RESUMO
INTRODUCTION: We examined the effects of gastrocnemius eccentric contractions (ECs) on the sciatic nerve in rats. METHODS: Rats were divided randomly into the following 3 groups: control, 180EC (ECs with 180°/s angular velocity), and 30EC (ECs with 30°/s angular velocity). Twenty ECs were induced by electrical stimulation of the gastrocnemius. On days 3, 7, and 10 after the ECs, nerve conduction velocity (NCV) was measured, and sciatic nerve branches were harvested for analysis. RESULTS: A significant decrease in NCV was observed between the control and day-7 180EC. Significant reduction in the levels of myelin sheath protein zero (p0) between day 7 and day 3 180EC and a significant increase of macrophage-related protein and tyrosine kinase receptor C were observed between day 7 180EC and day 7 30EC. CONCLUSIONS: ECs with fast angular velocities induce functional and structural damage in innervating nerve.
Assuntos
Músculo Esquelético/lesões , Músculo Esquelético/inervação , Nervo Isquiático/lesões , Animais , Western Blotting , Estimulação Elétrica , Proteína GAP-43/biossíntese , Proteína GAP-43/genética , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica , Contração Isométrica/fisiologia , Macrófagos/metabolismo , Masculino , Contração Muscular/fisiologia , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Proteína P0 da Mielina/biossíntese , Proteína P0 da Mielina/genética , Condução Nervosa/fisiologia , Ratos , Ratos Wistar , Receptor trkC/metabolismoRESUMO
Myelin protein zero (P0 or MPZ) is a major myelin protein (â¼30 kDa) expressed in the peripheral nervous system (PNS) in terrestrial vertebrates. Several groups have detected a P0-related 36-kDa (or 35-kDa) protein that is expressed in the PNS as an antigen for the serum IgG of patients with neuropathy. The molecular structure and function of this 36-kDa protein are, however, still unknown. We hypothesized that the 36-kDa protein may be derived from P0 mRNA by stop codon readthrough. We found a highly conserved region after the regular stop codon in predicted sequences from the 3'-UTR of P0 in higher animals. MS of the 36-kDa protein revealed that both P0 peptides and peptides deduced from the P0 3'-UTR sequence were found among the tryptic fragments. In transfected cells and in an in vitro transcription/translation system, the 36-kDa molecule was also produced from the identical mRNA that produced P0. We designated this 36-kDa molecule as large myelin protein zero (L-MPZ), a novel isoform of P0 that contains an additional domain at the C terminus. In the PNS, L-MPZ was localized in compact myelin. In transfected cells, just like P0, L-MPZ was localized at cell-cell adhesion sites in the plasma membrane. These results suggest that L-MPZ produced by the stop codon readthrough mechanism is potentially involved in myelination. Since this is the first finding of stop codon readthrough in a common mammalian protein, detailed analysis of L-MPZ expression will help to understand the mechanism of stop codon readthrough in mammals.
Assuntos
Códon de Terminação/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteína P0 da Mielina/biossíntese , Animais , Doença Crônica , Códon de Terminação/genética , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Feminino , Células HeLa , Humanos , Camundongos , Pessoa de Meia-Idade , Proteína P0 da Mielina/genética , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Células NIH 3T3 , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Ratos , Ratos WistarRESUMO
We show that normal peripheral nerve myelination depends on strict dosage of the most abundantly expressed myelin gene, myelin protein zero (Mpz). Transgenic mice containing extra copies of Mpz manifested a dose-dependent, dysmyelinating neuropathy, ranging from transient perinatal hypomyelination to arrested myelination and impaired sorting of axons by Schwann cells. Myelination was restored by breeding the transgene into the Mpz-null background, demonstrating that dysmyelination does not result from a structural alteration or Schwann cell-extrinsic effect of the transgenic P(0) glycoprotein. Mpz mRNA overexpression ranged from 30-700%, whereas an increased level of P(0) protein was detected only in nerves of low copy-number animals. Breeding experiments placed the threshold for dysmyelination between 30 and 80% Mpz overexpression. These data reveal new points in nerve development at which Schwann cells are susceptible to increased gene dosage, and suggest a novel basis for hereditary neuropathy.
