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1.
Cell Mol Life Sci ; 77(1): 161-177, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31161284

RESUMO

Peripheral nervous system development involves a tight coordination of neuronal birth and death and a substantial remodelling of the myelinating glia cytoskeleton to achieve myelin wrapping of its projecting axons. However, how these processes are coordinated through time is still not understood. We have identified engulfment and cell motility 1, Elmo1, as a novel component that regulates (i) neuronal numbers within the Posterior Lateral Line ganglion and (ii) radial sorting of axons by Schwann cells (SC) and myelination in the PLL system in zebrafish. Our results show that neuronal and myelination defects observed in elmo1 mutant are rescued through small GTPase Rac1 activation. Inhibiting macrophage development leads to a decrease in neuronal numbers, while peripheral myelination is intact. However, elmo1 mutants do not show defective macrophage activity, suggesting a role for Elmo1 in PLLg neuronal development and SC myelination independent of macrophages. Forcing early Elmo1 and Rac1 expression specifically within SCs rescues elmo1-/- myelination defects, highlighting an autonomous role for Elmo1 and Rac1 in radial sorting of axons by SCs and myelination. This uncovers a previously unknown function of Elmo1 that regulates fundamental aspects of PNS development.


Assuntos
Bainha de Mielina/metabolismo , Neurogênese , Neurônios/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Apoptose , Axônios/metabolismo , Axônios/ultraestrutura , Movimento Celular , Neurônios/metabolismo , Neurônios/ultraestrutura , Nervos Periféricos/crescimento & desenvolvimento , Nervos Periféricos/ultraestrutura , Células de Schwann/citologia , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura
2.
Tissue Cell ; 60: 60-69, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31582019

RESUMO

This study aimed to evaluate the histopathological and ultrastructural changes in sciatic nerve barriers after exposure to different doses of nicotine. Twenty-seven adult male rats were divided into 2 groups; group I served as control (n = 9) and group II that received nicotine (n = 18) was subdivided into two equal subgroups; group IIa and group IIb that were injected subcutaneously daily for one month with nicotine at a dose of 3 mg/kg and 6 mg/kg body weight, respectively. Specimens of sciatic nerve were processed for light and electron microscopy. Immunohistochemical expression of ZO-1 and vascular endothelium growth factor (VEGF) were investigated. Abundance of mRNA for VEGF was determined via qRT-PCR. Total antioxidant capacity (TAC), malondialdehyde (MDA), alanine transaminase (ALT) and aspartate transaminase (AST) were measured. Group IIb showed increased perineural fibrosis and myelin abnormalities. ZO-1 expression was significantly decreased. Schwann cells showed features of apoptosis and blood capillaries showed disrupted lining. High statistical difference in the level of mRNA expression of VEGF between group IIb and group I was found. There was decreased level of TAC and increased MDA, ALT and AST. A dose-dependent nicotine-induced oxidative stress on the sciatic nerve occurred via disruption of nerve barriers, altered VEGF and ZO-1 levels.


Assuntos
Nicotina/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Células de Schwann , Nervo Isquiático/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Alanina Transaminase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/metabolismo , Masculino , Ratos , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura , Nervo Isquiático/ultraestrutura
3.
Nat Commun ; 10(1): 1467, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30931926

RESUMO

In contrast to acute peripheral nerve injury, the molecular response of Schwann cells in chronic neuropathies remains poorly understood. Onion bulb structures are a pathological hallmark of demyelinating neuropathies, but the nature of these formations is unknown. Here, we show that Schwann cells induce the expression of Neuregulin-1 type I (NRG1-I), a paracrine growth factor, in various chronic demyelinating diseases. Genetic disruption of Schwann cell-derived NRG1 signalling in a mouse model of Charcot-Marie-Tooth Disease 1A (CMT1A), suppresses hypermyelination and the formation of onion bulbs. Transgenic overexpression of NRG1-I in Schwann cells on a wildtype background is sufficient to mediate an interaction between Schwann cells via an ErbB2 receptor-MEK/ERK signaling axis, which causes onion bulb formations and results in a peripheral neuropathy reminiscent of CMT1A. We suggest that diseased Schwann cells mount a regeneration program that is beneficial in acute nerve injury, but that overstimulation of Schwann cells in chronic neuropathies is detrimental.


