanced glycation end products (RAGE) induces proinflammatory response in diabetic nerve tissues. Macrophage infiltration is invoked in the pathogenesis of diabetic polyneuropathy (DPN), while the association between macrophage and RAGE activation and the downstream effects of macrophages remain to be fully clarified in DPN. This study explored the role of RAGE in the pathogenesis of DPN through the modified macrophages. Infiltrating proinflammatory macrophages impaired insulin sensitivity, atrophied the neurons in dorsal root ganglion, and slowed retrograde axonal transport (RAT) in the sciatic nerve of type 1 diabetic mice. RAGE-null mice showed an increase in the population of antiinflammatory macrophages, accompanied by intact insulin sensitivity, normalized ganglion cells, and RAT. BM transplantation from RAGE-null mice to diabetic mice protected the peripheral nerve deficits, suggesting that RAGE is a major determinant for the polarity of macrophages in DPN. In vitro coculture analyses revealed proinflammatory macrophage-elicited insulin resistance in the primary neuronal cells isolated from dorsal root ganglia. Applying time-lapse recording disclosed a direct impact of proinflammatory macrophage and insulin resistance on the RAT deficits in primary neuronal cultures. These results provide a potentially novel insight into the development of RAGE-related DPN.
Assuntos
Diabetes Mellitus Experimental , Neuropatias Diabéticas , Resistência à Insulina , Camundongos , Animais , Receptor para Produtos Finais de Glicação Avançada/genética , Diabetes Mellitus Experimental/complicações , MacrófagosRESUMO
Oxaliplatin (OHP) is a platinum-based agent that can cause peripheral neuropathy, an adverse effect in which the dorsal root ganglion (DRG) neurons are targeted. Zonisamide has exhibited neuroprotective activities toward adult rat DRG neurons in vitro and therefore, we aimed to assess its potential efficacy against OHP-induced neurotoxicity. Pretreatment with zonisamide (100 µM) alleviated the DRG neuronal death caused by OHP (75 µM) and the protective effects were attenuated by a co-incubation with 25 µM of the mitogen-activated protein kinase (MAPK; MEK/ERK) inhibitor, U0126, or the phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor, LY294002. Pretreatment with zonisamide also suppressed the OHP-induced p38 MAPK phosphorylation in lined DRG neurons, ND7/23, while the OHP-induced DRG neuronal death was alleviated by pretreatment with the p38 MAPK inhibitor, SB239063 (25 µM). Although zonisamide failed to protect the immortalized rat Schwann cells IFRS1 from OHP-induced cell death, it prevented neurite degeneration and demyelination-like changes, as well as the reduction of the serine/threonine-specific protein kinase (AKT) phosphorylation in DRG neuron-IFRS1 co-cultures exposed to OHP. Zonisamide's neuroprotection against the OHP-induced peripheral sensory neuropathy is possibly mediated by a stimulation of the MEK/ERK and PI3K/AKT signaling pathways and suppression of the p38 MAPK pathway in DRG neurons. Future studies will allow us to solidify zonisamide as a promising remedy against the neurotoxic adverse effects of OHP.
Assuntos
Gânglios Espinais , Doenças do Sistema Nervoso Periférico , Animais , Células Cultivadas , Técnicas de Cocultura , Gânglios Espinais/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Oxaliplatina/efeitos adversos , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Células de Schwann/metabolismo , Zonisamida/efeitos adversos , Zonisamida/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Pyruvate functions as a key molecule in energy production and as an antioxidant. The efficacy of pyruvate supplementation in diabetic retinopathy and nephropathy has been shown in animal models; however, its significance in the functional maintenance of neurons and Schwann cells under diabetic conditions remains unknown. We observed rapid and extensive cell death under high-glucose (> 10 mM) and pyruvate-starved conditions. Exposure of Schwann cells to these conditions led to a significant decrease in glycolytic flux, mitochondrial respiration and ATP production, accompanied by enhanced collateral glycolysis pathways (e.g., polyol pathway). Cell death could be prevented by supplementation with 2-oxoglutarate (a TCA cycle intermediate), benfotiamine (the vitamin B1 derivative that suppresses the collateral pathways), or the poly (ADP-ribose) polymerase (PARP) inhibitor, rucaparib. Our findings suggest that exogenous pyruvate plays a pivotal role in maintaining glycolysis-TCA cycle flux and ATP production under high-glucose conditions by suppressing PARP activity.
