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1.
Methods Mol Biol ; 2269: 245-254, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687684

RESUMO

Peripheral nerves have a limited ability to regenerate and current clinical approaches involving microsurgery give suboptimal recovery. Engineered tissues using aligned cellular collagen hydrogels can be used as in vitro models through the incorporation of human Schwann cells. However, primary human Schwann cells are difficult to obtain and can be challenging to culture. The ability to generate Schwann cells from human-induced pluripotent stem cells (hiPSCs) provides a more reliable cell source for modeling peripheral nerve tissue. Here, we describe protocols for generating hiPSC-derived Schwann cells and incorporating them into 3D engineered tissue culture models for peripheral nerve research.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Regeneração Nervosa , Nervos Periféricos/metabolismo , Células de Schwann/metabolismo , Engenharia Tecidual , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Nervos Periféricos/citologia
2.
Nat Commun ; 12(1): 1624, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712610

RESUMO

Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state. We profile ganglioneuromas and neuroblastomas, rich and poor in SC stroma, respectively, and peripheral nerves after injury, rich in repair SCs. Indeed, stromal SCs in ganglioneuromas and repair SCs share the expression of nerve repair-associated genes. Neuroblastoma cells, derived from aggressive tumors, respond to primary repair-related SCs and their secretome with increased neuronal differentiation and reduced proliferation. Within the pool of secreted stromal and repair SC factors, we identify EGFL8, a matricellular protein with so far undescribed function, to act as neuritogen and to rewire cellular signaling by activating kinases involved in neurogenesis. In summary, we report that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/fisiologia , Família de Proteínas EGF/genética , Família de Proteínas EGF/metabolismo , Neurogênese/fisiologia , Células de Schwann/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Plasticidade Celular/fisiologia , Proliferação de Células , Técnicas de Cocultura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regeneração Nervosa , Neuroblastoma/patologia , Neurogênese/genética , Traumatismos dos Nervos Periféricos , Transcriptoma , Adulto Jovem
3.
Biomed Pharmacother ; 133: 111062, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378965

RESUMO

Diabetic peripheral neuropathy (DPN) is the common complication of diabetes mellitus. Histone deacetylase (HDAC) inhibitor trichostatin A (TSA) is reported to ameliorate the peripheral nerves degeneration of DPN. However, the exact mechanism is still not well elucidated. Here, we first revealed that TSA promoted nerve conduction and brain derived neurotrophic factor (BDNF) expression in the sciatic nerves of diabetic mice. In line, TSA also reversed high glucose-reduced mature BDNF expression in vitro cultured rat Schwann cells (RSC96). Then unexpectedly, the downstream targets of TSA HDAC1 and HDAC5 were not involved in TSA-improved BDNF expression. Furthermore, unfolded protein response (UPR) chaperone GRP78 was revealed to be downregulated with high glucose stimulation in RSC96 cells, which was avoided with TSA treatment. Also, GRP78 upregulation mediated TSA-improved mature BDNF expression in high glucose-cultured RSC96 cells by binding with BDNF. As well, TSA treatment enhanced the binding of GRP78 with BDNF in RSC96 cells. Again, UPR-associated transcription factors XBP-1s and ATF6 were involved in TSA-increased GRP78 expression in high glucose-stimulated RSC96 cells. Finally, conditioned medium from high glucose-cultured RSC96 cells delayed neuron SH-SY5Y differentiation and that from TSA-treated high glucose-cultured RSC96 cells promoted SH-SY5Y cell differentiation. Taken together, our findings suggested that TSA increased BDNF expression to ameliorate DPN by improving XBP-1s/ATF6/GRP78 axis in Schwann cells.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neuropatias Diabéticas/tratamento farmacológico , Proteínas de Choque Térmico/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Células de Schwann/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , Fator 6 Ativador da Transcrição/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Linhagem Celular Tumoral , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Ratos , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Transdução de Sinais , Regulação para Cima , Proteína 1 de Ligação a X-Box/genética
4.
J Vis Exp ; (166)2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33346186