Assuntos
Doenças Desmielinizantes/congênito , Dosagem de Genes , Proteína P0 da Mielina/biossíntese , Proteína P0 da Mielina/genética , Doenças do Sistema Nervoso Periférico/congênito , Animais , Western Blotting , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/genética , Proteínas da Mielina/biossíntese , Proteínas da Mielina/genética , Especificidade de Órgãos , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura , Nervo Isquiático/metabolismo , Nervo Isquiático/ultraestruturaRESUMO
To investigate the role of erbB signaling in the interactions between peripheral axons and myelinating Schwann cells, we generated transgenic mice expressing a dominant-negative erbB receptor in these glial cells. Mutant mice have delayed onset of myelination, thinner myelin, shorter internodal length, and smaller axonal caliber in adulthood. Consistent with the morphological defects, transgenic mice also have slower nerve conduction velocity and defects in their responses to mechanical stimulation. Molecular analysis indicates that erbB signaling may contribute to myelin formation by regulating transcription of myelin genes. Analysis of sciatic nerves showed a reduction in the levels of expression of myelin genes in mutant mice. In vitro assays revealed that neuregulin-1 (NRG1) induces expression of myelin protein zero (P0). Furthermore, we found that the effects of NRG1 on P0 expression depend on the NRG1 isoform used. When NRG1 is presented to Schwann cells in the context of cell-cell contact, type III but not type I NRG1 regulates P0 gene expression. These results suggest that disruption of the NRG1-erbB signaling pathway could contribute to the pathogenesis of peripheral neuropathies with hypomyelination and neuropathic pain.
Assuntos
Fibras Nervosas Mielinizadas/metabolismo , Neuregulina-1/metabolismo , Proteínas Oncogênicas v-erbB/genética , Nervos Periféricos/crescimento & desenvolvimento , Células de Schwann/metabolismo , Sensação/genética , Animais , Axônios/metabolismo , Axônios/patologia , Comunicação Celular/genética , Diferenciação Celular/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Transgênicos , Proteína P0 da Mielina/biossíntese , Proteína P0 da Mielina/genética , Proteínas da Mielina/biossíntese , Proteínas da Mielina/genética , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Fibras Nervosas Mielinizadas/patologia , Condução Nervosa/genética , Neuregulina-1/genética , Neuregulina-1/farmacologia , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/fisiopatologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Células de Schwann/patologia , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/metabolismo , Nervo Isquiático/patologiaRESUMO
Pituitary gland is a well-known endocrine tissue. The hypothalamo-neurohypophysial system, containing arginine vasopressin and oxytocin, shows a reversible morphological reorganization of both neurons and glial cells during chronic physiological stimulations. Since many signal transducing and cell adhesion molecules (CAMs) are recovered in membrane microdomain (MD) fractions, MDs are considered as signaling platforms of cells. In order to know the molecular background for these endocrine systems, we characterized MD-components derived from rat pituitary and found specific enrichment of several proteins in the fraction. One of them was identified as myelin protein zero (P0) with mass analysis and this result was further confirmed by a result that a specific antibody to this protein reacted to the authentic P0 protein in the myelin fraction of rat sciatic nerve. P0 is one of type-I transmembrane CAMs and a major structural component of mammalian peripheral nerve myelin. In mammals, expression of P0 has been considered to be restricted to peripheral nervous system. This result however indicates that P0 expresses more widely and its enrichment in the MD-fraction from rat pituitary suggests the participation in cell-cell communications.