Assuntos
Nefropatias Diabéticas/patologia , Glucose/metabolismo , Hiperglicemia/complicações , Ácido Pirúvico/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Nefropatias Diabéticas/prevenção & controle , Modelos Animais de Doenças , Feminino , Glicólise/efeitos dos fármacos , Humanos , Hiperglicemia/sangue , Hiperglicemia/metabolismo , Indóis/farmacologia , Indóis/uso terapêutico , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Cultura Primária de Células , Ratos , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Células de Schwann/patologia , Tiamina/análogos & derivados , Tiamina/farmacologia , Tiamina/uso terapêuticoRESUMO
Besides its insulinotropic actions on pancreatic ß cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported; however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron-IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.
Assuntos
Neuropatias Diabéticas/tratamento farmacológico , Exenatida/farmacologia , Peptídeo 1 Semelhante ao Glucagon/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Animais , Movimento Celular/genética , Sobrevivência Celular/genética , Cromonas/farmacologia , Técnicas de Cocultura , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/patologia , Exenatida/genética , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Morfolinas/farmacologia , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Nervo Isquiático/lesõesRESUMO
Aldose reductase (AR) is a member of the reduced nicotinamide adenosine dinucleotide phosphate (NADPH)-dependent aldo-keto reductase superfamily. It is also the rate-limiting enzyme of the polyol pathway, catalyzing the conversion of glucose to sorbitol, which is subsequently converted to fructose by sorbitol dehydrogenase. AR is highly expressed by Schwann cells in the peripheral nervous system (PNS). The excess glucose flux through AR of the polyol pathway under hyperglycemic conditions has been suggested to play a critical role in the development and progression of diabetic peripheral neuropathy (DPN). Despite the intensive basic and clinical studies over the past four decades, the significance of AR over-activation as the pathogenic mechanism of DPN remains to be elucidated. Moreover, the expected efficacy of some AR inhibitors in patients with DPN has been unsatisfactory, which prompted us to further investigate and review the understanding of the physiological and pathological roles of AR in the PNS. Particularly, the investigation of AR and the polyol pathway using immortalized Schwann cells established from normal and AR-deficient mice could shed light on the causal relationship between the metabolic abnormalities of Schwann cells and discordance of axon-Schwann cell interplay in DPN, and led to the development of better therapeutic strategies against DPN.
Assuntos
Aldeído Redutase/metabolismo , Redes e Vias Metabólicas , Polímeros/metabolismo , Células de Schwann/metabolismo , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Animais , Diabetes Mellitus/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Humanos , Oxirredução , Sorbitol/metabolismoRESUMO
Patients with transthyretin (TTR)-type familial amyloid polyneuropathy (FAP) typically exhibit sensory dominant polyneuropathy and autonomic neuropathy. However, the molecular pathogenesis of the neuropathy remains unclear. In this study, we characterize the features of FAP TTR the substitution of lysine for glutamic acid at position 61 (E61K). This FAP was late-onset, with sensory dominant polyneuropathy, autonomic neuropathy, and cardiac amyloidosis. Interestingly, no amyloid deposits were found in the endoneurium of the four nerve specimens examined. Therefore, we examined the amyloidogenic properties of E61K TTR in vitro. Recombinant wild-type TTR, the substitution of methionine for valine at position 30 (V30M) TTR, and E61K TTR proteins were incubated at 37°C for 72 hr, and amyloid fibril formation was assessed using the thioflavin-T binding assay. Amyloid fibril formation by E61K TTR was less than that by V30M TTR, and similar to that by wild-type TTR. E61K TTR did not have an inhibitory effect on neurite outgrowth from adult rat dorsal root ganglion (DRG) neurons, but V30M TTR did. Furthermore, we studied the sural nerve of our patient by terminal deoxynucleotidyl transferase dUTP nick end labeling and electron microscopy. A number of apoptotic cells were observed in the endoneurium of the nerve by transferase dUTP nick end labeling. Chromatin condensation was confirmed in the nucleus of non-myelinating Schwann cells by electron microscopy. These findings suggest that E61K TTR is low amyloidogenic, in vitro and in vivo. However, TTR aggregates and amyloid fibrils in the DRG may cause sensory impairments in FAP because the DRG has no blood-nerve barrier. Moreover, Schwann cell apoptosis may contribute to the neurodegeneration.
