RESUMO
The transplantation of rodent Schwann cells (SCs) provides anatomical and functional restitution in a variety of spinal cord injury (SCI) models, supporting the recent translation of SCs to phase 1 clinical trials for human SCI. Whereas human (Hu)SCs have been examined experimentally in a complete SCI transection paradigm, to date the reported behavior of SCs when transplanted after a clinically relevant contusive SCI has been restricted to the use of rodent SCs. Here, in a xenotransplant, contusive SCI paradigm, the survival, biodistribution, proliferation and tumorgenicity as well as host responses to HuSCs, cultured according to a protocol analogous to that developed for clinical application, were investigated. HuSCs persisted within the contused nude rat spinal cord through 6 months after transplantation (longest time examined), exhibited low cell proliferation, displayed no evidence of tumorigenicity and showed a restricted biodistribution to the lesion. Neuropathological examination of the CNS revealed no adverse effects of HuSCs. Animals exhibiting higher numbers of surviving HuSCs within the lesion showed greater volumes of preserved white matter and host rat SC and astrocyte ingress as well as axon ingrowth and myelination. These results demonstrate the safety of HuSCs when employed in a clinically relevant experimental SCI paradigm. Further, signs of a potentially positive influence of HuSC transplants on host tissue pathology were observed. These findings show that HuSCs exhibit a favorable toxicity profile for up to 6 months after transplantation into the contused rat spinal cord, an important outcome for FDA consideration of their use in human clinical trials.
Assuntos
Regeneração Nervosa/fisiologia , Células de Schwann/fisiologia , Células de Schwann/transplante , Traumatismos da Medula Espinal/cirurgia , Adulto , Fatores Etários , Animais , Antígenos Nucleares/metabolismo , Proteínas de Ciclo Celular , Proliferação de Células/fisiologia , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Ratos , Ratos Nus , Receptor de Fator de Crescimento Neural/metabolismo , Traumatismos da Medula Espinal/mortalidade , Nervo Sural/citologia , Fatores de Tempo , Adulto JovemRESUMO
Myelinating Schwann cells (SCs) are highly plastic cells that are able to dedifferentiate and re-enter the cell cycle. However, the molecular signals controlling dedifferentiation are not completely understood. Because a connection between mitogenic signaling and myelin loss has been suggested, we investigated the role of cAMP, a strong inducer of the myelinating phenotype, and mitogenic factors activating receptor tyrosine kinases (RTKs) on SC dedifferentiation. We herein provide evidence indicating that cAMP was required to not only initiate but also maintain a state of differentiation because SCs rapidly dedifferentiated and became competent to resume proliferation upon the removal of cAMP stimulation. Surprisingly, isolated SCs could undergo multiple cycles of differentiation and dedifferentiation upon cAMP addition and removal, respectively, in the absence of mitogenic factors and without entering the cell cycle. Conversely, the activation of RTKs and the ERK cascade by a variety of growth factors, including neuregulin, was not sufficient to initiate dedifferentiation in the presence of cAMP. Importantly, a reduction of cAMP triggered dedifferentiation through a mechanism that required JNK, rather than ERK, activity and an induction of the expression of c-Jun, a transcriptional inhibitor of myelination. In summary, the reversible transition from an undifferentiated to a myelinating state was dependent on cAMP but independent of RTK signaling and cell cycle progression, further indicating that dedifferentiation and proliferation are uncoupled and differentially regulated events in SCs.
Assuntos
Desdiferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/farmacologia , MAP Quinase Quinase 4/metabolismo , Células de Schwann/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Desdiferenciação Celular/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Neurregulinas/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Células de Schwann/citologia , Transdução de Sinais/fisiologiaRESUMO
Motoneuron death can occur over several spinal levels with disease or trauma, resulting in muscle denervation. We tested whether cotransplantation of embryonic neurons with 1 or more neurotrophic factors into peripheral nerve improved axon regeneration, muscle fiber area, reinnervation, and function to a greater degree than cell transplantation alone. Sciatic nerves of adult Fischer rats were cut to denervate muscles; 1 week later, embryonic ventral spinal cord cells (days 14-15) were transplanted into the tibial nerve stump as the only source of neurons for muscle reinnervation. Factors that promote motoneuron survival (cardiotrophin 1; fibroblast growth factor 2; glial cell line-derived neurotrophic factor; insulin-like growth factor 1; leukemia inhibitory factor; and hepatocyte growth factor) were added to the transplant individually or in combinations. Inclusion of a single factor with the cells resulted in comparable myelinated axon counts, muscle fiber areas, and evoked electromyographic activity to cells alone 10 weeks after transplantation. Only cell transplantation with glial cell line-derived neurotrophic factor, hepatocyte growth factor, and insulin-like growth factor 1 significantly increased motoneuron survival, myelinated axon counts, muscle reinnervation, and evoked electromyographic activity compared with cells alone. Thus, immediate application of a specific combination of factors to dissociated embryonic neurons improves survival of motoneurons and the long-term function of reinnervated muscle.