Assuntos
Microdomínios da Membrana/metabolismo , Proteína P0 da Mielina/biossíntese , Hipófise/metabolismo , Transdução de Sinais/fisiologia , Animais , Anticorpos Monoclonais/química , Arginina Vasopressina/metabolismo , Imuno-Histoquímica , Microdomínios da Membrana/química , Microdomínios da Membrana/imunologia , Proteína P0 da Mielina/química , Proteína P0 da Mielina/imunologia , Proteína P0 da Mielina/isolamento & purificação , Ocitocina/metabolismo , Hipófise/química , Hipófise/citologia , Hipófise/imunologia , Ratos , Ratos Wistar , Nervo Isquiático/química , Nervo Isquiático/citologia , Nervo Isquiático/imunologia , Nervo Isquiático/metabolismoRESUMO
Axonal degeneration is the major cause of permanent neurological disability in individuals with inherited diseases of myelin. Axonal and neuronal changes that precede axonal degeneration, however, are not well characterized. We show here that dysmyelinated lower motor neurons retract and regenerate dysfunctional presynaptic terminals, leading to severe neurological disability before axonal degeneration. In addition, dysmyelination led to a decreased synaptic quantal content, an indicator of synaptic dysfunction. The amplitude and rise time of miniature endplate potentials were also increased, but these changes were primarily consistent with an increase in the passive membrane properties of the transgenic muscle fibers. Maintenance of synaptic connections should be considered as a therapeutic target for slowing progression of neurological disability in primary diseases of myelin.
Assuntos
Doenças Desmielinizantes/fisiopatologia , Neuropatia Hereditária Motora e Sensorial/fisiopatologia , Neurônios Motores/fisiologia , Regeneração Nervosa , Junção Neuromuscular/fisiopatologia , Terminações Pré-Sinápticas/fisiologia , Animais , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Avaliação da Deficiência , Modelos Animais de Doenças , Progressão da Doença , Eletromiografia , Neuropatia Hereditária Motora e Sensorial/genética , Neuropatia Hereditária Motora e Sensorial/patologia , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiopatologia , Atrofia Muscular/patologia , Proteína P0 da Mielina/biossíntese , Proteína P0 da Mielina/genética , Regeneração Nervosa/genética , Junção Neuromuscular/patologia , Terminações Pré-Sinápticas/patologia , Células de Schwann/metabolismo , Células de Schwann/patologia , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/patologiaRESUMO
Schwann cells are the myelinating glia of the peripheral nervous system, and their development is regulated by various growth factors, such as neuregulin, platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). However, the mechanism of intracellular signaling pathways following these ligand stimuli in Schwann cell differentiation remains elusive. Here, we demonstrate that in cultured Schwann cells, neuregulin and PDGF suppressed the expression of myelin-associated protein markers, whereas IGF-I promoted it. Although these ligands activated common downstream signaling pathways [i.e., extracellular signal-regulated kinase (Erk) and phosphatidylinositol-3-kinase (PI3K)-Akt pathways], the profiles of activation varied among ligands. To elucidate the function of these pathways and the mechanisms underlying Schwann cell differentiation, we used adenoviral vectors to selectively activate or inactivate these pathways. We found that the selective activation of Erk pathways suppressed Schwann cell differentiation, whereas that of PI3K pathways promoted it. Furthermore, lithium chloride, a modulator of glycogen synthase kinase-3beta (GSK-3beta) promoted Schwann cell differentiation, suggesting the involvement of GSK-3beta as a downstream molecule of PI3K-Akt pathways. Selective activation of PI3K pathways in Schwann cells by gene transfer also demonstrated increased myelination in in vitro Schwann cell-DRG neuron cocultures and in vivo allogenic nerve graft experiments. We conclude that signals mediated by PI3K-Akt are crucial for initiation of myelination and that the effects of growth factors are primarily dependent on the balance between Erk and PI3K-Akt activation. Our results also propose the possibility of augmenting Schwann cell functions by modulating intracellular signals in light of future cell therapies.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Bainha de Mielina/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Células de Schwann/enzimologia , Nervo Isquiático/transplante , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Células Cultivadas/fisiologia , Técnicas de Cocultura , Colforsina/farmacologia , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/genética , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/fisiologia , Glicogênio Sintase Quinase 3 beta , Fator de Crescimento Insulin-Like I/farmacologia , Cloreto de Lítio/farmacologia , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/fisiologia , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/genética , Proteína P0 da Mielina/biossíntese , Proteína P0 da Mielina/genética , Glicoproteína Associada a Mielina/biossíntese , Glicoproteína Associada a Mielina/genética , Neuregulina-1/farmacologia , Neurônios Aferentes/citologia , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/fisiologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/fisiologia , Nervo Isquiático/citologia , Transdução de Sinais/efeitos dos fármacos , Transplante Homólogo , Proteínas ras/fisiologiaRESUMO