Assuntos
Neuropatias Amiloides Familiares/genética , Amiloide/biossíntese , Pré-Albumina/genética , Substituição de Aminoácidos , Amiloide/genética , Amiloidose/patologia , Animais , Apoptose , Cristalografia por Raios X , Humanos , Mutação , Nervos Periféricos/patologia , Placa Amiloide/patologia , Pré-Albumina/química , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Células de Schwann/metabolismo , Nervo Sural/patologiaRESUMO
Zonisamide, an anti-epileptic and anti-Parkinson's disease drug, displays neurotrophic activity on cultured motor neurons and facilitates axonal regeneration after peripheral nerve injury in mice, but its underlying mechanisms remain unclear. In this study, zonisamide enhanced neurite outgrowth from cultured adult rat dorsal root ganglion (DRG) neurons in a concentration-dependent manner (1 µM < 10 µM < 100 µM), and its activity was significantly attenuated by co-treatment with a phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor LY294002 or a mitogen-activated protein kinase (MAPK) inhibitor U0126. In agreement with these findings, 100 µM zonisamide for 1 h induced phosphorylation of AKT and ERK1/2, key molecules of PI3K and MAPK signaling pathways, respectively in mouse neuroblastoma × rat DRG neuron hybrid cells ND7/23. In contrast, zonisamide failed to promote proliferation or migration of immortalized Fischer rat Schwann cells 1 (IFRS1). These findings suggest that the beneficial effects of zonisamide on peripheral nerve regeneration may be attributable to its direct actions on neurons through PI3K and MAPK pathways, rather than the stimulation of Schwann cells.
Assuntos
Anticonvulsivantes/farmacologia , Gânglios Espinais/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Zonisamida/farmacologia , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Gânglios Espinais/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Ratos , Ratos Wistar , Células de Schwann/citologia , Células de Schwann/metabolismo , Relação Estrutura-AtividadeRESUMO
Glycolaldehyde (GA) is a highly reactive hydroxyaldehyde and one of the glycolytic metabolites producing advanced glycation endproducts (AGEs), but its toxicity toward neurons and Schwann cells remains unclear. In the present study, we found that GA exhibited more potent toxicity than other AGE precursors (glyceraldehyde, glyoxal, methylglyoxal and 3-deoxyglucosone) against immortalized IFRS1 adult rat Schwann cells and ND7/23 neuroblastoma × neonatal rat dorsal root ganglion (DRG) neuron hybrid cells. GA affected adult rat DRG neurons and ND7/23 cells more severely than GA-derived AGEs, and exhibited concentration- and time-dependent toxicity toward ND7/23 cells (10 < 100 < 250 < 500 µM; 6 h < 24 h). Treatment with 500 µM GA significantly up-regulated the phosphorylation of c-jun N-terminal kinase (JNK) and p-38 mitogen-activated kinase (p-38 MAPK) in ND7/23 cells. Furthermore, GA-induced ND7/23 cell death was significantly inhibited due to co-treatment with 10 µM of the JNK inhibitor SP600125 or the p-38 MAPK inhibitor SB239063. These findings suggest the involvement of JNK and p-38 MAPK-signaling pathways in GA-induced neuronal cell death and that enhanced GA production under diabetic conditions might be involved in the pathogenesis of diabetic neuropathy.
Assuntos
Acetaldeído/análogos & derivados , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Acetaldeído/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Feminino , Ratos , Ratos Wistar , Células Receptoras Sensoriais/metabolismoRESUMO
A large variety of drugs have been reported to cause peripheral neuropathies as dose-limiting adverse effects; however, most of them primarily affect axons and/or neuronal cell bodies rather than Schwann cells and/or myelin sheaths. In this chapter, we focus on the drugs that seem to elicit the neuropathies with schwannopathy and/or myelinopathy-predominant phenotypes, such as amiodarone, dichloroacetate, and tumor necrosis factor-α antagonists. Although the pathogenesis of demyelination induced by these drugs remain largely obscure, the recent in vivo and in vitro studies have implicated the involvement of metabolic abnormalities and impaired autophagy in Schwann cells and immune system disorders in the disruption of neuron-Schwann cell contact and interactions.