RESUMO

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Junção Neuromuscular/citologia , Animais , Curare , Matriz Extracelular/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Junção Neuromuscular/ultraestrutura , Células de Schwann/citologia , Células de Schwann/metabolismo
5.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374622

RESUMO

In diabetic peripheral neuropathy (DPN), metabolic disorder by hyperglycemia progresses in peripheral nerves. In addition to the direct damage to peripheral neural axons, the homeostatic mechanism of peripheral nerves is disrupted by dysfunction of the blood-nerve barrier (BNB) and Schwann cells. The disruption of the BNB, which is a crucial factor in DPN development and exacerbation, causes axonal degeneration via various pathways. Although many reports revealed that hyperglycemia and other important factors, such as dyslipidemia-induced dysfunction of Schwann cells, contributed to DPN, the molecular mechanisms underlying BNB disruption have not been sufficiently elucidated, mainly because of the lack of in vitro studies owing to difficulties in establishing human cell lines from vascular endothelial cells and pericytes that form the BNB. We have developed, for the first time, temperature-sensitive immortalized cell lines of vascular endothelial cells and pericytes originating from the BNB of human sciatic nerves, and we have elucidated the disruption to the BNB mainly in response to advanced glycation end products in DPN. Recently, we succeeded in developing an in vitro BNB model to reflect the anatomical characteristics of the BNB using cell sheet engineering, and we established immortalized cell lines originating from the human BNB. In this article, we review the pathologic evidence of the pathology of DPN in terms of BNB disruption, and we introduce the current in vitro BNB models.


Assuntos
Barreira Hematoneural/metabolismo , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/metabolismo , Animais , Membrana Basal/metabolismo , Biomarcadores , Barreira Hematoneural/patologia , Neuropatias Diabéticas/patologia , Suscetibilidade a Doenças , Humanos , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Hipóxia/metabolismo , Imuno-Histoquímica , Microvasos/metabolismo , Pericitos/metabolismo , Nervos Periféricos/irrigação sanguínea , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Células de Schwann/metabolismo
6.
Yakugaku Zasshi ; 140(11): 1381-1388, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33132274

RESUMO

Epalrestat (EPS), approved in Japan, is currently the only aldose reductase inhibitor that is available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH) in rat Schwann cells. GSH, the most abundant non-protein thiol antioxidant in cells, is important for protection against oxidative stress. Oxidative stress is associated with the development and progression of many pathological conditions, such as atherosclerosis, diabetes, and neurodegeneration. In this study, we tested the hypothesis that EPS enhances resistance to oxidative stress, by using rat Schwann cells. To determine whether EPS protects Schwann cells from oxidative stress, we performed experiments by using radical generators, drugs, and heavy metals as the source of oxidative stress. EPS reduced the cytotoxicity induced by 2,2-azobis-[2-(2-imidazolin-2-yl) propane] dihydrochloride, 6-hydroxydopamine, cisplatin, palmitate, cadmium chloride, and manganese (II) sulfate, indicating that EPS plays a role in protecting cells from oxidative stress. We suggest that EPS has the potential to prevent the development and progression of disorders caused by oxidative stress.


Assuntos
Antioxidantes/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Metais Pesados/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Rodanina/análogos & derivados , Células de Schwann/metabolismo , Tiazolidinas/farmacologia , Aldeído Redutase/antagonistas & inibidores , Animais , Células Cultivadas , Inibidores Enzimáticos , Ratos , Rodanina/farmacologia
7.
Nat Commun ; 11(1): 4514, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908139