AIM: Using proteomics, we aimed to identify the proteins differentially regulated by dexamethasone in the mouse cochlea based on mass-spectrometry data. BACKGROUND: Glucocorticoid therapy is widely used for many forms of sensorineural hearing loss; however, the molecular mechanism of its action in the cochlea remains poorly understood. METHODS: Dexamethasone or control saline was intratympanically applied to the cochleae of mice. Twelve hours after application, proteins differentially regulated by dexamethasone in the cochlea were analyzed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-mass spectrometry. Next, dexamethasone-dependent regulation of these proteins was verified in the cochleae of mice with noise-induced hearing loss (NIHL) and systemic administration of dexamethasone by western blotting. Immunolocalizations of these proteins were examined in cochleae with NIHL. RESULTS: A total of 247 proteins with a greater than 95% confidence interval of protein identification were found, and 11 differentially expressed proteins by dexamethasone were identified by the iTRAQ-mass spectrometry. One protein, myelin protein zero (Mpz), was upregulated (1.870 ± 0.201-fold change, p < 0.01) at 6 hours post-systemic dexamethasone and noise exposure in a mouse model of NIHL. Heat shock protein 70 (Hsp70) was downregulated (0.511 ± 0.274-fold change, p < 0.05) at 12 hours post-systemic dexamethasone. Immunohistochemistry confirmed Mpz localization to the efferent and afferent processes of the spiral neurons, whereas Hsp70 showed a more ubiquitous expression pattern in the cochlea. CONCLUSION: Both Mpz and Hsp70 have been reported to be closely associated with sensorineural hearing loss in humans. Dexamethasone significantly modulated the expression levels of these proteins in the cochleae of mice.
Assuntos
Anti-Inflamatórios/farmacologia , Cóclea/metabolismo , Surdez/genética , Dexametasona/farmacologia , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Proteína P0 da Mielina/biossíntese , Proteína P0 da Mielina/genética , Proteômica/métodos , Administração Tópica , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Cóclea/efeitos dos fármacos , Surdez/tratamento farmacológico , Dexametasona/administração & dosagem , Dexametasona/uso terapêutico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Perda Auditiva Provocada por Ruído/genética , Perda Auditiva Provocada por Ruído/metabolismo , Perda Auditiva Neurossensorial/tratamento farmacológico , Perda Auditiva Neurossensorial/genética , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos C57BL , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Membrana TimpânicaRESUMO
Insulin-like growth factor-I (IGF-I) in vivo or in the presence of other permissive factors can promote myelination in the central nervous system. In the current study, we examine the role of IGF-I in the myelination of peripheral nerves. In rat cocultures of dorsal root ganglia (DRG) and Schwann cells (SC) grown in serum- and insulin-free defined medium, IGF-I induces a dose dependent upregulation in myelin proteins such as P0, corresponding to maximal SC ensheathment. Furthermore, IGF-I is essential in promoting a dose-dependent, long-term myelination of DRG sensory axons. In the absence of IGF-I, axons and SC survive, but fail to myelinate. In the presence of 10 nM IGF-I, 59% of axons are myelinated at 21 days, whereas in the absence of IGF-I myelination fails to occur. Maximum SC ensheathment occurs 48 hours after addition of IGF-I. If IGF-I is withdrawn at 48 hours, axon segregation by SC persists, however, most axons and SC do not exhibit a one-to-one relationship and little myelination is observed. IGF-I is important in myelination and is critical not only for initial SC ensheathment of the axon and upregulation of myelin proteins, but also for sustained myelination. Furthermore, IGF-I associated axonal size is not the sole determinant for myelination.
Assuntos
Gânglios Espinais/citologia , Fator de Crescimento Insulin-Like I/farmacologia , Bainha de Mielina/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Células de Schwann/fisiologia , Animais , Células Cultivadas , Feto/citologia , Gânglios Espinais/embriologia , Microscopia Eletrônica , Proteína P0 da Mielina/biossíntese , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/metabolismo , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/ultraestrutura , Ratos , Ratos Sprague-Dawley , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestruturaRESUMO
We describe a family carrying the Thr148Met mutation in the P0 gene. Contrary to other neuropathies caused by myelin gene defects, no demyeliantion could be found in our biopsies. Based on follow up examinations, extensive morphometry and immunohistochemical analysis we suggest that the mild hypomyelination documented in our family secondarily causes axonal degeneration and axonal loss of large and small fibers which predominates the clinical picture.
