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1.
Neurochem Res ; 44(9): 2230-2236, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31486011

RESUMO

Upper limb nerve injuries are common, and their treatment poses a challenge for physicians and surgeons. Experimental models help in minimum exploration of the functional characteristics of peripheral nerve injuries of forelimbs. This study was conducted to characterize the functional recovery (1, 3, 7, 10, 14, and 21 days) after median and ulnar nerve crush in mice and analyze the histological and biochemical markers of nerve regeneration (after 21 days). Sensory-functional impairments appeared after 1 day. The peripheral nerve morphology, the nerve structure, and the density of myelin proteins [myelin protein zero (P0) and peripheral myelin protein 22 (PMP22)] were analyzed after 21 days. Cold allodynia and fine motor coordination recovery occurred on the 10th day, and grip strength recovery was observed on the 14th day after injury. After 21 days, there was partial myelin sheath recovery. PMP22 recovery was complete, whereas P0 recovery was not. Results suggest that there is complete functional recovery even with partial remyelination of median and ulnar nerves in mice.


Assuntos
Nervo Mediano/fisiopatologia , Recuperação de Função Fisiológica , Remielinização , Nervo Ulnar/fisiopatologia , Animais , Masculino , Nervo Mediano/lesões , Nervo Mediano/metabolismo , Camundongos , Proteína P0 da Mielina/metabolismo , Proteínas da Mielina/metabolismo , Compressão Nervosa , Nervo Ulnar/lesões , Nervo Ulnar/metabolismo
2.
Int J Mol Med ; 44(2): 759-767, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173167

RESUMO

Enterovirus 71 (EV71) accounts for the majority of hand, foot and mouth disease­related deaths due to fatal neurological complications. EV71 structural viral protein 1 (VP1) promotes viral replication by inducing autophagy in neuron cells, but the effect of VP1 on myelin cells is unclear. The present study aimed to investigate the role and mechanism of VP1 in autophagy of mouse Schwann cells. An EV71 VP1­expressing vector (pEGFP­C3­VP1) was generated and transfected into mouse Schwann cells. Transmission electron microscopy and western blot analysis for microtubule­associated protein 1 light chain 3 α (LC3) II (an autophagy marker) were used to assess autophagy. Reverse transcription­quantitative PCR and immunofluorescence were performed to determine the expression of peripheral myelin protein 22 (PMP22). Small interfering RNA against PMP22 was used to investigate the role of PMP22 in mouse Schwann cell autophagy. Salubrinal [a selective endoplasmic reticulum (ER) stress inhibitor] was used to determine whether PMP22 expression was affected by ER stress. The present results indicated that VP1 promoted mouse Schwann cell autophagy. Overexpression of VP1 upregulated PMP22. PMP22 deficiency downregulated LC3II and thus inhibited autophagy. Furthermore, PMP22 expression was significantly suppressed by salubrinal. In conclusion, VP1 promoted mouse Schwann cell autophagy through upregulation of ER stress­mediated PMP22 expression. Therefore, the VP1/ER stress/PMP22 autophagy axis may be a potential therapeutic target for EV71 infection­induced fatal neuronal damage.


Assuntos
Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Proteínas da Mielina/metabolismo , Células de Schwann/virologia , Proteínas Estruturais Virais/metabolismo , Animais , Autofagia , Linhagem Celular , Estresse do Retículo Endoplasmático , Infecções por Enterovirus/virologia , Humanos , Camundongos , Células de Schwann/metabolismo , Células de Schwann/patologia
3.
Glia ; 67(9): 1775-1792, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31184779