RESUMO

The velocity of nerve conduction is moderately enhanced by larger axonal diameters and potently sped up by myelination of axons. Myelination thus allows rapid impulse propagation with reduced axonal diameters; however, no myelin-dependent mechanism has been reported that restricts radial growth of axons. By label-free proteomics, STED-microscopy and cryo-immuno electron-microscopy we here identify CMTM6 (chemokine-like factor-like MARVEL-transmembrane domain-containing family member-6) as a myelin protein specifically localized to the Schwann cell membrane exposed to the axon. We find that disruption of Cmtm6-expression in Schwann cells causes a substantial increase of axonal diameters but does not impair myelin biogenesis, radial sorting or integrity of axons. Increased axonal diameters correlate with accelerated sensory nerve conduction and sensory responses and perturbed motor performance. These data show that Schwann cells utilize CMTM6 to restrict the radial growth of axons, which optimizes nerve function.


Assuntos
Axônios/metabolismo , Proteínas da Mielina/metabolismo , Nervos Periféricos/citologia , Células de Schwann/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Axônios/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Masculino , Camundongos , Camundongos Knockout , Bainha de Mielina/metabolismo , Condução Nervosa , Nervos Periféricos/metabolismo , Nervos Periféricos/ultraestrutura , Proteômica , Células de Schwann/citologia , Células de Schwann/ultraestrutura , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/ultraestrutura
8.
PLoS One ; 15(9): e0238208, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881928

RESUMO

INTRODUCTION: Peripheral nerve injury (PNI) often leads to significant functional loss in patients and poses a challenge to physicians since treatment options for improving functional outcomes are limited. Recent studies suggest that erythropoietin and glucocoticoids have beneficial effects as mediators of neuro-regenerative processes. We hypothesized that combination treatment with erythropoietin and glucocoticoids would have a synergistic effect on functional outcome after PNI. MATERIALS AND METHODS: Sciatic nerve crush injury was simulated in ten-week-old male C57BL/6 mice. The mice were divided into four groups according to the type of drugs administered (control, erythropoietin, dexamethasone, and erythropoietin with dexamethasone). Motor functional recovery was monitored by walking track analysis at serial time points up to 28 days after injury. Morphological analysis of the nerve was performed by immunofluorescent staining for neurofilament (NF) heavy chain and myelin protein zero (P0) in cross-sectional and whole-mount nerve preparations. Additionally, morphological analysis of the muscle was performed by Hematoxylin and eosin staining. RESULTS: Combination treatment with erythropoietin and dexamethasone significantly improved the sciatic functional index at 3, 7, 14, and 28 days after injury. Fluorescence microscopy of cross sectional nerve revealed that the combination treatment increased the ratio of P0/NF-expressing axons. Furthermore, confocal microscopy of the whole-mount nerve revealed that the combination treatment increased the fluorescence intensity of P0 expression. The cross-sectional area and minimum Feret's diameter of the muscle fibers were significantly larger in the mice which received combination treatment than those in the controls. CONCLUSION: Our results demonstrated that combination treatment with erythropoietin and dexamethasone accelerates functional recovery and reduces neurogenic muscle atrophy caused by PNI in mice, which may be attributed to the preservation of myelin and Schwann cell re-myelination. These findings may provide practical therapeutic options for patients with acute PNI.


Assuntos
Dexametasona/uso terapêutico , Eritropoetina/uso terapêutico , Músculos/metabolismo , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Nervo Isquiático/metabolismo , Doença Aguda , Animais , Axônios/metabolismo , Dexametasona/farmacologia , Modelos Animais de Doenças , Quimioterapia Combinada , Eritropoetina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Músculos/patologia , Atrofia Muscular/patologia , Atrofia Muscular/prevenção & controle , Proteína P0 da Mielina/metabolismo , Traumatismos dos Nervos Periféricos/patologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Células de Schwann/citologia , Células de Schwann/metabolismo , Nervo Isquiático/patologia
9.
Nat Commun ; 11(1): 4133, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807777