Assuntos
Sobrevivência Celular/fisiologia , Neurônios Motores/fisiologia , Músculo Esquelético/fisiopatologia , Fatores de Crescimento Neural/farmacologia , Neurônios/transplante , Neuropatia Ciática/terapia , Transplante de Células-Tronco , Animais , Axônios/fisiologia , Eletromiografia , Potencial Evocado Motor , Feminino , Denervação Muscular , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/inervação , Fibras Nervosas Mielinizadas/fisiologia , Fatores de Crescimento Neural/metabolismo , Regeneração Nervosa/fisiologia , Junção Neuromuscular/fisiopatologia , Neurônios/fisiologia , Ratos , Ratos Endogâmicos F344 , Nervo Isquiático/lesões , Nervo Isquiático/fisiologia , Neuropatia Ciática/fisiopatologia , Medula Espinal/embriologiaRESUMO
The expression of myelination-associated genes (MGs) can be induced by cyclic adenosine monophosphate (cAMP) elevation in isolated Schwann cells (SCs). To further understand the effect of known SC mitogens in the regulation of SC differentiation, we studied the response of SCs isolated from adult nerves to combined cAMP, growth factors, including neuregulin, and serum. In adult SCs, the induction of MGs by cAMP coincided with the loss of genes expressed in non-myelin-forming SCs and with a change in cell morphology from a bipolar to an expanded epithelial-like shape. Prolonged treatment with high doses of cAMP-stimulating agents, as well as low cell density, was required for the induction of SC differentiation. Stimulation with serum, neuregulin alone, or other growth factors including PDGF, IGF and FGF, increased SC proliferation but did not induce the expression of MGs or the associated morphological change. Most importantly, when these factors were administered in combination with cAMP-stimulating agents, SC proliferation was synergistically increased without reducing the differentiating activity of cAMP. Even though the initiation of DNA synthesis and the induction of differentiation were mostly incompatible events in individual cells, SCs were able to differentiate under conditions that also supported active proliferation. Overall, the results indicate that in the absence of neurons, cAMP can trigger SC re-differentiation concurrently with, but independently of, growth factor signaling.
Assuntos
AMP Cíclico/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Bainha de Mielina/genética , Células de Schwann/fisiologia , Animais , Contagem de Células , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , DNA/biossíntese , Fatores de Crescimento de Fibroblastos/metabolismo , Gânglios Espinais/fisiologia , Expressão Gênica , Neurregulinas/metabolismo , Neurônios/fisiologia , Fenótipo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ratos , Células de Schwann/citologia , Soro/metabolismoRESUMO
In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Células de Schwann/metabolismo , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Epinefrina/metabolismo , Fibroblastos/metabolismo , Humanos , Dados de Sequência Molecular , Neuregulina-1 , Homologia de Sequência de AminoácidosRESUMO
N-cadherin and beta-catenin are involved in cell adhesion and cell cycle in tumor cells and neural crest. Both are expressed at key stages of Schwann cell (SC) development, but little is known about their function in the SC lineage. We studied the role of these molecules in adult rat derived SC-embryonic dorsal root ganglion cocultures by using low-Ca(2+) conditions and specific blocking antibodies to interfere with N-cadherin function and by using small interfering RNA (siRNA) to decrease beta-catenin expression in both SC-neuron cocultures and adult rat-derived SC monocultures. N-cadherin blocking conditions decreased SC-axon association and reduced axon-induced SC proliferation. In SC monocultures, beta-catenin reduction diminished the proliferative response of SCs to the mitogen beta1-heregulin, and, in SC-DRG cocultures, beta-catenin reduction inhibited axon-contact-dependent SC proliferation. Stimulation of SC cultures with beta1-heregulin increased total beta-catenin protein amount, phosphorylation of GSK-3beta and beta-catenin presence in nuclear extracts. In conclusion, our findings suggest a previously unrecognized contribution of beta-catenin and N-cadherin to axon-induced SC proliferation.