Assuntos
Doenças Desmielinizantes/genética , Neuregulina-1/genética , Comunicação Parácrina , Células de Schwann/metabolismo , Nervo Sural/metabolismo , Animais , Animais Geneticamente Modificados , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Diabetes Mellitus Tipo 1/complicações , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/metabolismo , Neuropatias Diabéticas/patologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Atividade Motora , Proteínas da Mielina/genética , Neuregulina-1/metabolismo , Neurite Autoimune Experimental/genética , Neurite Autoimune Experimental/metabolismo , Neurite Autoimune Experimental/patologia , Neuroglia/metabolismo , Ratos , Receptor ErbB-2/metabolismo , Células de Schwann/ultraestrutura , Nervo Isquiático/lesões , Transdução de Sinais , Nervo Sural/ultraestrutura , Nervo Tibial
4.
Eur J Paediatr Neurol ; 23(2): 254-261, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30626539

RESUMO

AIMS: To define the neurological and neuropathological alterations caused by SYNE1 mutations. METHODS: We describe 5 patients (3 males, 2 females; age 3-24 years) from 3 families. The diagnostic work-up included three muscle biopsies and two nerve biopsies in three of the cases. RESULTS: Three different phenotypes were discerned. Two patients showed progressive ataxia, mental retardation, neuropathy and radially deviated thumbs (spinocerebellar ataxia, SCAR, type 8 phenotype). Two patients had mild congenital myopathy with restrictive lung disease, clubfeet and thumb anomalies (myopathic arthrogryposis). One patient had congenital myopathy with dilated cardiomyopathy and adducted thumbs (Emery-Dreifuss Muscular Dystrophy, EDMD, type 4). Light microscopy of the three muscle biopsies revealed chronic non-necrotizing myopathy without rimmed vacuoles in all cases combined with neurogenic atrophy in one case. The two nerve biopsies showed predominantly axonal neuropathy with demyelinating features. Nuclear alterations, most notably lobulation and focal widening of the space between inner and outer leaflet of the nuclear envelope, were a prominent consistent feature of myonuclei and Schwann cell nuclei in each of the three muscle specimens and one nerve specimen that could be examined by electron microscopy. CONCLUSION: Thumb abnormalities and nuclear envelope alterations are characteristic for SYNE 1 mutations. Schwann cell nuclei are affected, indicating that such nuclear envelope changes in glial cells contribute to the neurodegenerative phenotype in human nesprinopathies.


Assuntos
Proteínas do Tecido Nervoso/genética , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/genética , Polegar/anormalidades , Adolescente , Criança , Pré-Escolar , Proteínas do Citoesqueleto , Feminino , Humanos , Masculino , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Mutação , Membrana Nuclear/patologia , Fenótipo , Células de Schwann/patologia , Células de Schwann/ultraestrutura , Adulto Jovem
5.
Int J Med Sci ; 16(1): 8-16, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30662323

RESUMO

Background: Due to its high antioxidant activity, baicalein, a kind of flavonoid present in Radical Scutellariae, has various pharmacological effects. However, the protective effect against oxidative stress in Schwann cells, which plays an important role in peripheral neuropathy, has not yet been studied. In this study, the effects of baicalein on hydrogen peroxide (H2O2)-induced DNA damage and apoptosis in RT4-D6P2T Schwann cells were evaluated. Methods: Cell viability assay was performed using MTT assay and colony formation assay. Apoptosis was assessed by flow cytometry analysis and DNA fragmentation assay. The effects on DNA damage and ATP content were analyzed by comet method and luminometer. In addition, changes in protein expression were observed by Western blotting. Results: Our results show that baicalein significantly inhibits H2O2-induced cytotoxicity through blocking reactive oxygen species (ROS) generation. We also demonstrate that baicalein is to block H2O2-induced DNA damage as evidenced by inhibition of DNA tail formation and γH2AX phosphorylation. Moreover, baicalein significantly attenuated H2O2-induced apoptosis and mitochondrial dysfunction, and restored inhibition of ATP production. The suppression of apoptosis by baicalein in H2O2-stimulated cells was associated with reduction of increased Bax/Bcl-2 ratio, activation of caspase-9 and -3, and degradation of poly (ADP-ribose) polymerase. Conclusions: These results demonstrate that baicalein eliminates H2O2-induced apoptosis through conservation of mitochondrial function by the removal of ROS. Therefore, it is suggested that baicalein protects Schwann cells from oxidative stress, and may be beneficial for the prevention and treatment of peripheral neuropathy induced by oxidative stress.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Flavanonas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Células de Schwann/fisiologia , Antioxidantes/uso terapêutico , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Flavanonas/uso terapêutico , Regulação da Expressão Gênica , Genes bcl-2 , Humanos , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Células de Schwann/ultraestrutura , Proteína X Associada a bcl-2
6.
Glia ; 67(5): 950-966, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30637802