RESUMO

Chromatin organization is critical for cell growth, differentiation, and disease development, however, its functions in peripheral myelination and myelin repair remain elusive. In this report, we demonstrate that the CCCTC-binding factor (CTCF), a crucial chromatin organizer, is essential for Schwann cell myelination and myelin regeneration after nerve injury. Inhibition of CTCF or its deletion blocks Schwann cell differentiation at the pro-myelinating stage, whereas overexpression of CTCF promotes the myelination program. We find that CTCF establishes chromatin interaction loops between enhancer and promoter regulatory elements and promotes expression of a key pro-myelinogenic factor EGR2. In addition, CTCF interacts with SUZ12, a component of polycomb-repressive-complex 2 (PRC2), to repress the transcriptional program associated with negative regulation of Schwann cell maturation. Together, our findings reveal a dual role of CTCF-dependent chromatin organization in promoting myelinogenic programs and recruiting chromatin-repressive complexes to block Schwann cell differentiation inhibitors to control peripheral myelination and repair.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Bainha de Mielina/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Fator de Ligação a CCCTC/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Proteína 2 de Resposta de Crescimento Precoce/genética , Camundongos , Bainha de Mielina/genética , Complexo Repressor Polycomb 2/genética , Ratos , Células de Schwann/metabolismo
10.
PLoS Genet ; 16(8): e1008752, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790717

RESUMO

Myelin is composed of plasma membrane spirally wrapped around axons and compacted into dense sheaths by myelin-associated proteins. Myelin is elaborated by neuroepithelial derived oligodendrocytes in the central nervous system (CNS) and by neural crest derived Schwann cells in the peripheral nervous system (PNS). While some myelin proteins accumulate in only one lineage, myelin basic protein (Mbp) is expressed in both. Overlapping the Mbp gene is Golli, a transcriptional unit that is expressed widely both within and beyond the nervous system. A super-enhancer domain within the Golli/Mbp locus contains multiple enhancers shown previously to drive reporter construct expression specifically in oligodendrocytes or Schwann cells. In order to determine the contribution of each enhancer to the Golli/Mbp expression program, and to reveal if functional interactions occur among them, we derived mouse lines in which they were deleted, either singly or in different combinations, and relative mRNA accumulation was measured at key stages of early development and at maturity. Although super-enhancers have been shown previously to facilitate interaction among their component enhancers, the enhancers investigated here demonstrated largely additive relationships. However, enhancers demonstrating autonomous activity strictly in one lineage, when missing, were found to significantly reduce output in the other, thus revealing cryptic "stealth" activity. Further, in the absence of a key oligodendrocyte enhancer, Golli accumulation was markedly and uniformly attenuated in all cell types investigated. Our observations suggest a model in which enhancer-mediated DNA-looping and potential super-enhancer properties underlie Golli/Mbp regulatory organization.


Assuntos
Elementos Facilitadores Genéticos , Proteína Básica da Mielina/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina/metabolismo , Neurogênese , Oligodendroglia/citologia , Oligodendroglia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células de Schwann/citologia , Células de Schwann/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo
11.
Mem Inst Oswaldo Cruz ; 115: e200075, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32696914

RESUMO

BACKGROUND: Although Mycobacterium leprae (ML) is well characterised as the causative agent of leprosy, the pathophysiological mechanisms underlying peripheral nerve damage still need further understanding. In vitro and in vivo studies have yielded insights into molecular mechanisms of ML interaction with Schwann cells (SC), indicating the regulation of genes and proteins crucial to neural plasticity. OBJECTIVES: We aimed to investigate the effect of ML on neurotrophins expression in human SC (hSC) and mice sciatic nerves to better understand their role in leprosy neuropathy, and aiming to contribute to future therapeutic approaches. METHODS: We evaluated mRNA and protein expression of BDNF, NGF, NT-3, NT-4 in hSC from amputation nerve fragments, as well as in athymic nude mice, infected by ML for eight months. FINDINGS AND MAIN CONCLUSIONS: Our in vitro results showed a trend to decline in NGF and BDNF mRNA in ML-treated hSC, compared to controls. The immunodetection of BDNF and NT-4 was significantly downregulated in ML-treated hSC. Conversely, ML-infected mice demonstrated upregulation of NT-3, compared to non-infected animals. Our findings indicate that ML may be involved in neurotrophins regulation, suggesting that a pathogen-related imbalance of these growth factors may have a role in the neural impairment of leprosy.