Assuntos
Axônios/metabolismo , Caderinas/metabolismo , Proliferação de Células , Células de Schwann/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Axônios/efeitos dos fármacos , Western Blotting , Comunicação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Embrião de Mamíferos , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Neuregulina-1/farmacologia , RNA Interferente Pequeno , Ratos , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , TransfecçãoRESUMO
Myelin in the mammalian nervous system has a high concentration of galactolipids [galactosylceramide (GalCer) and sulfatide] with 2-hydroxy fatty acids. We recently reported that fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is the major fatty acid 2-hydroxylase in the mouse brain. In this report, we show that FA2H also plays a major role in the formation of 2-hydroxy galactolipids in the peripheral nervous system. FA2H mRNA and FA2H activity in the neonatal rat sciatic nerve increased rapidly during developmental myelination. The contents of 2-hydroxy fatty acids were approximately 5% of total galactolipid fatty acids at 4 days of age and increased to 60% in GalCer and to 35% in sulfatides at 60 days of age. The chain length of galactolipid fatty acids also increased significantly during myelination. FA2H expression in cultured rat Schwann cells was highly increased in response to dibutyryl cyclic AMP, which stimulates Schwann cell differentiation and upregulates myelin genes, such as UDP-galactose:ceramide galactosyltransferase and protein zero. These observations indicate that FA2H is a myelination-associated gene. FA2H-directed RNA interference (RNAi) by short-hairpin RNA expression resulted in a reduction of cellular 2-hydroxy fatty acids and 2-hydroxy GalCer in D6P2T Schwannoma cells, providing direct evidence that FA2H-dependent fatty acid 2-hydroxylation is required for the formation of 2-hydroxy galactolipids in peripheral nerve myelin. Interestingly, FA2H-directed RNAi enhanced the migration of D6P2T cells, suggesting that, in addition to their structural role in myelin, 2-hydroxy lipids may greatly influence the migratory properties of Schwann cells.
Assuntos
Galactolipídeos/metabolismo , Oxigenases de Função Mista/metabolismo , Bainha de Mielina/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Animais , Células Cultivadas , Oxigenases de Função Mista/genética , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-DawleyRESUMO
We sought to directly compare growth and myelination of local and supraspinal axons by implanting into the injured spinal cord Schwann cells (SCs) transduced ex vivo with adenoviral (AdV) or lentiviral (LV) vectors encoding a bifunctional neurotrophin molecule (D15A). D15A mimics actions of both neurotrophin-3 and brain-derived neurotrophic factor. Transduced SCs were injected into the injury center 1 week after a moderate thoracic (T8) adult rat spinal cord contusion. D15A expression and bioactivity in vitro; D15A levels in vivo; and graft volume, SC number, implant axon number and cortico-, reticulo-, raphe-, coerulo-spinal and sensory axon growth were determined for both types of vectors employed to transduce SCs. ELISAs revealed that D15A-secreting SC implants contained significantly higher levels of neurotrophin than non-transduced SC and AdV/GFP and LV/GFP SC controls early after implantation. At 6 weeks post-implantation, D15A-secreting SC grafts exhibited 5-fold increases in graft volume, SC number and myelinated axon counts and a 3-fold increase in myelinated to unmyelinated (ensheathed) axon ratios. The total number of axons within grafts of LV/GFP/D15A SCs was estimated to be over 70,000. Also 5-HT, DbetaH, and CGRP axon length was increased up to 5-fold within D15A grafts. In sum, despite qualitative differences using the two vectors, increased neurotrophin secretion by the implanted D15A SCs led to the presence of a significantly increased number of axons in the contusion site. These results demonstrate the therapeutic potential for utilizing neurotrophin-transduced SCs to repair the injured spinal cord.