RESUMO

Oligodendrocytes (OLs) provide the myelin sheath surrounding axons that propagates action potentials in the central nervous system (CNS). The metabolism of myelinated membranes and proteins is strictly regulated in the OLs and is closely associated with OL differentiation and maturation. The ubiquitination-associated proteasome and endosomal system have not yet been well studied during OL differentiation and maturation. Here, we determined the functions of the Lys63-linked ubiquitination (K63Ub) and K63-specific deubiquitination (DUB) systems regulated by BRCA1/BRCA2-containing complex subunit 3 (BRCC3) during OL differentiation. The competitive inhibition of K63Ub by overexpression of mutant ubiquitin (K63R) in oligodendrocyte precursor cells (OPCs) indicated that the two major CNS myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), were upregulated in OLs derived from K63R OPCs. In contrast, the knockdown of BRCC3 (BRCC3-KD) through the application of lentivirus-mediated shRNA delivery system into OPCs suppressed OL differentiation by decreasing MBP expression and PLP production. Further immunoprecipitation assays revealed higher levels of sphingolipid GalC, MBP, and PLP, which were associated with K63Ub-immunoprecipitants and detected in endosome/lysosomal compartments, in BRCC3-KD OLs than those in OLs transfected with the scrambled shRNA (scramble OLs). The differentiation of OLs from BRCC3-KD OPCs was impaired in the demyelinating corpus callosum of rats receiving a cuprizone-containing diet. In the demyelinating tissues from human patients suffering from multiple sclerosis, we detected a decreased number of BRCC3-expressing OLs at the lesion site, accompanied by a greater number of OLs expressing EEA1 and K63Ub at high levels. Altogether, the counterbalance of the K63Ub machinery and BRCC3-triggered DUB machinery are important for the cellular trafficking of myelin proteins and OL differentiation.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Neurogênese/fisiologia , Oligodendroglia/metabolismo , Ubiquitinação/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Endossomos/metabolismo , Endossomos/patologia , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Proteínas da Mielina/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Células Precursoras de Oligodendrócitos/patologia , Oligodendroglia/patologia , Ratos Sprague-Dawley
4.
Methods Mol Biol ; 1936: 37-63, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820892

RESUMO

Molecular characterization of myelin is a prerequisite for understanding the normal structure of the axon/myelin-unit in the healthy nervous system and abnormalities in myelin-related disorders. However, reliable molecular profiles necessitate very pure myelin membranes, in particular when considering the power of highly sensitive "omics"-data acquisition methods. Here, we recapitulate the history and recent applications of myelin purification. We then provide our laboratory protocols for the biochemical isolation of a highly pure myelin-enriched fraction from mouse brains and for its proteomic analysis. We also supply methodological modifications when investigating posttranslational modifications, RNA, or myelin from peripheral nerves. Notably, technical advancements in solubilizing myelin are beneficial for gel-based and gel-free myelin proteome analyses. We conclude this article by exemplifying the exceptional power of label-free proteomics in the mass-spectrometric quantification of myelin proteins.


Assuntos
Proteínas da Mielina/metabolismo , Proteômica/métodos , Animais , Centrifugação com Gradiente de Concentração , Espectrometria de Massas , Camundongos , Processamento de Proteína Pós-Traducional
5.
Basic Clin Pharmacol Toxicol ; 125(2): 152-165, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30916885