Assuntos
Doenças Desmielinizantes/induzido quimicamente , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Bainha de Mielina/patologia , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Células de Schwann/patologia , Amiodarona/efeitos adversos , Axônios , Ácido Dicloroacético/efeitos adversos , Humanos , Bainha de Mielina/efeitos dos fármacos , Células de Schwann/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
The hypocretin/orexin neuropeptide system coordinates the regulation of various physiological processes. Our previous study reported that a reduction in the expression of pleomorphic adenoma genelike 1 (Plagl1), which encodes a C2H2 zincfinger transcription factor, occurs in hypocretin neuronablated transgenic mice, suggesting that PLAGL1 is coexpressed in hypocretin neurons and regulates hypocretin transcription. The present study examined whether canonical preprohypocretin transcription is functionally modulated by PLAGL1. Double immunostaining indicated that the majority of hypocretin neurons were positive for PLAGL1 immunoreactivity in the nucleus. Notably, PLAGL1 immunoreactivity in hypocretin neurons was altered in response to several conditions affecting hypocretin function. An uneven localization of PLAGL1 was detected in the nuclei of hypocretin neurons following sleep deprivation. Chromatin immunoprecipitation revealed that endogenous PLAGL1 may bind to a putative PLAGL1binding site in the proximal region of the hypocretin gene, in the murine hypothalamus. In addition, electroporation of the PLAGL1 expression vector into the fetal hypothalamus promoted hypothalamic hypocretin transcription. These results suggested that PLAGL1 may regulate hypothalamic hypocretin transcription.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Orexinas/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Embrião de Mamíferos/citologia , Genes Supressores de Tumor , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Regiões Promotoras Genéticas/genética , Ligação ProteicaRESUMO
Amiodarone hydrochloride (AMD), an anti-arrhythmic agent, has been shown to cause peripheral neuropathy; however, its pathogenesis remains unknown. We examined the toxic effects of AMD on an immortalized adult rat Schwann cell line, IFRS1, and cocultures of IFRS1 cells and adult rat dorsal root ganglion neurons or nerve growth factor-primed PC12 cells. Treatment with AMD (1, 5, and 10 µm) induced time- and dose-dependent cell death, accumulation of phospholipids and neutral lipids, upregulation of the expression of gangliosides, and oxidative stress (increased nuclear factor E2-related factor in nuclear extracts and reduced GSH/GSSG ratios) in IFRS1 cells. It also induced the upregulation of LC3-II and p62 expression, with phosphorylation of p62, suggesting that deficient autolysosomal degradation is involved in AMD-induced IFRS1 cell death. Furthermore, treatment of the cocultures with AMD induced detachment of IFRS1 cells from neurite networks in a time- and dose-dependent manner. These findings suggest that AMD-induced lysosomal storage accompanied by enhanced oxidative stress and impaired lysosomal degradation in Schwann cells might be a cause of demyelination in the peripheral nervous system.
Assuntos
Doenças Desmielinizantes/metabolismo , Lisossomos/metabolismo , Estresse Oxidativo , Células de Schwann/metabolismo , Amiodarona/toxicidade , Animais , Células Cultivadas , Inibidores Enzimáticos/toxicidade , Feminino , Gânglios Espinais/citologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Células PC12 , Fosfolipídeos/metabolismo , Ratos , Ratos Wistar , Células de Schwann/efeitos dos fármacosRESUMO
Glucagon-like peptide-1 (GLP-1) is thought to preserve neurons and glia following axonal injury and neurodegenerative disorders. We investigated the neurotrophic and neuroprotective properties of exendin (Ex)-4, a synthetic GLP-1 receptor (GLP-1R) agonist, on adult rat dorsal root ganglion (DRG) neurons and PC12 cells. GLP-1R was predominantly localized on large and small peptidergic neurons in vivo and in vitro, suggesting the involvement of GLP-1 in both the large and small sensory fiber functions. Ex-4 dose-dependently (1 ≤ 10 ≤ 100 nM) promoted neurite outgrowth and neuronal survival at 2 and 7 days in culture, respectively. Treatment with 100 nM Ex-4 restored the reduced neurite outgrowth and viability of DRG neurons caused by the insulin removal from the medium and suppressed the activity of RhoA, an inhibitory regulator for peripheral nerve regeneration, in PC12 cells. Furthermore, these effects were attenuated by co-treatment with phosphatidylinositol-3'-phosphate kinase (PI3K) inhibitor, LY294002. These findings imply that Ex-4 enhances neurite outgrowth and neuronal survival through the activation of PI3K signaling pathway, which negatively regulates RhoA activity. Ex-4 and other GLP-1R agonists may compensate for the reduced insulin effects on neurons, thereby being beneficial for the treatment of diabetic neuropathy.