Assuntos
Axônios/patologia , Axônios/fisiologia , Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/fisiopatologia , Proteína P0 da Mielina/genética , Adulto , Substituição de Aminoácidos , Biópsia , Contagem de Células , Doença de Charcot-Marie-Tooth/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Proteína P0 da Mielina/biossíntese , Bainha de Mielina/patologia , Glicoproteína Associada a Mielina/biossíntese , Condução Nervosa , Nervo Sural/metabolismo , Nervo Sural/patologia , Nervo Sural/ultraestruturaRESUMO
The data here reported show that the gene expression of the glycoprotein Po and of the myelin basic protein, the major components of myelin in the peripheral nervous system, dramatically decreases with ageing in the sciatic nerve of normal male rats. A one-month treatment with dihydroprogesterone, the 5alpha-reduced derivative of progesterone, is able to partially restore the fall in Po gene expression occurring in the sciatic nerve of aged male rats, without significantly modifying the gene expression of the myelin basic protein. In cultures of neonatal Schwann cells (the peripheral nervous system elements involved in the synthesis of myelin), the addition of progesterone and of dihydroprogesterone significantly increases Po gene expression; the 3alpha-reduced metabolite of dihydroprogesterone, tetrahydroprogesterone proved to be even more effective. These data suggest that the effect of progesterone is linked to its conversion into dihydroprogesterone and especially into tetrahydroprogesterone, since Schwann cells possess the 5alpha-reductase-3alpha-hydroxysteroid dehydrogenase system. The data provide the first demonstration that ageing decreases the gene expression of two major components of the peripheral myelin in the sciatic nerve; they also show that this phenomenon may be partially reversed by progesterone derivatives, which might act by stimulating Po gene expression in the Schwann cells.
Assuntos
20-alfa-Di-Hidroprogesterona/farmacologia , Envelhecimento/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Básica da Mielina/genética , Proteína P0 da Mielina/genética , Pregnanolona/farmacologia , Nervo Isquiático/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Animais , Animais Recém-Nascidos , Northern Blotting , Células Cultivadas , Masculino , Proteína Básica da Mielina/biossíntese , Proteína P0 da Mielina/biossíntese , Oligodendroglia , Ratos , Ratos Sprague-Dawley , Células de Schwann , Nervo Isquiático/efeitos dos fármacosRESUMO
Exposure of developing rats to a diet containing elemental tellurium systemically inhibits cholesterol synthesis at the level of squalene epoxidase. At high tellurium exposure levels (> 0.1% in the diet), there is an associated segmental demyelination of the PNS. Low levels of dietary tellurium (0.0001%) led to in vivo inhibition of squalene epoxidase activity in sciatic nerve, and inhibition increased with increasing exposure levels. With increasing dose and increasing exposure times, there was an increasing degree of demyelination and increasing down-regulation of mRNA levels for myelin P0 protein, ceramide galactosyltransferase (rate-limiting enzyme in cerebroside synthesis), and HMG-CoA reductase (rate-limiting enzyme in cholesterol synthesis). Because these were all down-regulated in parallel, we conclude there is coordinate regulation of the entire program for myelin synthesis in Schwann cells. An anomaly was that at early time points and low tellurium levels, mRNA levels for HMG-CoA reductase were slightly elevated, presumably in response to tellurium-induced sterol deficits. We suggest the eventual down-regulation relates to a separate mechanism by which Schwann cells regulate cholesterol synthesis, related to the need for coordinate synthesis of myelin components. Levels of mRNA for the low-affinity nerve growth factor receptor (indicator of alterations in axon-Schwann cell interactions) and for lysozyme (marker for phagocytic macrophages) were both up-regulated in a dose- and time-dependent manner which correlated with the presence of segmental demyelination. Levels of mRNA coding for myelin-related proteins were down-regulated at low tellurium exposure levels, without demyelination or up-regulation of nerve growth factor receptor. This suggests the down-regulation is related to the tellurium-induced cholesterol deficit, and not to the loss of axonal contact associated with early stages of demyelination or to the entry of activated macrophages.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Proteína P0 da Mielina/biossíntese , Proteínas da Mielina/biossíntese , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Telúrio/farmacologia , Acetatos/metabolismo , Ração Animal , Animais , Colesterol/biossíntese , Hidroximetilglutaril-CoA Redutases/biossíntese , Cinética , Masculino , Oxigenases/antagonistas & inibidores , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Células de Schwann/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Esqualeno/metabolismo , Esqualeno Mono-Oxigenase , Telúrio/administração & dosagem , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