RESUMO

Carbon monoxide (CO) poisoning can lead to many serious neurological symptoms. Currently, there are no effective therapies for CO poisoning. In this study, rats exposed to CO received hyperbaric oxygen therapy, and those in the Fasudil group were given additional Fasudil injection once daily. We found that the escape latency in CO poisoning group (CO group) was significantly prolonged, the T1 /Ttotal was obviously decreased, and the mean escape time and the active escape latency were notably extended compared with those in normal control group (NC group, P < 0.05). After administration of Fasudil, the escape latency was significantly shortened, T1 /Ttotal was gradually increased as compared with CO group (>1 week, P < 0.05). Ultrastructural damage of neurons and blood-brain barrier of rats was serious in CO group, while the structural and functional integrity of neuron and mitochondria maintained relatively well in Fasudil group. Moreover, we also noted that the expressions of neurite outgrowth inhibitor (Nogo), oligodendrocyte-myelin glycoprotein (OMgp) and Rock in brain tissue were significantly increased in CO group, and the elevated levels of the three proteins were still observed at 2 months after CO poisoning. Fasudil markedly reduced their expressions compared with those of CO group (P < 0.05). In summary, the activation of Nogo-OMgp/Rho signalling pathway is associated with brain injury in rats with CO poisoning. Fasudil can efficiently down-regulate the expressions of Nogo, OMgp and Rock proteins, paving a way for the treatment of acute brain damage after CO poisoning.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Encéfalo/efeitos dos fármacos , Intoxicação por Monóxido de Carbono/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Encéfalo/patologia , Monóxido de Carbono/toxicidade , Intoxicação por Monóxido de Carbono/etiologia , Intoxicação por Monóxido de Carbono/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Proteínas Ligadas por GPI/metabolismo , Humanos , Masculino , Proteínas da Mielina/metabolismo , Proteínas Nogo/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Ratos , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
6.
Glia ; 67(5): 870-883, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30623969

RESUMO

In multiple sclerosis, demyelination occurs as a consequence of chronic autoimmunity in the central nervous system causing progressive neurological impairment in patients. After a demyelinating event, new myelin sheaths are formed by adult oligodendroglial progenitor cells; a process called remyelination. However, remyelination often fails in multiple sclerosis due to insufficient recruitment and differentiation of oligodendroglial precursor cells. A pivotal role for the two-pore-domain potassium (K2P ) channel, TASK1, has already been proven for an animal model of multiple sclerosis. However, the mechanisms underlying the TASK1-mediated effects are still elusive. Here, we tested the role of TASK1 channels in oligodendroglial differentiation and remyelination after cuprizone-induced demyelination in male mice. We found TASK1 channels to be functionally expressed on primary murine and human, pluripotent stem cell-derived oligodendrocytes. Lack of TASK1 channels resulted in an increase of mature oligodendrocytes in vitro as well as a higher number of mature oligodendrocytes and accelerated developmental myelination in vivo. Mechanistically, Task1-deficient cells revealed a higher amount of phosphorylated WNK1, a kinase known to be involved in the downstream signaling of the myelination regulator LINGO-1. Furthermore, we analyzed the effect of genetic TASK1 ablation or pharmacological TASK1 inhibition on disease-related remyelination. Neither channel inhibition nor lack of TASK1 channels promoted remyelination after pathological demyelination. In summary, we conclude that functional TASK1 channels participate in the modulation of differentiating oligodendroglial cells in a previously unknown manner. However, while being involved in developmental myelination our data suggest that TASK1 channels have no major effect on remyelination.


Assuntos
Diferenciação Celular/genética , Doenças Desmielinizantes/patologia , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Anestésicos Locais/farmacologia , Animais , Animais Recém-Nascidos , Bupivacaína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores da Monoaminoxidase/toxicidade , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Proteínas da Mielina/ultraestrutura , Proteínas do Tecido Nervoso/genética , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Células Precursoras de Oligodendrócitos/fisiologia , Células Precursoras de Oligodendrócitos/ultraestrutura , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/fisiologia , Oligodendroglia/ultraestrutura , Canais de Potássio de Domínios Poros em Tandem/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/farmacologia
7.
PLoS One ; 14(1): e0209752, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30650121