RESUMO

Direct conversion is considered a promising approach to obtain tissue-specific cells for cell therapies; however, this strategy depends on exogenous gene expression that may cause undesired adverse effects such as tumorigenesis. By optimizing the Schwann cell induction system, which was originally developed for trans-differentiation of bone marrow mesenchymal stem cells into Schwann cells, we established a system to directly convert adult human skin fibroblasts into cells comparable to authentic human Schwann cells without gene introduction. Serial treatments with beta-mercaptoethanol, retinoic acid, and finally a cocktail of basic fibroblast growth factor, forskolin, platelet-derived growth factor-AA, and heregulin-ß1 (EGF domain) converted fibroblasts into cells expressing authentic Schwann cell markers at an efficiency of approximately 75%. Genome-wide gene expression analysis suggested the conversion of fibroblasts into the Schwann cell-lineage. Transplantation of induced Schwann cells into severed peripheral nerve of rats facilitated axonal regeneration and robust functional recovery in sciatic function index comparable to those of authentic human Schwann cells. The contributions of induced Schwann cells to myelination of regenerated axons and re-formation of neuromuscular junctions were also demonstrated. Our data clearly demonstrated that cells comparable to functional Schwann cells feasible for the treatment of neural disease can be induced from adult human skin fibroblasts without gene introduction. This direct conversion system will be beneficial for clinical applications to peripheral and central nervous system injuries and demyelinating diseases.


Assuntos
Diferenciação Celular/fisiologia , Fibroblastos/fisiologia , Traumatismos dos Nervos Periféricos/cirurgia , Recuperação de Função Fisiológica/fisiologia , Células de Schwann/fisiologia , Células de Schwann/transplante , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Locomoção/fisiologia , Masculino , Microscopia Eletrônica , Proteína P0 da Mielina/metabolismo , Traumatismos dos Nervos Periféricos/fisiopatologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/ultraestrutura , Soro/fisiologia , Pele/citologia , Fatores de Tempo , Tretinoína/farmacologia
7.
Glia ; 67(4): 571-581, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30378179

RESUMO

Myelinating Schwann cells undergo irreversible demyelination in many demyelinating neuropathies that show complete demyelination of the internode. Dedifferentiation, reprogramming, and myelin clearance processes-which are specifically discussed in this article-appear to be shared by various demyelinating peripheral conditions, such as Wallerian degeneration, immune-mediated, and toxic demyelinating diseases. We propose to introduce the concept of the "demyelinating Schwann cell (DSC)" as a novel cell phenotype, which has specific properties required for myelin sheath clearance. We anticipate that the introduction of the DSC concept will provide a significant advance in understanding the pathophysiological mechanisms of demyelinating peripheral neuropathies.


Assuntos
Polirradiculoneuropatia/patologia , Células de Schwann/patologia , Animais , Autofagia , Humanos , Fagocitose , Células de Schwann/ultraestrutura , Degeneração Walleriana/patologia
8.
Acta Biomater ; 85: 310-319, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30586648

RESUMO

The development of bioactive soft materials that can guide cell behavior and have biomimetic mechanical properties is an active and challenging topic in regenerative medicine. A common strategy to create a bioactive soft material is the integration of biomacromolecules with polymers. However, limited by their complex structures and sensitivity to temperature and chemicals, it is relatively difficult to maintain the bioactivity of biomacromolecules during their preparation, storage, and application. Here, a new kind of bioactive soft material based on the molecular integration of metal ions and polymers is designed and exemplified by a hybrid of magnesium ion (Mg2+) and poly(glycerol-sebacate-maleate) (PGSM-Mg). Mg2+ was firmly incorporated into PGSM molecules through a complexation interaction as evidenced by X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR). The PGSM matrix provided the soft nature and facile processing of the hybrid, which could serve as an injectable material and be fabricated into elastic porous three-dimensional (3D) scaffolds. The Mg2+ immobilized in the PGSM chain conferred neuroactivity to the resultant hybrid. PGSM-Mg exhibited adequate biodegradability and a sustained release of Mg2+. PGSM-Mg 3D scaffolds promoted the adhesion and proliferation of Schwann cells (SCs) more effectively than poly(lactic-co-glycolic acid) (PLGA) scaffolds. Furthermore, SCs on PGSM-Mg scaffolds expressed significantly more neural specific genes than those on PLGA, PGS, and PGSM, including nerve growth factor (NGF) and neurotrophic factor-3 (NTF3). All these results indicated that Mg2+ immobilized through molecular integration could efficiently regulate the bioactivity of polymers. In view of the wide availability, diverse bioactivity, and high stability of metal ions, the strategy of molecular coupling of metal ions and polymers is expected to be a new general approach to construct bioactive soft materials. STATEMENT OF SIGNIFICANCE: Bioactive soft materials are designed on the basis of the molecular integration of metal ions and polymers. Immobilized metal ions offer a new way to endow bioactivity to polymers. Different from biomolecules such as proteins and genes, metal ions are quite stable and can resist harsh processing conditions. Further, the polymeric matrix provides the soft nature and facile processing of the hybrid. Different from stiff metal-containing inorganic materials, the hybrid is a biomimetic soft material and can be readily processed just like its polymer precursor under mild conditions. In view of the diversity of metal ions and polymers, this strategy is expected to be a new powerful and general approach to construct bioactive soft materials for a wide range of biomedical applications.