The present results show that androgens are able to modulate the Po gene expression in different models. In particular, we have shown that: (1) the messenger for the androgen receptor (AR) is present in the rat sciatic nerve but not in cultured Schwann cells; (2) castration induces a decrease of Po mRNA levels in the sciatic nerve of male rats, which is counteract by the subsequent treatment with dihydrotestosterone (DHT), the 5alpha-reduced metabolite of testosterone; (3) castration is also able to significantly decrease in the sciatic nerve the activity of the enzyme 5alpha-reductase (which converts testosterone into DHT); and (4) DHT is able to stimulate Po gene expression in cultured Schwann cells. These observations seem to indicate that androgens may exert their effect on Po gene expression via indirect mechanisms; modulation of neuronal influences reaching the Schwann cells through the binding of the androgen to the AR present in neurons may be postulated. However, alternative mechanisms may also be taken in consideration. The data presented suggest indeed that androgens might act on Schwann cells via the progesterone receptor (PR) rather than the AR. It has been observed that: (1) the messenger for PR is present in Schwann cells; (2) DHT may activate the transcriptional activity of a PR-responsive gene by binding to the PR; and (3) putative steroid responsive elements have been described in this paper to be present in the Po promoter region.
Assuntos
Di-Hidrotestosterona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína P0 da Mielina/biossíntese , Células de Schwann/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Testosterona/farmacologia , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Animais , Células Cultivadas , Masculino , Proteína P0 da Mielina/genética , Orquiectomia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Androgênicos/efeitos dos fármacos , Receptores de Progesterona/biossíntese , Receptores de Progesterona/efeitos dos fármacos , Receptores de Progesterona/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Schwann/metabolismo , Nervo Isquiático/citologia , Nervo Isquiático/metabolismo , Especificidade por SubstratoRESUMO
Insulin-like growth factor-I (IGF-I) promotes the proliferation and differentiation of Schwann cells (SC). We use SC/dorsal root ganglion neuron (DRG) cocultures to examine the effects of IGF-I on the interaction between axons and SC. As SC extend processes toward the axon in the presence of IGF-I, these processes attach to and ensheath axons. Continued IGF-I exposure leads to enhanced P0 expression and long-term myelination. No myelination occurs in the absence of IGF-I. These data imply that IGF-I is critical not only for SC attachment and ensheathment of axons but also for long-term myelination.
Assuntos
Gânglios Espinais/fisiologia , Fator de Crescimento Insulin-Like I/farmacologia , Bainha de Mielina/fisiologia , Neurônios/fisiologia , Nervos Periféricos/fisiologia , Células de Schwann/fisiologia , Animais , Animais Recém-Nascidos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Proteína P0 da Mielina/biossíntese , Proteína P0 da Mielina/genética , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/ultraestrutura , Neurônios/citologia , Nervos Periféricos/citologia , Ratos , Ratos Sprague-Dawley , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacosRESUMO
In mammals, P(0) expression is thought to be restricted to the peripheral nervous system (PNS), whereas in some other vertebrates it is expressed not only in the PNS but also in the central nervous system (CNS). Previously we reported that P(0) is expressed in the rat spinal cord and that its glycosylation state changes with age. In this report, we determine, by immunohistochemical and immunocytochemical analyses, that the neurons in the spinal cord express P(0). Furthermore, our data show that the number of neurons expressing P(0) decreases and their sizes become smaller with age. Thus, the results emphasize the importance of neurons expressing P(0) in the spinal cord in the formation and maintenance of the neural network.