Assuntos
Axônios/fisiologia , Transplante de Células , Fatores de Crescimento Neural/metabolismo , Células de Schwann/fisiologia , Traumatismos da Medula Espinal/cirurgia , Transdução Genética/métodos , Análise de Variância , Animais , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Comportamento Animal , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Transplante de Células/métodos , Dopamina beta-Hidroxilase/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Regeneração Nervosa/fisiologia , Ratos , Ratos Endogâmicos F344 , Nervo Isquiático/citologia , Serotonina/metabolismoRESUMO
Motoneuron death leads to muscle denervation and atrophy. Transplantation of embryonic neurons into peripheral nerves results in reinnervation and provides a strategy to rescue muscles from atrophy independent of neuron replacement in a damaged or diseased spinal cord. But the count of regenerating axons always exceeds the number of motor units in this model, so target-derived trophic factor levels may limit reinnervation. Our aim was to examine whether long-term infusion of fibroblast growth factor-6 (FGF-6) into denervated medial gastrocnemius muscles improved the function of muscles reinnervated from neurons transplanted into nerve of adult Fischer rats. Factor delivery (10 microg, 4 weeks) began after sciatic nerve transection. After a week of nerve degeneration, 1 million embryonic day 14-15 ventral spinal cord cells were transplanted into the distal tibial stump as a neuron source. Ten weeks later, neurons that expressed motoneuron markers survived in the nerves. More myelinated axons were in nerves to saline-treated muscles than in FGF-6-treated muscles. However, each group showed comparable reductions in muscle fiber atrophy because of reinnervation. Mean reinnervated fiber area was 43%-51% of non-denervated fibers. Denervated fiber area averaged 11%. FGF-6-treated muscles were more fatigable than other reinnervated muscles but had stronger motor units and fewer type I fibers than did saline-treated muscles. FGF-6 thus influenced function by changing the type of fiber reinnervated by transplanted neurons. Deficits in FGF-6 may also contribute to the increase in type I fibers in muscles reinnervated from peripheral axons, suggesting that the effects of FGF-6 on fiber type are independent of the neuron source used for reinnervation.
Assuntos
Fator 6 de Crescimento de Fibroblastos/farmacologia , Fadiga Muscular/fisiologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/embriologia , Músculo Esquelético/inervação , Neurônios/transplante , Nervos Periféricos/embriologia , Nervos Periféricos/fisiologia , Proteínas Proto-Oncogênicas/farmacologia , Animais , Atrofia , Axônios/fisiologia , Contagem de Células , Sobrevivência Celular/fisiologia , Transplante de Células , Feminino , Imuno-Histoquímica , Neurônios Motores/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Músculo Esquelético/citologia , Fibras Nervosas Mielinizadas/fisiologia , Regeneração Nervosa/fisiologia , Junção Neuromuscular/fisiologia , Ratos , Ratos Endogâmicos F344RESUMO
The elevation of intracellular cAMP synergistically enhances the neuregulin-dependent proliferation of cultured Schwann cells (SCs); however, the mechanism by which this occurs has not been completely defined. To better understand this mechanism, we investigated the effect of cAMP on the activation of the extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)-Akt (PKB) pathways by heregulin, a member of the neuregulin family. Using primary cultures of adult SCs, we demonstrated that the adenylyl cyclase activator, forskolin, enhanced heregulin-dependent SC proliferation by reducing the time required for S-phase entry. When cAMP levels were increased, using either forskolin or a cell permeable analogue of cAMP, the heregulin-induced phosphorylation of ERK was converted from transient to sustained and the heregulin-induced phosphorylation of Akt was synergistically increased. Consistent with these observations, studies in which inhibitors of MEK, the upstream stimulating ERK kinase, and PI3-K were administered at different times following the onset of stimulation indicated that sustained high levels of both MEK/ERK and PI3-K/Akt activity before S-phase initiation were essential for S-phase entry. Overall, these novel results indicate that in neuregulin-stimulated SCs the activation of cAMP-mediated pathways accelerates G1-S progression by prolonging ERK activation and concurrently enhancing Akt activation.