Assuntos
Materiais Biocompatíveis/farmacologia , Magnésio/farmacologia , Neurônios/efeitos dos fármacos , Polímeros/química , Animais , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Decanoatos/síntese química , Decanoatos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Glicerol/análogos & derivados , Glicerol/síntese química , Glicerol/química , Íons , Polímeros/síntese química , Ratos , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestrutura , Temperatura , Tecidos Suporte/química
9.
Cell Mol Biol (Noisy-le-grand) ; 64(14): 66-71, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30511623

RESUMO

Polylactide-co-glycolide acid (PLGA) is known as a biodegradable and biocompatible polymer. This polymer has been highly used in tissue engineering. In this study, the biological behavior of Schwann cells (Rat) was investigated in co-culture with L lysine/gelatine coated PLGA nano-fiber. In this study, PLGA was dissolved in a hexafluoro propanol based solvent and nanofiber prepared by an electronic method. They were coated with gelatin and poly-L-lysine individually. These polymer properties were investigated by Scanning Electron Microscopy (SEM) analysis and contact angle measurement. After extraction of rat Schwann cells, the cells were cultured in three groups of nano-fiber; nano-fiber PLGA, nano-fiber gelatine coated PLGA and nano-fiber poly-L-lysine coated PLGA. Cell death and Cell proliferation were evaluated by Acridine orange staining (living cell with a green nucleus and dead cell with an orange nucleus) and morphology was investigated by SEM in 2, 4 and 6 days. The diameter of electronic nanofiber PLGA was between 270 to 700 nm. Average contact angles of PLGA, PLGA coated with gelatine, coated with poly-L-lysine and PLGA were 40.12, 64.58 and 107.66degrees, respectively. The findings showed a significant reduction of cell proliferation in PLGA nanofiber ( it was important than PLGA without nano-fiber (P <0.05)). But, this amount was increased in nanofiber which coated with poly-L-lysine and gelatine. PLGA nanofiber-poly-L-lysine was more biocompatible than PLGA nanofiber-gelatine and this comparison was done with rat Schwann cells.


Assuntos
Nanofibras/química , Regeneração Nervosa/fisiologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Células de Schwann/metabolismo , Animais , Animais Recém-Nascidos , Morte Celular , Proliferação de Células , Forma Celular , Nanofibras/ultraestrutura , Ratos , Células de Schwann/ultraestrutura , Tecidos Suporte/química
10.
ASN Neuro ; 10: 1759091418803282, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30419760