Assuntos
Envelhecimento , Proteína P0 da Mielina/biossíntese , Neurônios/metabolismo , Medula Espinal/metabolismo , Animais , Contagem de Células , Tamanho Celular , Imuno-Histoquímica , Ratos , Medula Espinal/citologiaRESUMO
The rat dorsal root ganglia (DRG) model is a long-standing in vitro model for analysis of myelination in the peripheral nervous system. For performing systematic, high throughput analysis with transgenic animals, a simplified BL6 mouse protocol is indispensable. Here we present a stable and reliable protocol for myelinating co-cultures producing a high myelin ratio using cells from C57BL/6 mice. As an easy accessible and operable method, Sudan staining proved to be efficient in myelin detection for fixed cultures. Green fatty acid stain turned out to be highly reliable for analysis of the dynamic biological processes of myelination in vital cultures. Once myelinated we were able to induce demyelination by the addition of forskolin into the model system. In addition, we provide an optimised rat DRG protocol with significantly improved myelin ratio and a comparison of the protocols presented. Our results strengthen the value of ex vivo myelination models in neurobiology.
Assuntos
Bainha de Mielina/fisiologia , Animais , Compostos Azo , Western Blotting , Compostos de Boro , Células Cultivadas/fisiologia , Colforsina/farmacologia , Corantes , Doenças Desmielinizantes , Corantes Fluorescentes , Gânglios Espinais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Microscopia de Fluorescência , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/genética , Proteína P0 da Mielina/biossíntese , Proteína P0 da Mielina/genética , Bainha de Mielina/ultraestrutura , Naftalenos , Ácidos Palmíticos , Cultura Primária de Células , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Células de Schwann/metabolismo , Nervo Isquiático/citologia , Especificidade da Espécie , Coloração e Rotulagem/métodosRESUMO
BACKGROUND: Electrical stimulation (ES) has been shown to promote nerve regeneration when it was applied to the proximal nerve stump. However, the possible beneficial effect of establishing a local electrical environment between a large nerve defect on nerve regeneration has not been reported in previous studies. The present study attempted to establish a local electrical environment between a large nerve defect, and examined its effect on nerve regeneration and functional recovery. METHODOLOGY/FINDINGS: In the present study, a conductive scaffold was constructed and used to bridge a 15 mm sciatic nerve defect in rats, and intermittent ES (3 V, 20 Hz) was applied to the conductive scaffold to establish an electrical environment at the site of nerve defect. Nerve regeneration and functional recovery were examined after nerve injury repair and ES. We found that axonal regeneration and remyelination of the regenerated axons were significantly enhanced by ES which was applied to conductive scaffold. In addition, both motor and sensory functional recovery was significantly improved and muscle atrophy was partially reversed by ES localized at the conductive scaffold. Further investigations showed that the expression of S-100, BDNF (brain-derived neurotrophic factor), P0 and Par-3 was significantly up-regulated by ES at the conductive scaffold. CONCLUSIONS/SIGNIFICANCE: Establishing an electrical environment with ES localized at the conductive scaffold is capable of accelerating nerve regeneration and promoting functional recovery in a 15 mm nerve defect in rats. The findings provide new directions for exploring regenerative approaches to achieve better functional recovery in the treatment of large nerve defect.