RESUMO

The most common type of Charcot-Marie-Tooth disease is caused by a duplication of PMP22 leading to dysmyelination, axonal loss and progressive muscle weakness (CMT1A). Currently, no approved therapy is available for CMT1A patients. A novel polytherapeutic proof-of-principle approach using PXT3003, a low-dose combination of baclofen, naltrexone and sorbitol, slowed disease progression after long-term dosing in adult Pmp22 transgenic rats, a known animal model of CMT1A. Here, we report an early postnatal, short-term treatment with PXT3003 in CMT1A rats that delays disease onset into adulthood. CMT1A rats were treated from postnatal day 6 to 18 with PXT3003. Behavioural, electrophysiological, histological and molecular analyses were performed until 12 weeks of age. Daily oral treatment for approximately 2 weeks ameliorated motor deficits of CMT1A rats reaching wildtype levels. Histologically, PXT3003 corrected the disturbed axon calibre distribution with a shift towards large motor axons. Despite dramatic clinical amelioration, only distal motor latencies were improved and correlated with phenotype performance. On the molecular level, PXT3003 reduced Pmp22 mRNA overexpression and improved the misbalanced downstream PI3K-AKT / MEK-ERK signalling pathway. The improved differentiation status of Schwann cells may have enabled better long-term axonal support function. We conclude that short-term treatment with PXT3003 during early development may partially prevent the clinical and molecular manifestations of CMT1A. Since PXT3003 has a strong safety profile and is currently undergoing a phase III trial in CMT1A patients, our results suggest that PXT3003 therapy may be a bona fide translatable therapy option for children and young adolescent patients suffering from CMT1A.


Assuntos
Baclofeno/farmacologia , Doença de Charcot-Marie-Tooth/tratamento farmacológico , Naltrexona/farmacologia , Sorbitol/farmacologia , Animais , Axônios/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Combinação de Medicamentos , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Debilidade Muscular/metabolismo , Proteínas da Mielina/efeitos dos fármacos , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Condução Nervosa , Fosfatidilinositol 3-Quinases/metabolismo , Estudo de Prova de Conceito , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Células de Schwann/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
8.
Glia ; 67(5): 967-984, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30667096

RESUMO

Protein tyrosine phosphatase receptor type Z (PTPRZ) maintains oligodendrocyte precursor cells (OPCs) in an undifferentiated state. The inhibition of PTPase by its ligand pleiotrophin (PTN) promotes OPC differentiation; however, the substrate molecules of PTPRZ involved in the differentiation have not yet been elucidated in detail. We herein demonstrated that the tyrosine phosphorylation of AFAP1L2, paxillin, ERBB4, GIT1, p190RhoGAP, and NYAP2 was enhanced in OPC-like OL1 cells by a treatment with PTN. AFAP1L2, an adaptor protein involved in the PI3K-AKT pathway, exhibited the strongest response to PTN. PTPRZ dephosphorylated AFAP1L2 at tyrosine residues in vitro and in HEK293T cells. In OL1 cells, the knockdown of AFAP1L2 or application of a PI3K inhibitor suppressed cell differentiation as well as the PTN-induced phosphorylation of AKT and mTOR. We generated a knock-in mouse harboring a catalytically inactive Cys to Ser (CS) mutation in the PTPase domain. The phosphorylation levels of AFAP1L2, AKT, and mTOR were higher, and the expression of oligodendrocyte markers, including myelin basic protein (MBP) and myelin regulatory factor (MYRF), was stronger in CS knock-in brains than in wild-type brains on postnatal day 10; however, these differences mostly disappeared in the adult stage. Adult CS knock-in mice exhibited earlier remyelination after cuprizone-induced demyelination through the accelerated differentiation of OPCs. These phenotypes in CS knock-in mice were similar to those in Ptprz-deficient mice. Therefore, we conclude that the PTN-PTPRZ signal stimulates OPC differentiation partly by enhancing the tyrosine phosphorylation of AFAP1L2 in order to activate the PI3K-AKT pathway.