RESUMO

Charcot-Marie-Tooth Disorder Type 4B (CMT4B) is a demyelinating peripheral neuropathy caused by mutations in myotubularin-related (MTMR) proteins 2, 13, or 5 (CMT4B1/2/3), which regulate phosphoinositide turnover and endosomal trafficking. Although mouse models of CMT4B2 exist, an in vitro model would make possible pharmacological and reverse genetic experiments needed to clarify the role of MTMR13 in myelination. We have generated such a model using Schwann cell-dorsal root ganglion (SC-DRG) explants from Mtmr13-/- mice. Myelin sheaths in mutant cultures contain outfoldings highly reminiscent of those observed in the nerves of Mtmr13-/- mice and CMT4B2 patients. Mtmr13-/- SC-DRG explants also contain reduced Mtmr2, further supporting a role of Mtmr13 in stabilizing Mtmr2. Elevated PI(3,5)P2 has been implicated as a cause of myelin outfoldings in Mtmr2-/- models. In contrast, the role of elevated PI3P or PI(3,5)P2 in promoting outfoldings in Mtmr13-/- models is unclear. We found that over-expression of MTMR2 in Mtmr13-/- SC-DRGs moderately reduced the prevalence of myelin outfoldings. Thus, a manipulation predicted to lower PI3P and PI(3,5)P2 partially suppressed the phenotype caused by Mtmr13 deficiency. We also explored the relationship between CMT4B2-like myelin outfoldings and kinases that produce PI3P and PI(3,5)P2 by analyzing nerve pathology in mice lacking both Mtmr13 and one of two specific PI 3-kinases. Intriguingly, the loss of vacuolar protein sorting 34 or PI3K-C2ß in Mtmr13-/- mice had no impact on the prevalence of myelin outfoldings. In aggregate, our findings suggest that the MTMR13 scaffold protein likely has critical functions other than stabilizing MTMR2 to achieve an adequate level of PI 3-phosphatase activity.


Assuntos
Neurônios/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Células de Schwann/metabolismo , Animais , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Técnicas de Cocultura , Doenças Desmielinizantes/metabolismo , Embrião de Mamíferos , Feminino , Gânglios Espinais/citologia , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Básica da Mielina/metabolismo , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/genética , Células de Schwann/ultraestrutura , Nervo Isquiático/ultraestrutura
11.
Int J Nanomedicine ; 13: 6265-6277, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30349249

RESUMO

Background: Spinal cord injury (SCI) is a traumatic disease of the central nervous system, accompanied with high incidence and high disability rate. Tissue engineering scaffold can be used as therapeutic systems to provide effective repair for SCI. Purpose: In this study, a novel tissue engineering scaffold has been synthesized in order to explore the effect of nerve repair on SCI. Patients and methods: Polycaprolactone (PCL) scaffolds loaded with actived Schwann cells (ASCs) and induced pluripotent stem cells -derived neural stem cells (iPSC-NSCs), a combined cell transplantation strategy, were prepared and characterized. The cell-loaded PCL scaffolds were further utilized for the treatment of SCI in vivo. Histological observation, behavioral evaluation, Western-blot and qRT-PCR were used to investigate the nerve repair of Wistar rats after scaffold transplantation. Results: The iPSCs displayed similar characteristics to embryonic stem cells and were efficiently differentiated into neural stem cells in vitro. The obtained PCL scaffolds werê0.5 mm in thickness with biocompatibility and biodegradability. SEM results indicated that the ASCs and (or) iPS-NSCs grew well on PCL scaffolds. Moreover, transplantation reduced the volume of lesion cavity and improved locomotor recovery of rats. In addition, the degree of spinal cord recovery and remodeling maybe closely related to nerve growth factor and glial cell-derived neurotrophic factor. In summary, our results demonstrated that tissue engineering scaffold treatment could increase tissue remodeling and could promote motor function recovery in a transection SCI model. Conclusion: This study provides preliminary evidence for using tissue engineering scaffold as a clinically viable treatment for SCI in the future.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Poliésteres/química , Células de Schwann/citologia , Traumatismos da Medula Espinal/terapia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Axônios/patologia , Comportamento Animal , Separação Celular , Técnicas de Cocultura , Sangue Fetal/citologia , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Leucócitos Mononucleares/citologia , Camundongos , Fatores de Crescimento Neural/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/ultraestrutura , Ratos Wistar , Células de Schwann/ultraestrutura , Medula Espinal/patologia
12.
Glia ; 66(11): 2487-2502, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30306639

RESUMO

The transition of differentiated Schwann cells to support of nerve repair after injury is accompanied by remodeling of the Schwann cell epigenome. The EED-containing polycomb repressive complex 2 (PRC2) catalyzes histone H3K27 methylation and represses key nerve repair genes such as Shh, Gdnf, and Bdnf, and their activation is accompanied by loss of H3K27 methylation. Analysis of nerve injury in mice with a Schwann cell-specific loss of EED showed the reversal of polycomb repression is required and a rate limiting step in the increased transcription of Neuregulin 1 (type I), which is required for efficient remyelination. However, mouse nerves with EED-deficient Schwann cells display slow axonal regeneration with significantly low expression of axon guidance genes, including Sema4f and Cntf. Finally, EED loss causes impaired Schwann cell proliferation after injury with significant induction of the Cdkn2a cell cycle inhibitor gene. Interestingly, PRC2 subunits and CDKN2A are commonly co-mutated in the transition from benign neurofibromas to malignant peripheral nerve sheath tumors (MPNST's). RNA-seq analysis of EED-deficient mice identified PRC2-regulated molecular pathways that may contribute to the transition to malignancy in neurofibromatosis.