Assuntos
Mycobacterium leprae , Fatores de Crescimento Neural/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Animais , Humanos , Camundongos , Camundongos Nus
12.
Nucleic Acids Res ; 48(16): 8959-8976, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32672815

RESUMO

Schwann cells are the nerve ensheathing cells of the peripheral nervous system. Absence, loss and malfunction of Schwann cells or their myelin sheaths lead to peripheral neuropathies such as Charcot-Marie-Tooth disease in humans. During Schwann cell development and myelination chromatin is dramatically modified. However, impact and functional relevance of these modifications are poorly understood. Here, we analyzed histone H2B monoubiquitination as one such chromatin modification by conditionally deleting the Rnf40 subunit of the responsible E3 ligase in mice. Rnf40-deficient Schwann cells were arrested immediately before myelination or generated abnormally thin, unstable myelin, resulting in a peripheral neuropathy characterized by hypomyelination and progressive axonal degeneration. By combining sequencing techniques with functional studies we show that H2B monoubiquitination does not influence global gene expression patterns, but instead ensures selective high expression of myelin and lipid biosynthesis genes and proper repression of immaturity genes. This requires the specific recruitment of the Rnf40-containing E3 ligase by Egr2, the central transcriptional regulator of peripheral myelination, to its target genes. Our study identifies histone ubiquitination as essential for Schwann cell myelination and unravels new disease-relevant links between chromatin modifications and transcription factors in the underlying regulatory network.


Assuntos
Proteína 2 de Resposta de Crescimento Precoce/fisiologia , Neuropatia Hereditária Motora e Sensorial/metabolismo , Histonas/metabolismo , Sistema Nervoso Periférico/metabolismo , Células de Schwann/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Sistema Nervoso Periférico/patologia , Ratos , Células de Schwann/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
13.
Nat Commun ; 11(1): 3420, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647127

RESUMO

Remyelination of the peripheral and central nervous systems (PNS and CNS, respectively) is a prerequisite for functional recovery after lesion. However, this process is not always optimal and becomes inefficient in the course of multiple sclerosis. Here we show that, when acetylated, eukaryotic elongation factor 1A1 (eEF1A1) negatively regulates PNS and CNS remyelination. Acetylated eEF1A1 (Ac-eEF1A1) translocates into the nucleus of myelinating cells where it binds to Sox10, a key transcription factor for PNS and CNS myelination and remyelination, to drag Sox10 out of the nucleus. We show that the lysine acetyltransferase Tip60 acetylates eEF1A1, whereas the histone deacetylase HDAC2 deacetylates eEF1A1. Promoting eEF1A1 deacetylation maintains the activation of Sox10 target genes and increases PNS and CNS remyelination efficiency. Taken together, these data identify a major mechanism of Sox10 regulation, which appears promising for future translational studies on PNS and CNS remyelination.


Assuntos
Fator 1 de Elongação de Peptídeos/metabolismo , Remielinização/genética , Ativação Transcricional/genética , Acetilação , Envelhecimento/metabolismo , Animais , Desdiferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Lisina Acetiltransferase 5/metabolismo , Camundongos , Modelos Biológicos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Fatores de Transcrição SOXE/metabolismo , Fator de Transcrição STAT3/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Teofilina/farmacologia , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos
14.
Life Sci ; 256: 117959, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32531375