Assuntos
Gânglios Espinais/fisiologia , Insulina/fisiologia , Neurônios/fisiologia , Fármacos Neuroprotetores/farmacologia , Peptídeos/farmacologia , Peçonhas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Exenatida , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/fisiologia , Morfolinas/farmacologia , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Células PC12 , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Ratos Wistar , Receptores de Glucagon/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Familial amyloidotic polyneuropathy (FAP) is one of the transthyretin (TTR) amyloidoses characterized by extracellular amyloid deposits and peripheral nerve involvement. Recently, we found significant expression of the TTR gene in Schwann cells of the peripheral nervous system. We hypothesized that local expression of variant TTR in Schwann cells may contribute to neurodegeneration in FAP. Schwann cells derived from the dorsal root ganglia (DRG) of transgenic mice expressing variant human TTR in a mouse null background were cultured long term to obtain spontaneously immortalized cell lines. We established an immortalized Schwann cell line, TgS1, derived from the transgenic mice. TgS1 cells synthesized variant TTR and secreted it into the medium. As sensory neuropathy usually arises early in FAP, we examined the effect of the conditioned medium derived from TgS1 cells on neurite outgrowth from DRG sensory neurons. Conditioned medium derived from TgS1 cells inhibited neurite outgrowth from the sensory neurons. TTR deposition in the DRG of aged transgenic mice was investigated by immunohistochemistry. TTR aggregates were observed in the cytoplasm of Schwann cells and satellite cells. Proteasome inhibition induced TTR aggregates as aggresomes in TgS1 cells. In conclusion, local variant TTR gene expression in Schwann cells might trigger neurodegeneration in FAP. We established a spontaneously immortalized Schwann cell line derived from familial amyloidotic polyneuropathy transgenic mice. Conditioned medium from the cells contained variant transthyretin (TTR), and inhibited neurite outgrowth of neurons. TTR aggregates were observed in the Schwann cells and satellite cells of aged mice. Proteasome inhibition induced TTR aggregates as aggresomes in the cultured cells. These results support the hypothesis that Schwann cells contribute to neurodegeneration in familial amyloidotic polyneuropathy (FAP).
Assuntos
Neuropatias Amiloides Familiares/metabolismo , Degeneração Neural/metabolismo , Pré-Albumina/biossíntese , Células de Schwann/metabolismo , Neuropatias Amiloides Familiares/patologia , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Degeneração Neural/patologia , Células de Schwann/patologiaRESUMO
Galectin-1 (GAL-1), a member of a family of ß-galactoside binding animal lectins, is predominantly expressed in isolectin B4 (IB4)-binding small non-peptidergic (glial cell line-derived neurotrophic factor (GDNF)-responsive) sensory neurons in the sections of adult rat dorsal root ganglia (DRG), but its functional role and the regulatory mechanisms of its expression in the peripheral nervous system remain unclear. In the present study, both recombinant nerve growth factor (NGF) and GDNF (50 ng/ml) promoted neurite outgrowth from cultured adult rat DRG neurons, whereas GDNF, but not NGF, significantly increased the number of IB4-binding neurons and the relative protein expression of GAL-1 in the neuron-enriched culture of DRG. The GAL-1 expression in immortalized adult rat Schwann cells IFRS1 and DRG neuron-IFRS1 cocultures was unaltered by treatment with GDNF, which suggests that GDNF/GAL-1 signaling axis is more related to neurite outgrowth, rather than neuron-Schwann cell interactions. The GDNF-induced neurite outgrowth and GAL-1 upregulation were attenuated by anti-GDNF family receptor (RET) antibody and phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor LY294002, suggesting that the neurite-outgrowth promoting activity of GDNF may be attributable, at least partially, to the upregulation of GAL-1 through RET-PI3K pathway. On the contrary, no significant differences were observed between GAL-1 knockout and wild-type mice in DRG neurite outgrowth in the presence or absence of GDNF. Considerable immunohistochemical colocalization of GAL-3 with GAL-1 in DRG sections and GDNF-induced upregulation of GAL-3 in cultured DRG neurons imply the functional redundancy between these galectins.