Assuntos
Proliferação de Células/fisiologia , Regulação da Expressão Gênica/genética , Regeneração Nervosa/efeitos dos fármacos , Complexo Repressor Polycomb 2/metabolismo , Células de Schwann/fisiologia , Neuropatia Ciática/fisiopatologia , Animais , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Regeneração Nervosa/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurregulinas/metabolismo , Proteína Oncogênica v-akt/metabolismo , Complexo Repressor Polycomb 2/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
13.
Glia ; 66(12): 2632-2644, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30295958

RESUMO

Proper function of the nervous system depends on myelination. In peripheral nerves, Schwann cells (SCs) myelinate axons and the miRNA biogenesis pathway is required for developmental myelination and myelin maintenance. However, regulatory roles of this pathway at different stages of myelination are only partially understood. We addressed the requirement of the core miRNA biogenesis pathway components Dgcr8, Drosha, and Dicer in developing and adult SCs using mouse mutants with a comparative genetics and transcriptomics approach. We found that the microprocessor components Dgcr8 and Drosha are crucial for axonal radial sorting and to establish correct SC numbers upon myelination. Transcriptome analyses revealed a requirement of the microprocessor to prevent aberrantly increased expression of injury-response genes. Those genes are predicted targets of abundant miRNAs in sciatic nerves (SNs) during developmental myelination. In agreement, Dgcr8 and Dicer are required for proper maintenance of the myelinated SC state, where abundant miRNAs in adult SNs are predicted to target injury-response genes. We conclude that the miRNA biogenesis pathway in SCs is crucial for preventing inappropriate activity of injury-response genes in developing and adult SCs.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , MicroRNAs/metabolismo , Células de Schwann/patologia , Neuropatia Ciática/patologia , Neuropatia Ciática/prevenção & controle , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Conexinas/genética , Conexinas/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Microscopia Eletrônica , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura , Fatores de Transcrição/metabolismo
14.
Ultrastruct Pathol ; 42(5): 377-408, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30339059

RESUMO

Young male Zucker rats with a leptin receptor mutation are obese, have a non-insulin-dependent diabetes mellitus (NIDDM), and other endocrinopathies. Tibial branches of the sciatic nerve reveal a progressive demyelination that progresses out of the Schwann cells (SCs) where electron-contrast deposits are accumulated while the minor lines or intermembranous SC contacts display exaggerated spacings. Cajal bands contain diversely contrasted vesicles adjacent to the abaxonal myelin layer with blemishes; they appear dispatched centripetally out of many narrow electron densities, regularly spaced around the myelin annulus. These anomalies widen and yield into sectors across the stacked myelin layers. Throughout the worse degradations, the adaxonal membrane remains along the axonal neuroplasm. This peripheral neuropathy with irresponsive leptin cannot modulate hypothalamic-pituitary-adrenal axis and SC neurosteroids, thus exacerbates NIDDM condition. Additionally, the ultrastructure of the progressive myelin alterations may have unraveled a peculiar, centripetal mode of trafficking maintenance of the peripheral nervous system myelin, while some adhesive glycoproteins remain between myelin layers, somewhat hindering the axon mutilation. Heading title: Peripheral neuropathy and myelin.


Assuntos
Doenças Desmielinizantes/genética , Neuropatias Diabéticas/patologia , Receptores para Leptina/genética , Nervo Isquiático/patologia , Nervo Isquiático/ultraestrutura , Animais , Diabetes Mellitus Tipo 2 , Masculino , Mutação , Bainha de Mielina/ultraestrutura , Fibras Nervosas Mielinizadas/ultraestrutura , Ratos , Ratos Zucker , Células de Schwann/ultraestrutura
15.
J Neurol Sci ; 394: 99-106, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30243104