RESUMO

Resveratrol has the ability to promote functional recovery after sciatic nerve crush injury (SNCI), though the mechanism through which this occurs in not fully understood. Resveratrol can promote autophagy, a key process in Wallerian degeneration; thus, we hypothesized that resveratrol could promote recovery from SNCI by promoting Schwann cell autophagy and acceleration of Wallerian degeneration. Motor function recovery was assessed by calculating Sciatic Function Indexes (SFIs) at days 7, 14, 21, 28 post SNCI. Autophagy and myelin clearance were assessed by microtubule-associated protein light chain 3B (LC3B) and myelin protein zero (MPZ) immunofluorescence and Western blot analysis on the fourth day after SNCI. The autophagy of Schwann cells following resveratrol administration was quantified by immunofluorescence in RSC96 cells. Immunofluorescence and Transmission electron microscopy (TEM) were also used in Resveratrol treated sciatic nerve four days post-SNCI to find LC3B positive areas and typical double membrane structures represent for autophagy. The SNCI+resveratrol (crush+Res) groups recovered faster than the SNCI+vehicles (crush+V) group. On day four, almost all of the myelin had regenerated in the crush+Res rats, while the crush+V group's myelin remained intact and the expression levels of LC3-II/I was the highest. On day 28 post-injury, both the control and crush+Res groups' myelin neurofibers reached peak numbers as did the thickness of the myelin sheath. Both in vitro and in vivo immunofluorescence showed that LC3B was colocalized with Schwann cells. This is the first study to observe that resveratrol can promote recovery from SCNI by accelerating the myelin clearance process by promoting autophagy of Schwann cells.


Assuntos
Autofagia/efeitos dos fármacos , Lesões por Esmagamento/fisiopatologia , Compressão Nervosa , Recuperação de Função Fisiológica/efeitos dos fármacos , Resveratrol/farmacologia , Células de Schwann/patologia , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Lesões por Esmagamento/patologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Atividade Motora/efeitos dos fármacos , Proteína P0 da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/patologia , Regeneração Nervosa/efeitos dos fármacos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Nervo Isquiático/efeitos dos fármacos
15.
Nat Commun ; 11(1): 2123, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358558

RESUMO

Mammals differ in their regeneration potential after traumatic injury, which might be caused by species-specific regeneration programs. Here, we compared murine and human Schwann cell (SC) response to injury and developed an ex vivo injury model employing surgery-derived human sural nerves. Transcriptomic and lipid metabolism analysis of murine SCs following injury of sural nerves revealed down-regulation of lipogenic genes and regulator of lipid metabolism, including Pparg (peroxisome proliferator-activated receptor gamma) and S1P (sphingosine-1-phosphate). Human SCs failed to induce similar adaptations following ex vivo nerve injury. Pharmacological PPARg and S1P stimulation in mice resulted in up-regulation of lipid gene expression, suggesting a role in SCs switching towards a myelinating state. Altogether, our results suggest that murine SC switching towards a repair state is accompanied by transcriptome and lipidome adaptations, which are reduced in humans.


Assuntos
Metabolismo dos Lipídeos/fisiologia , Células de Schwann/citologia , Células de Schwann/metabolismo , Animais , Feminino , Humanos , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , Bainha de Mielina/metabolismo , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , PPAR gama/metabolismo , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(23): 12856-12867, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32439707