Assuntos
Axônios/fisiologia , Estimulação Elétrica , Bainha de Mielina/metabolismo , Regeneração Nervosa/fisiologia , Potenciais de Ação , Animais , Axônios/patologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Proteínas de Transporte/biossíntese , Quitosana/química , Modelos Animais de Doenças , Eletrofisiologia/métodos , Masculino , Proteína P0 da Mielina/biossíntese , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas S100/biossíntese , Nervo Isquiático/patologia , Regulação para CimaRESUMO
Previous studies have demonstrated that electrical stimulation (ES) enhances axonal regeneration following central and peripheral nerve injury. However, the effect of ES on peripheral remyelination after nerve damage has been investigated less, and the mechanism underlying its action remains unclear. In the present study, neuron/Schwann cell (SC) co-cultures in vitro and crush-injured sciatic nerves in rats were subjected to 1 h of continuous ES (20 Hz, 100 micros, 3 V). Electron microscopy and nerve morphometry were performed to investigate the extent of regenerated nerve myelination. The expression profiles of P0, Par-3 and brain-derived neurotrophic factor (BDNF) in vitro and in vivo were examined by western blotting. We reported that 20 Hz ES increased the number of regenerated and myelinated axons at 4 and 8 weeks after injury. P0 level in the ES-treated groups, as well as myelin sheath thickness, were enhanced compared with the controls. The earlier peak Par-3 in the ES-treated groups indicated earlier initiation of SC myelination. Moreover, the similar results were achieved in the cell co-culture. Additionally, brief ES significantly elevated BDNF expression in co-cultured cells and nerve tissues. In conclusion, ES of the site of nerve injury potentiates axonal regrowth and myelin maturation during peripheral nerve regeneration. Further, the therapeutic actions of ES on myelination that is mediated via enhanced BDNF signals, which driving the promyelination effect on SCs at the onset of myelination.
Assuntos
Terapia por Estimulação Elétrica , Bainha de Mielina/patologia , Fatores de Crescimento Neural/biossíntese , Nervo Isquiático/metabolismo , Animais , Axônios/ultraestrutura , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Proteínas de Transporte/biossíntese , Células Cultivadas , Técnicas de Cocultura , Proteína P0 da Mielina/biossíntese , Bainha de Mielina/metabolismo , Regeneração Nervosa , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Células de Schwann/metabolismo , Células de Schwann/patologia , Nervo Isquiático/lesões , Nervo Isquiático/patologiaRESUMO
The interleukin-6 (IL-6) family of cytokines is thought to be involved in the development and regeneration of peripheral nerves; however, their roles in myelination remain unclear. In this study, we examined the effects of IL-6 on the expression of genes for compact myelin proteins using Schwann cell cultures prepared by multiple explantation of adult rat sciatic nerves. In semi-quantitative reverse transcription-polymerase chain reaction analysis, stimulation of Schwann cells with IL-6 significantly increased the mRNA level of peripheral myelin protein 22 (PMP22), but not those of myelin protein zero and myelin basic protein. The increase in PMP22 mRNA was markedly suppressed by AG490, a Janus kinase 2 (JAK2) inhibitor, but not significantly by PD098059, a mitogen-activated protein kinase inhibitor. Immunocytochemical staining revealed that IL-6 enhanced immunoreactivities for the phosphorylated forms of both JAK2 and signal transducer and activator of transcription 3 (STAT3), as well as that for PMP22. These results indicate that IL-6 can enhance PMP22 production in Schwann cells via a JAK2-dependent pathway by probably activating STAT3 and thus may contribute to myelination.
Assuntos
Interleucina-6/fisiologia , Janus Quinase 2/fisiologia , Proteínas da Mielina/biossíntese , Células de Schwann/metabolismo , Animais , Células Cultivadas , Receptor gp130 de Citocina/fisiologia , Interleucina-6/farmacologia , Janus Quinase 2/antagonistas & inibidores , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/genética , Proteína P0 da Mielina/biossíntese , Proteína P0 da Mielina/genética , Proteínas da Mielina/genética , Fosforilação , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Nervo Isquiático/citologia , Nervo Isquiático/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Differential targeting of myelin proteins to multiple, biochemically and functionally distinct Schwann cell plasma membrane domains is essential for myelin formation. In this study, we investigated whether the myelin protein P0 contains targeting signals using Madin-Darby canine kidney (MDCK) cells. By confocal microscopy, P0 was localized to MDCK cell basolateral membranes. C-terminal deletion resulted in apical accumulation, and stepwise deletions defined a 15-mer region that was required for basolateral targeting. Alanine substitutions within this region identified the YAML sequence as a functional tyrosine-based targeting signal, with the ML sequence serving as a secondary leucine-based signal. Replacement of the P0 ectodomain with green fluorescent protein altered the distribution of constructs lacking the YAML signal. Coexpression of the myelin-associated glycoprotein did not alter P0 distribution in MDCK cells. The results indicate that P0 contains a hierarchy of targeting signals, which may contribute to P0 localization in myelinating Schwann cells and the pathogenesis in human disease.