RESUMO

INTRODUCTION: We evaluated the morphology of amyloid fibrils in the peripheral nervous system using biopsy or autopsy specimens from hereditary transthyretin amyloidosis patients. The impact of amyloid fibril formation on neighboring tissues was also investigated. METHODS: Sural nerve biopsy specimens from 34 patients were examined using electron microscopy. Twenty-eight patients had Val30Met mutations, and the remaining 6 patients had non-Val30Met mutations (i.e., Glu54Lys, Pro24Ser, Thr49Ala, Val71Ala, Val94Gly, and Ala97Gly). The patients with the Val30Met mutation included a case from Brazil (supposedly of Portuguese origin), 6 early-onset cases from endemic foci in Japan, and 21 late-onset cases from non-endemic areas in Japan. RESULTS: Long amyloid fibers were abundant in the early-onset Val30Met cases from the Japanese endemic foci and Brazil, whereas the amyloid fibrils were generally short in the late-onset Val30Met and non-Val30Met cases. The amyloid fibrils seemed to mature from dotty structures among amorphous electron-dense extracellular materials and pull surrounding tissues during the maturation process. The distortion of Schwann cells close to amyloid fibril masses was conspicuous, particularly in cases with long amyloid fibrils. Atrophy was conspicuous in non-myelinating Schwann cells and bands of Büngner (i.e., Schwann cell subunits that previously contained myelinated axons), particularly those completely surrounded by amyloid fibrils. In contrast, the myelinated fibers tended to be only partially surrounded by amyloid fibrils and morphologically preserved due to their large size. Only a few demyelinated axons were found. CONCLUSION: Pre-fibrillar amyloid precursors appear to play a pivotal role during the initial phase of amyloid fibril formation. The mechanical distortion and subsequent atrophy of Schwann cells resulting from the elongation of amyloid fibrils may be related to small-fiber predominant loss, which is a classical characteristic of amyloid neuropathy. Although rather rare, the destruction of myelin (i.e., demyelination) resulting from amyloid deposition may relate to nerve conduction abnormalities mimicking chronic inflammatory demyelinating polyneuropathy.


Assuntos
Neuropatias Amiloides Familiares/patologia , Amiloide/ultraestrutura , Nervo Sural/patologia , Nervo Sural/ultraestrutura , Adulto , Amiloide/genética , Neuropatias Amiloides Familiares/genética , Brasil , Feminino , Humanos , Japão , Masculino , Mutação/genética , Pré-Albumina/genética , Células de Schwann/patologia , Células de Schwann/ultraestrutura
16.
J Neurosci ; 38(40): 8650-8665, 2018 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-30143570

RESUMO

Terminal or perisynaptic Schwann cells (TPSCs) are nonmyelinating, perisynaptic glial cells at the neuromuscular junction (NMJ) that respond to neural activity by increasing intracellular calcium (Ca2+) and regulate synaptic function. The onset of activity-induced TPSC Ca2+ responses, as well as whether axonal Schwann cells (ASCs) along the nerve respond to nerve stimulation during development, is unknown. Here, we show that phrenic nerve stimulation in developing male and female mice elicited Ca2+ responses in both ASCs and TPSCs at embryonic day 14. ASC responses were lost in a proximo-distal gradient over time, but could continue to be elicited by bath application of neurotransmitter, suggesting that a loss of release rather than a change in ASC competence accounted for this response gradient. Similar to those of early postnatal TPSCs, developing ASC/TPSC responses were mediated by purinergic P2Y1 receptors. The loss of ASC Ca2+ responses was correlated to the proximo-distal disappearance of synaptophysin immunoreactivity and synaptic vesicles in phrenic axons. Accordingly, developing ASC Ca2+ responses were blocked by botulinum toxin. Interestingly, the loss of ASC Ca2+ responses was also correlated to the proximo-distal development of myelination. Finally, compared with postnatal TPSCs, neonatal TPSCs and ASCs displayed Ca2+ signals in response to lower frequencies and shorter durations of nerve stimulation. Together, these results with GCaMP3-expressing Schwann cells provide ex vivo evidence that both axons and presynaptic terminals initially exhibit activity-induced vesicular release of neurotransmitter, but that the subsequent loss of axonal synaptic vesicles accounts for the postnatal restriction of vesicular release to the NMJ.SIGNIFICANCE STATEMENT Neural activity regulates multiple aspects of development, including myelination. Whether the excitation of developing neurons in vivo results in the release of neurotransmitter from both axons and presynaptic terminals is unclear. Here, using mice expressing the genetically encoded calcium indicator GCaMP3 in Schwann cells, we show that both terminal/perisynaptic Schwann cells at the diaphragm neuromuscular junction and axonal Schwann cells along the phrenic nerve exhibit activity-induced calcium responses early in development, mediated by the vesicular release of ATP from the axons of motor neurons acting on P2Y1 receptors. These ex vivo findings corroborate classic in vitro studies demonstrating transmitter release by developing axons, and thus represent a tool to study the mechanisms and significance of this process during embryonic development.