Assuntos
Ciclo Celular/fisiologia , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteína Oncogênica v-akt/metabolismo , Células de Schwann/enzimologia , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Fase G1/fisiologia , Genes cdc/efeitos dos fármacos , Genes cdc/fisiologia , Humanos , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/enzimologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Proteínas do Tecido Nervoso/efeitos dos fármacos , Neuregulina-1 , Proteína Oncogênica v-akt/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Fase S/efeitos dos fármacos , Fase S/fisiologia , Células de Schwann/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
This study investigated the effects of prolonged cold preservation and Schwann-cell injection on nerve regeneration through peripheral nerve allografts. Forty rats were randomized to the following groups: group I, isograft; group II, allograft; group III, isograft + Schwann cells; group IV, 6-week cold-preserved allograft; and group V, 6-week cold-preserved allograft with Schwann cells. Nerves from all animals were harvested at 4 weeks after surgery for histological and histomorphometric analysis. Untreated allograft recipients demonstrated poor nerve regeneration and histological evidence of rejection. The remaining four groups showed robust regeneration without evidence of rejection. In a short nerve allograft model, prolonged cold preservation of allografts supported robust nerve regeneration, but the addition of cultured Schwann cells conferred no additional benefit for nerve regeneration. Further work in large animals is needed to establish the role for exogenous Schwann cells in nerve allotransplantation.
Assuntos
Temperatura Baixa , Regeneração Nervosa , Células de Schwann/transplante , Nervo Tibial/fisiopatologia , Nervo Tibial/transplante , Preservação de Tecido , Animais , Masculino , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos Lew , Nervo Tibial/lesões , Transplante Homólogo , Transplante IsogênicoRESUMO
Demyelination contributes to the physiological and behavioral deficits after contusive spinal cord injury (SCI). Therefore, remyelination may be an important strategy to facilitate repair after SCI. We show here that rat embryonic day 14 spinal cord-derived glial-restricted precursor cells (GRPs), which differentiate into both oligodendrocytes and astrocytes, formed normal-appearing central myelin around axons of cultured DRG neurons and had enhanced proliferation and survival in the presence of neurotrophin 3 (NT3) and brain-derived neurotrophin factor (BDNF). We infected GRPs with retroviruses expressing the multineurotrophin D15A (with both BDNF and NT3 activities) and then transplanted them into the contused adult thoracic spinal cord at 9 d after injury. Expression of D15A in the injured spinal cord is five times higher in animals receiving D15A-GRP grafts than ones receiving enhanced green fluorescent protein (EGFP)-GRP or DMEM grafts. Six weeks after transplantation, the grafted GRPs differentiated into mature oligodendrocytes expressing both myelin basic protein (MBP) and adenomatus polyposis coli (APC). Ultrastructural analysis showed that the grafted GRPs formed morphologically normal-appearing myelin sheaths around the axons in the ventrolateral funiculus (VLF) of spinal cord. Expression of D15A significantly increased the percentage of APC+ oligodendrocytes of grafted GRPs (15-30%). Most importantly, 8 of 12 rats receiving grafts of D15A-GRPs recovered transcranial magnetic motor-evoked potential responses, indicating that conduction through the demyelinated VLF axons was restored. Such electrophysiological recovery was not observed in rats receiving grafts of EGFP-GRPs, D15A-NIH3T3 cells, or an injection of an adenovirus expressing D15A. Recovery of hindlimb locomotor function was also significantly enhanced only in the D15A-GRP-grafted animals at 4 and 5 weeks after transplantation. Therefore, combined treatment with neurotrophins and GRP grafts can facilitate functional recovery after traumatic SCI and may prove to be a useful therapeutic strategy to repair the injured spinal cord.