RESUMO

The conventional outflow pathway is a complex tissue responsible for maintaining intraocular pressure (IOP) homeostasis. The coordinated effort of multiple cells with differing responsibilities ensures healthy outflow function and IOP maintenance. Dysfunction of one or more resident cell types results in ocular hypertension and risk for glaucoma, a leading cause of blindness. In this study, single-cell RNA sequencing was performed to generate a comprehensive cell atlas of human conventional outflow tissues. We obtained expression profiles of 17,757 genes from 8,758 cells from eight eyes of human donors representing the outflow cell transcriptome. Upon clustering analysis, 12 distinct cell types were identified, and region-specific expression of candidate genes was mapped in human tissues. Significantly, we identified two distinct expression patterns (myofibroblast- and fibroblast-like) from cells located in the trabecular meshwork (TM), the primary structural component of the conventional outflow pathway. We also located Schwann cell and macrophage signatures in the TM. The second primary component structure, Schlemm's canal, displayed a unique combination of lymphatic/blood vascular gene expression. Other expression clusters corresponded to cells from neighboring tissues, predominantly in the ciliary muscle/scleral spur, which together correspond to the uveoscleral outflow pathway. Importantly, the utility of our atlas was demonstrated by mapping glaucoma-relevant genes to outflow cell clusters. Our study provides a comprehensive molecular and cellular classification of conventional and unconventional outflow pathway structures responsible for IOP homeostasis.


Assuntos
Humor Aquoso/metabolismo , Glaucoma/patologia , Pressão Intraocular/fisiologia , Miofibroblastos/metabolismo , Malha Trabecular/metabolismo , Glaucoma/genética , Humanos , Macrófagos/metabolismo , RNA-Seq , Células de Schwann/metabolismo , Análise de Célula Única , Malha Trabecular/citologia
17.
PLoS One ; 15(5): e0233651, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469980

RESUMO

Transference of RNAs and ribosomes from Schwann cell-to-axon was demonstrated in normal and regenerating peripheral nerves. Previously, we have shown that RNAs transfer is dependent on F-actin cytoskeleton and Myosin Va. Here, we explored the contribution of microtubules to newly synthesized RNAs transport from Schwann cell nuclei up to nodal microvilli in sciatic nerves. Results using immunohistochemistry and quantitative confocal FRET analysis indicate that Schwann cell-derived RNAs co-localize with microtubules in Schwann cell cytoplasm. Additionally, transport of Schwann cell-derived RNAs is nocodazole and colchicine sensitive demonstrating its dependence on microtubule network integrity. Moreover, mRNAs codifying neuron-specific proteins are among Schwann cell newly synthesized RNAs population, and some of them are associated with KIF1B and KIF5B microtubules-based motors.


Assuntos
Axônios/metabolismo , Microtúbulos/metabolismo , RNA/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Animais , Masculino , Bainha de Mielina/metabolismo , Regeneração Nervosa , RNA/análise , Transporte de RNA , Ratos , Ratos Sprague-Dawley
18.
Wei Sheng Yan Jiu ; 49(1): 14-18, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32290908

RESUMO

OBJECTIVE: To investigate the expression of S100ß protein and mRNA of Schwann cells(SC) in sciatic nerves of 2, 5-hexanedione(HD) intoxicated rats. METHODS: Nine-week old SPF male Wistar rats were administered at daily dosing of 100 and 300 mg/kg by intraperitoneal injection for continuous 8 weeks(five times every week). Age-matched control rats received an equivalent volume of normal saline. Ten rats in each group were sacrificed and sciatic nerves were excised for S100ß determination, with excised sciatic nerves from another three rats for morphological observation through electron microscope. At the end of the exposure, the other 8-week treated animals were allowed to naturally recover for 8 weeks and sciatic nerves were excised at the end of the test. S100ß protein contents were determined by immunohistochemistry method, and mRNA expression was observed by real-time quantitative polymerase chain reaction(PCR). RESULTS: HD intoxication with 300 mg/kg was associated with severe neurological deficits of paralysis in hindlimbs, accompanied with evident movement gait abnormalities for 100 mg/kg dosage. The morphological abnormalities in myelin sheath of sciatic nerves were observed through electron microscope after HD-exposure. The S100ß contents in 100 mg/kg and 300 mg/kg groups remained relatively unaffected with 92% and 79% of the control respectively after HD-intoxication, and a increase to 149%(P<0. 05) and 119% after a recovery of 8 weeks was accompanied with. As to S100ß mRNA, HD-intoxication was associated with decreased expression to 0. 65(P<0. 05) and 0. 56 times(P<0. 05) of the control, and 1. 46 and 0. 87 times for 8-week recovery individually. CONCLUSION: The S100ß protein and mRNA levels were influenced by HD exposure, and the result suggested that S100ß might be involved in HD-induced peripheral axonopathy.