Assuntos
Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Citocinas/metabolismo , Oligodendroglia/fisiologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/diagnóstico por imagem , Modelos Animais de Doenças , Células HEK293 , Humanos , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Proteínas da Mielina/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Detecção de Sinal Psicológico/efeitos dos fármacos , Detecção de Sinal Psicológico/fisiologia , Transfecção , Microtomografia por Raio-X
9.
Transl Psychiatry ; 9(1): 13, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30664625

RESUMO

There is a strong genetic association between germline PTEN mutation and autism spectrum disorder (ASD), making Pten-mutant models exemplary for the study of ASD pathophysiology. We developed the Ptenm3m4 mouse, where Pten is largely restricted from the nucleus, which recapitulates patient-like, autism-related phenotypes: behavioral changes, macrocephaly, and white matter abnormalities. This study aimed to investigate the contribution of oligodendrocyte (OL) lineage differentiation and functional changes in myelination to the white matter phenotype. OL lineage differentiation and myelination in Ptenm3m4 mice was studied using immunohistochemical and electron microscopic analyses. We also used primary oligodendrocyte progenitor cells (OPCs) to determine the effect of the Ptenm3m4 mutation on OPC proliferation, migration and maturation. Finally, we assessed the myelinating competency of mutant OLs via co-culture with wildtype dorsal root ganglia (DRG) neurons. The in vivo analyses of Ptenm3m4/m3m4 murine brains showed deficits in proteolipid protein (Plp) trafficking in myelinating OLs. Despite the increased expression of myelin proteins in the brain, myelin deposition was observed to be abnormal, often occurring adjacent to, rather than around axons. Mutant primary OPCs showed enhanced proliferation and migration. Furthermore, mutant OPCs matured precociously, exhibiting aberrant myelination in vitro. Mutant OPCs, when co-cultured with wildtype DRG neurons, showed an inability to properly ensheath axons. Our findings provide evidence that the Ptenm3m4 mutation disrupts the differentiation and myelination programs of developing OLs. OL dysfunction in the Ptenm3m4 model explains the leukodystrophy phenotype, a feature commonly associated with autism, and highlights the growing importance of glial dysfunction in autism pathogenesis.


Assuntos
Transtorno do Espectro Autista/fisiopatologia , Encéfalo/fisiopatologia , Neuroglia/citologia , PTEN Fosfo-Hidrolase/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Mutação de Sentido Incorreto , Proteínas da Mielina/metabolismo , Neurogênese , Células Precursoras de Oligodendrócitos/citologia , PTEN Fosfo-Hidrolase/genética
10.
Int J Mol Sci ; 20(1)2019 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-30609838

RESUMO

Remyelination is a central aspect of new multiple sclerosis (MS) therapies, in which one aims to alleviate disease symptoms by improving axonal protection. However, a central problem is mediators expressed in MS lesions that prevent effective remyelination. Bone morphogenetic protein4 (BMP4) inhibits the development of mature oligodendrocytes in cell culture and also blocks the expression of myelin proteins. Additionally, numerous studies have shown that Noggin (SYM1)-among other physiological antagonists of BMP4-plays a prominent role in myelin formation in the developing but also the adult central nervous system. Nonetheless, neither BMP4 nor Noggin have been systematically studied in human MS lesions. In this study, we demonstrated by transcript analysis and immunohistochemistry that BMP4 is expressed by astrocytes and microglia/macrophages in association with inflammatory infiltrates in MS lesions, and that astrocytes also express BMP4 in chronic inactive lesions that failed to remyelinate. Furthermore, the demonstration of an increased expression of Noggin in so-called shadow plaques (i.e., remyelinated lesions with thinner myelin sheaths) in comparison to chronically inactive demyelinated lesions implies that antagonizing BMP4 is associated with successful remyelination in MS plaques in humans. However, although BMP4 is strongly overexpressed in inflammatory lesion areas, its levels are also elevated in remyelinated lesion areas, which raises the possibility that BMP4 signaling itself may be required for remyelination. Therefore, remyelination might be influenced by a small number of key factors. Manipulating these molecules, i.e., BMP4 and Noggin, could be a promising therapeutic approach for effective remyelination.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Transporte/metabolismo , Esclerose Múltipla/patologia , Remielinização , Adulto , Idoso , Astrócitos/citologia , Astrócitos/metabolismo , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Proteína Morfogenética Óssea 4/genética , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Masculino , Microglia/citologia , Microglia/metabolismo , Pessoa de Meia-Idade , Proteínas da Mielina/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Substância Branca/metabolismo , Substância Branca/patologia
11.
Glia ; 67(4): 634-649, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30637801