Assuntos
Neuroglia/citologia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/terapia , Medula Espinal/citologia , Transplante de Células-Tronco/métodos , Animais , Diferenciação Celular , Gânglios Espinais/citologia , Sobrevivência de Enxerto/fisiologia , Atividade Motora/fisiologia , Bainha de Mielina/fisiologia , Regeneração Nervosa/fisiologia , Condução Nervosa/fisiologia , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Medula Espinal/embriologia , Medula Espinal/fisiologiaRESUMO
Forskolin and heregulin synergistically drive human Schwann cell (HSC) proliferation in vitro, but the role of forskolin is not completely understood. To learn how forskolin might affect receptor levels in HSC cultured from adult nerve roots, we first studied expression and localization of HER2 and HER3 in intact roots, using Western blotting and light and electron microscopic immunocytochemistry. We then determined the effect of forskolin and heregulin on receptor expression in HSC cultured from nerve roots using Western blotting and RNase protection assays. HER2 and HER3 were expressed in nonmyelinating Schwann cells in roots and in cultured HSCs before exposure to forskolin. HER2, but not HER3, was also expressed in endoneurial fibroblasts and in cultured nerve root-derived fibroblasts. Treatment with forskolin for 24 h consistently increased HER2 and HER3 protein levels in HSCs but did not alter HER2 and HER3 mRNA levels. In addition, 24-h treatment with heregulin alone decreased HER2 and HER3 protein levels, an effect not previously described. When both heregulin and forskolin were present, HER2 and HER3 protein levels were similar to initial control values. The effect of forskolin on receptor levels was mimicked by dibutyryl-cAMP and receptor levels in both untreated and forskolin treated HSCs were decreased by treatment with the protein kinase A inhibitor H-89. Following pretreatment of HSCs with forskolin, increased receptor levels were correlated with increased rates of thymidine incorporation into HSCs. These results suggest that forskolin/heregulin synergy might derive, at least in part, from post-transcriptional effects leading to increased steady-state receptor levels.
Assuntos
Colforsina/farmacologia , RNA Mensageiro/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Células de Schwann/metabolismo , Raízes Nervosas Espinhais/metabolismo , Bucladesina/farmacologia , Proliferação de Células , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Neuregulina-1/metabolismo , Neuregulina-1/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/metabolismo , Receptor ErbB-2/efeitos dos fármacos , Receptor ErbB-2/genética , Receptor ErbB-3/efeitos dos fármacos , Receptor ErbB-3/genética , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestrutura , Raízes Nervosas Espinhais/efeitos dos fármacos , Raízes Nervosas Espinhais/ultraestrutura , Timidina/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Bone morphogenetic proteins (BMPs) have an important role in neuronal and astrocytic differentiation of embryonic and adult neural stem cells (NSCs). Here, we show that BMP6, BMP7, GDF5, and GDF6 instructively differentiate E12, E14, and E17 rat cortical NSCs into a variety of neural crest lineages. Clonal analysis shows that BMP7-treated NSCs develop mostly into smooth muscle and peripheral glia. We observed a rapid induction of premigratory neural crest markers like p75NTR, and AP-2 alpha followed by Msx1, Msx2, and Slug, transcription factors that participate in neural crest development. These results suggest that NSCs cultured in vitro in the presence of FGF2 display expanded developmental potential.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular/fisiologia , Sistema Nervoso Central/citologia , Crista Neural/citologia , Crista Neural/metabolismo , Animais , Antígenos de Diferenciação/biossíntese , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/fisiologia , Células Cultivadas , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/embriologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Células Clonais/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Idade Gestacional , Crista Neural/efeitos dos fármacos , Crista Neural/embriologia , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Fatores de Transcrição/biossíntese , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
The molecular mechanisms underlying the contact behavior of Schwann cells (SCs) and SC-axon association are poorly understood. SC-SC and SC-axon interactions were studied using purified adult rat SCs and cocultures of SCs with embryonic dorsal root ganglion neurons. After contact of SCs with axons, SCs start to extend processes in alignment with axons. This unique alignment was quantitated using a new assay. SC-axon alignment and SC-SC band formation were disrupted in medium containing low extracellular calcium, indicating the involvement of calcium-dependent adhesion molecules. N-cadherin expression was strong in developing rat sciatic nerves but weak in adult sciatic nerves. In purified adult-derived rat SCs, N-cadherin expression was increased by mitogens (neuregulins) and decreased by high cell density. High-resolution confocal images show intense N-cadherin signals in SC process tips. Subcellular N-cadherin was accumulated in bands at intercellular junctions between SCs and was clustered at axon-SC contact sites. Blocking antibodies (rabbit and guinea pig IgG directed against the first extracellular domain of N-cadherin) and cyclic pentapeptides (including the HAV motif) were used to perturb N-cadherin function. All blocking agents reduced the number of N-cadherin-positive SC-SC junctions and perturbed axon-aligned growth of SC processes. Averaging over all N-cadherin-perturbation experiments, in controls 67-86% of SCs exhibited axon-aligned process growth, whereas in treated cultures only 41% of the SCs aligned with axons. These results are evidence that in mammals N-cadherin is important for formation of SC-SC junctions and SC process growth in alignment with axons.