Assuntos
Hexanonas/toxicidade , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Células de Schwann/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Animais , Masculino , Ratos , Ratos Wistar , Células de Schwann/metabolismo , Nervo Isquiático/patologia , Testes de Toxicidade
19.
Sci Rep ; 10(1): 6396, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286429

RESUMO

Neuropathic pain caused by peripheral nerve injuries significantly affects sensory perception and quality of life. Accumulating evidence strongly link cholesterol with development and progression of Obesity and Diabetes associated-neuropathies. However, the exact mechanisms of how cholesterol/lipid metabolism in peripheral nervous system (PNS) contributes to the pathogenesis of neuropathy remains poorly understood. Dysregulation of LXR pathways have been identified in many neuropathic models. The cholesterol sensor, LXR α/ß, expressed in sensory neurons are necessary for proper peripheral nerve function. Deletion of LXR α/ß from sensory neurons lead to pain-like behaviors. In this study, we identified that LXR α/ß expressed in sensory neurons regulates neuronal Neuregulin 1 (Nrg1), protein involved in cell-cell communication. Using in vivo cell-specific approaches, we observed that loss of LXR from sensory neurons altered genes in non-neuronal cells located in the sciatic nerve (potentially representing Schwann cells (SC)). Our data suggest that neuronal LXRs may regulate non-neuronal cell function via a Nrg1-dependent mechanism. The decrease in Nrg1 expression in DRG neurons of WD-fed mice may suggest an altered Nrg1-dependent neuron-SC communication in Obesity. The communication between neurons and non-neuronal cells such as SC could be a new biological pathway to study and understand the molecular and cellular mechanism underlying Obesity-associated neuropathy and PNS dysfunction.


Assuntos
Dieta Ocidental , Receptores X do Fígado/metabolismo , Neuregulina-1/genética , Nervo Isquiático/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais , Animais , Orientação de Axônios , Receptores ErbB/metabolismo , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Camundongos , Neuregulina-1/metabolismo , Células de Schwann/metabolismo , Transcrição Genética
20.
Neuron ; 106(5): 806-815.e6, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32209430

RESUMO

During development of the peripheral nervous system (PNS), Schwann-cell-secreted gliomedin induces the clustering of Na+ channels at the edges of each myelin segment to form nodes of Ranvier. Here we show that bone morphogenetic protein-1 (BMP1)/Tolloid (TLD)-like proteinases confine Na+ channel clustering to these sites by negatively regulating the activity of gliomedin. Eliminating the Bmp1/TLD cleavage site in gliomedin or treating myelinating cultures with a Bmp1/TLD inhibitor results in the formation of numerous ectopic Na+ channel clusters along axons that are devoid of myelin segments. Furthermore, genetic deletion of Bmp1 and Tll1 genes in mice using a Schwann-cell-specific Cre causes ectopic clustering of nodal proteins, premature formation of heminodes around early ensheathing Schwann cells, and altered nerve conduction during development. Our results demonstrate that by inactivating gliomedin, Bmp1/TLD functions as an additional regulatory mechanism to ensure the correct spatial and temporal assembly of PNS nodes of Ranvier.


Assuntos
Proteína Morfogenética Óssea 1/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Bainha de Mielina/metabolismo , Nós Neurofibrosos/metabolismo , Metaloproteases Semelhantes a Toloide/genética , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Proteína Morfogenética Óssea 1/metabolismo , Camundongos , Camundongos Knockout , Condução Nervosa , Sistema Nervoso Periférico , Transporte Proteico , Células de Schwann/metabolismo , Metaloproteases Semelhantes a Toloide/metabolismo
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