Assuntos
Sinalização do Cálcio , Junção Neuromuscular/embriologia , Terminações Pré-Sinápticas/metabolismo , Células de Schwann/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura , Nervo Frênico/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Células de Schwann/ultraestrutura , Vesículas Sinápticas/ultraestrutura
17.
Neural Dev ; 13(1): 17, 2018 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-30089513

RESUMO

BACKGROUND: In the peripheral nervous system (PNS), specialized glial cells called Schwann cells produce myelin, a lipid-rich insulating sheath that surrounds axons and promotes rapid action potential propagation. During development, Schwann cells must undergo extensive cytoskeletal rearrangements in order to become mature, myelinating Schwann cells. The intracellular mechanisms that drive Schwann cell development, myelination, and accompanying cell shape changes are poorly understood. METHODS: Through a forward genetic screen in zebrafish, we identified a mutation in the atypical guanine nucleotide exchange factor, dock1, that results in decreased myelination of peripheral axons. Rescue experiments and complementation tests with newly engineered alleles confirmed that mutations in dock1 cause defects in myelination of the PNS. Whole mount in situ hybridization, transmission electron microscopy, and live imaging were used to fully define mutant phenotypes. RESULTS: We show that Schwann cells in dock1 mutants can appropriately migrate and are not decreased in number, but exhibit delayed radial sorting and decreased myelination during early stages of development. CONCLUSIONS: Together, our results demonstrate that mutations in dock1 result in defects in Schwann cell development and myelination. Specifically, loss of dock1 delays radial sorting and myelination of peripheral axons in zebrafish.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Sistema da Linha Lateral/citologia , Mutação/genética , Células de Schwann/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas rac de Ligação ao GTP/genética , Fatores Etários , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Sistema da Linha Lateral/embriologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microinjeções , Microscopia Eletrônica de Transmissão , Proteína Básica da Mielina/metabolismo , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/embriologia , RNA Mensageiro/metabolismo , Células de Schwann/ultraestrutura , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
18.
Methods Mol Biol ; 1791: 3-13, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30006697

RESUMO

Transmission electron microscopy (TEM) can provide excellent high-resolution images of cellular structures. A critical step with this technique is sample preparation. Here we describe our protocol for the preparation of semithin and ultrathin sections of mouse peripheral nerves in order to visualize the structure and pathological features of peripheral myelin with TEM.


Assuntos
Microscopia Eletrônica , Bainha de Mielina/ultraestrutura , Nervos Periféricos/ultraestrutura , Animais , Camundongos , Microscopia Eletrônica/métodos , Microscopia Eletrônica de Transmissão/métodos , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura
19.
Methods Mol Biol ; 1791: 115-129, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30006705

RESUMO

Our understanding of the processes controlling peripheral nervous system myelination have been significantly benefited by the development of an in vitro myelinating culture system in which primary Schwann cells are cocultured together with primary sensory neurons. In this chapter, we describe the protocol currently used in our laboratories to establish Schwann cells neuronal myelinating cocultures. We also include a detailed description of the various substrates that can be used to establish it.


Assuntos
Técnicas de Cocultura , Células do Corno Posterior/citologia , Cultura Primária de Células , Células de Schwann/citologia , Animais , Biomarcadores , Feminino , Camundongos , Células do Corno Posterior/metabolismo , Células do Corno Posterior/ultraestrutura , Gravidez , Cultura Primária de Células/métodos , Ratos , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura
20.
Methods Mol Biol ; 1791: 287-292, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30006719

RESUMO

To study the signaling mechanism of the development and maintenance of myelinating Schwann cells (SCs) in the peripheral nervous system, in vivo techniques for SC-selective gene manipulation are useful. The present protocol describes an in vivo electroporation method that allows efficient transfection of myelinating SCs in rodent sciatic nerves. This technique allows us to introduce the genes of interest into myelinating SCs by simply applying electric pulses to the sciatic nerve after plasmid DNA injection.


Assuntos
Eletroporação , Técnicas de Transferência de Genes , Bainha de Mielina/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Animais , Plasmídeos/administração & dosagem , Plasmídeos/genética , Ratos , Roedores , Células de Schwann/ultraestrutura , Nervo Isquiático/ultraestrutura , Transfecção
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