RESUMO

Proteolipid protein (PLP) is the most abundant integral membrane protein in central nervous system (CNS) myelin. Expression of the Plp-gene in oligodendrocytes is not essential for the biosynthesis of myelin membranes but required to prevent axonal pathology. This raises the question whether the exceptionally high level of PLP in myelin is required later in life, or whether high-level PLP expression becomes dispensable once myelin has been assembled. Both models require a better understanding of the turnover of PLP in myelin in vivo. Thus, we generated and characterized a novel line of tamoxifen-inducible Plp-mutant mice that allowed us to determine the rate of PLP turnover after developmental myelination has been completed, and to assess the possible impact of gradually decreasing amounts of PLP for myelin and axonal integrity. We found that 6 months after targeting the Plp-gene the abundance of PLP in CNS myelin was about halved, probably reflecting that myelin is slowly turned over in the adult brain. Importantly, this reduction by 50% was sufficient to cause the entire spectrum of neuropathological changes previously associated with the developmental lack of PLP, including myelin outfoldings, lamellae splittings, and axonal spheroids. In comparison to axonopathy and gliosis, the infiltration of cytotoxic T-cells was temporally delayed, suggesting a corresponding chronology also in the genetic disorders of PLP-deficiency. High-level abundance of PLP in myelin throughout adult life emerges as a requirement for the preservation of white matter integrity.


Assuntos
Axônios/metabolismo , Sistema Nervoso Central/citologia , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/metabolismo , Animais , Axônios/ultraestrutura , Citocinas/genética , Citocinas/metabolismo , Antagonistas de Estrogênios/farmacologia , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Imuno-Histoquímica , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Proteínas da Mielina/ultraestrutura , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/ultraestrutura , Bainha de Mielina/ultraestrutura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/ultraestrutura , RNA Mensageiro/metabolismo , Tamoxifeno/farmacologia
12.
J Neurol Neurosurg Psychiatry ; 90(1): 58-67, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30018047

RESUMO

Much has been achieved in terms of understanding the complex clinical and genetic heterogeneity of Charcot-Marie-Tooth neuropathy (CMT). Since the identification of mutations in the first CMT associated gene, PMP22, the technological advancement in molecular genetics and gene technology has allowed scientists to generate diverse animal models expressing monogenetic mutations that closely resemble the CMT phenotype. Additionally, one can now culture patient-derived neurons in a dish using cellular reprogramming and differentiation techniques. Nevertheless, despite the fact that finding a disease-causing mutation offers a precise diagnosis, there is no cure for CMT at present. This review will shed light on the exciting advancement in CMT disease modelling, the breakthroughs, pitfalls, current challenges for scientists and key considerations to move the field forward towards successful therapies.


Assuntos
Doença de Charcot-Marie-Tooth/fisiopatologia , Modelos Animais de Doenças , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Doença de Charcot-Marie-Tooth/genética , Humanos , Camundongos , Modelos Biológicos , Mutação , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo
13.
Mar Biotechnol (NY) ; 21(1): 52-64, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30443836

RESUMO

The protein level of muscle-specific human NogoA is abnormally upregulated in amyotrophic lateral sclerosis (ALS) mice and patients. On the other hand, while the presence of miR-206 in muscle cells delays onset and death in ALS, the relationship between these two phenomena remains unclear. Mammalian NogoA protein, also known as Reticulon 4a (Rtn4a), plays an important role in inhibiting the outgrowth of motor neurons. Our group previously identified zebrafish rtn4al as the target gene of miR-206 and found that knockdown of miR-206 increases rtn4al mRNA and Rtn4al protein in zebrafish embryos. It can be concluded from these results that neurite outgrowth of motor neurons is inhibited by Rtn4a1, which is entirely consistent with overexpression of either human NogoA or zebrafish homolog Rtn4al. Since an animal model able to express NogoA/rtn4al at the mature stage is unavailable, we generated a zebrafish transgenic line, Tg(Zα:TetON-Rtn4al), which conditionally and specifically overexpresses Rtn4al in the muscle tissue. After doxycycline induction, adult zebrafish displayed denervation at neuromuscular junction during the first week, then muscle disintegration and split myofibers during the third week, and, finally, significant weight loss in the sixth week. These results suggest that this zebrafish transgenic line, representing the inducible overexpression of Rtn4a1 in muscle, may provide an alternative animal model with which to study ALS because it exhibits ALS-like phenotype.