Assuntos
Galectina 3/fisiologia , Gânglios Espinais/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Neuritos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Western Blotting , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos WistarRESUMO
Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 µg/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 µg/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.
Assuntos
Bainha de Mielina/metabolismo , Neurônios/citologia , Células de Schwann/citologia , Animais , Diferenciação Celular , Técnicas de Cocultura , Fatores de Crescimento Neural/farmacologia , Neuregulina-1/metabolismo , Neuritos/fisiologia , Neurônios/metabolismo , Células PC12 , Ratos , Células de Schwann/metabolismoRESUMO
We established spontaneously immortalized Schwann cell lines from long-term cultures of adult Fischer 344 rat dorsal root ganglia (DRG) and peripheral nerves. One of these cell lines, designated immortalized Fischer rat Schwann cells 1 (IFRS1), showed spindle-shaped morphology; immunoreactivity for S100, p75 neurotrophin receptor (p75(NTR) ), glial fibrillary acidic protein (GFAP), laminin, and vimentin; and mRNA expression of neurotrophic factors (NGF, GDNF, and CNTF), neurotrophin receptors (p75(NTR) , truncated TrkB, and TrkC), cell adhesion molecules (L1, NCAM, and N-cadherin), myelin proteins [P0, PMP22, and myelin-associated glycoprotein (MAG)], transcription factors (Krox20, Sox10, and Oct6), neuregulin-1 receptors (ErbB2 and ErbB3), and an orphan G protein-coupled receptor (Gpr126). Conditioned medium (CM) obtained from IFRS1 cells exhibited potent biological activity for the promotion of neuronal survival and neurite outgrowth of cultured adult rat DRG neurons. Furthermore, light and electron microscopic analyses revealed that IFRS1 cells were capable of myelinating neurites while in coculture with adult rat DRG neurons. These findings indicate that IFRS1 cells possess some biological properties of mature Schwann cells and that the coculture system with adult DRG neurons and IFRS1 cells can be a useful tool for the study of peripheral nerve degeneration and regeneration.
Assuntos
Comunicação Celular/fisiologia , Neurônios/fisiologia , Células de Schwann/fisiologia , Animais , Compostos Azo , Linhagem Celular Transformada/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Gânglios Espinais/citologia , Regulação da Expressão Gênica/fisiologia , Óperon Lac/genética , Microscopia Eletrônica de Transmissão/métodos , Proteína Básica da Mielina/metabolismo , Proteína P0 da Mielina/metabolismo , Naftalenos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios/efeitos dos fármacos , Nervos Periféricos/citologia , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Células de Schwann/química , Células de Schwann/ultraestrutura , Transfecção/métodos , Tubulina (Proteína)/metabolismoRESUMO
Mouse FBJ virus-induced osteosarcoma FBJ-S1 cells rich in GD1a are not readily metastatic, whereas FBJ-LL cells with low levels of GD1a are highly metastatic. GD1a was previously shown to suppress metastasis of mouse FBJ cells and to upregulate caveolin-1 and stromal interaction molecule 1 expression. The present study demonstrates that matrix metalloproteinase-9 (MMP-9) expression renders FBJ-LL cells invasive. MMP-9 is inversely regulated by GD1a, based upon four observations: MMP-9 mRNA content was 5 times higher in FBJ-LL cells than FBJ-S1 cells; a GD1a-re-expressing FBJ-LL cell variant produced through beta1,4GalNAcT-1 cDNA transfection expressed lower levels of MMP-9; exogenous addition of GD1a to FBJ-LL cells decreased MMP-9 production in a dose- and time-dependent manner; and treatment of GD1a-rich cells with D-PDMP or siRNA targeting St3gal2 decreased GD1a expression, but augmented MMP-9 expression. This is the first report demonstrating that GD1a negatively regulates expression of MMP-9 at the transcriptional level.
Assuntos
Gangliosídeos/metabolismo , Regulação da Expressão Gênica/genética , Metaloproteinase 9 da Matriz/genética , Transcrição Gênica/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Relação Dose-Resposta a Droga , Gangliosídeos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Morfolinas/farmacologia , Invasividade Neoplásica/genética , Metástase Neoplásica , Osteossarcoma/enzimologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacosRESUMO
GD1a was previously shown responsible for regulating cell motility, cellular adhesiveness to vitronectin, phosphorylation of c-Met and metastatic ability of mouse FBJ osteosarcoma cells. To determine the particular molecules regulated by GD1a, FBJ cells were assessed for tumor-related gene expression by semi-quantitative RT-PCR. Caveolin-1 and stromal interaction molecule 1 (Stim1) expression in FBJ-S1 cells, rich in GD1a, were found to be 6 and 4 times as much, respectively, than in FBJ-LL cells devoid of GD1a. Enhanced production of caveolin-1 in protein was confirmed by Western blotting. A low-metastatic FBJ-LL cell variant, having high GD1a expression through beta1-4GalNAcT-1 (GM2/GD2 synthase) cDNA transfection (Hyuga S, et al, Int J Cancer 83: 685-91, 1999), showed enhanced production of caveolin-1 and Stim1 in mRNA and protein, compared to mock-transfectant M5. Incubation of FBJ-M5 cells with exogenous GD1a augmented the expression of caveolin-1 in mRNA and protein and Stim1 in mRNA as well. Treatment of FBJ-S1 with fumonisin B1, an inhibitor of N-acylsphinganine synthesis, for 15 days caused the complete depletion of gangliosides and suppressed the expression of caveolin-1 and Stim1. St3gal5 siRNA transfected cells showed decreased expression of caveolin-1 and Stim1 mRNA, as well as St3gal5 mRNA. These findings clearly indicate ganglioside GD1a to be involved in the regulation of the transformation suppressor genes, caveolin-1 and Stim1. Moreover, treatment with GD1a of mouse melanoma B16 cells and human hepatoma HepG2 cells brought about elevated expression of caveolin-1 and Stim1.
Assuntos
Caveolina 1/biossíntese , Caveolina 1/genética , Gangliosídeos/fisiologia , Regulação da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Regulação para Cima/fisiologia , Animais , Canais de Cálcio , Bovinos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Molécula 1 de Interação EstromalRESUMO
Sialyl-Lewis x (sLeX), one of the major selectin ligands, is expressed on T and B cells in a differentiation or activation stage-specific manner. We have demonstrated before that sLeX expression and core 2 beta 1,6-N-acetylglucosaminyltransferase (C2GnT) were simultaneously regulated during precursor B (pre-B) cell differentiation. Three C2GnT family genes, designated C2GnT-1, -2, and -3, were previously identified, but their roles have not been fully examined. In this study, we have investigated the roles of C2GnTs in the regulation of sLeX expression level during pre-B cell differentiation comparing with alpha 1,3fucosyltransferase-VII (FucT-VII) and alpha 2,3sialyltransferase-IV (ST3Gal-IV). Overexpression of not FucT-VII and ST3Gal-IV but C2GnT-1 blocked the down-regulation of sLeX expression by differentiation induction. Neither C2GnT-2 nor -3 but C2GnT-1 transcript was mainly expressed in B lineage cell lines and bone marrow-derived B lineage cells. Significant down-regulation of C2GnT-1 of the three C2GnTs was observed in KM3 cells during differentiation. The expression of C2GnT-1 correlated well to sLeX expression and differentiation stage. Furthermore, introduction of short interfering RNA against C2GnT-1 markedly reduced C2GnT-1 expression and resulted in down-regulation of sLeX expression. These results suggest that not the other glycosyltransferases but C2GnT-1 regulates sLeX expression level during differentiation of pre-B cells, providing the cells with substrate of sLeX structure biosynthesis.