Assuntos
Esclerose Amiotrófica Lateral/genética , Modelos Animais de Doenças , Neurônios Motores/metabolismo , Proteínas da Mielina/genética , Junção Neuromuscular/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Animais Geneticamente Modificados , Doxiciclina/farmacologia , Embrião não Mamífero , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Morfolinos/genética , Morfolinos/metabolismo , Neurônios Motores/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Proteínas da Mielina/agonistas , Proteínas da Mielina/antagonistas & inibidores , Proteínas da Mielina/metabolismo , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Proteínas Nogo/agonistas , Proteínas Nogo/genética , Proteínas Nogo/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Plasmídeos/química , Plasmídeos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/agonistas , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
14.
Neurotox Res ; 35(3): 495-504, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30406926

RESUMO

Currently, silver nanoparticles (AgNPs) are frequently used in a wide range of medical and consumer products. Substantial usage of AgNPs is considered to create substantive risks to both the environment and the human health. Since there is increasing evidence that the main mechanism of toxicity of AgNPs relates to oxidative stress, in the current study we investigate oxidative stress-related biochemical parameters in myelin isolated from adult rat brain subjected to a low dose of AgNPs. Animals were exposed for 2 weeks to 0.2 mg/kg b.w. of small (10 nm) AgNPs stabilized in citrate buffer or silver citrate established as a control to compare the effects of particulate and ionic forms of silver. We observe enhanced peroxidation of lipids and decreased concentrations of protein and non-protein -SH groups in myelin membranes. Simultaneously, expression of superoxide dismutase, a free radical scavenger, is increased whereas the process of protein glutathionylation, being a cellular protective mechanism against irreversible oxidation, is found to be inefficient. Results indicate that oxidative stress-induced alterations in myelin membranes may be the cause of ultrastructural disturbances in myelin sheaths.


Assuntos
Nanopartículas Metálicas/toxicidade , Bainha de Mielina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Prosencéfalo/efeitos dos fármacos , Prata/toxicidade , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ácido Cítrico/efeitos adversos , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Masculino , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Estresse Oxidativo/fisiologia , Prosencéfalo/metabolismo , RNA Mensageiro/metabolismo , Ratos Wistar , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/metabolismo , Fatores de Tempo
15.
Mol Neurobiol ; 56(6): 4231-4248, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30298339

RESUMO

Increasing findings suggest that demyelination may play an important role in the pathophysiology of brain injury, but the exact mechanisms underlying such damage are not well known. Mechanical tensile strain of brain tissue occurs during traumatic brain injury. Several studies have investigated the cellular and molecular events following a static tensile strain of physiological magnitude on individual cells such as oligodendrocytes. However, the pathobiological impact of high-magnitude mechanical strain on oligodendrocytes and myelinated fibers remains under investigated. In this study, we reported that an applied mechanical tensile strain of 30% on mouse organotypic culture of cerebellar slices induced axonal injury and elongation of paranodal junctions, two hallmarks of brain trauma. It was also able to activate MAPK-ERK1/2 signaling, a stretch-induced responsive pathway. The same tensile strain applied to mouse oligodendrocytes in primary culture induced a profound damage to cell morphology, partial cell loss, and a decrease of myelin protein expression. The lower tensile strain of 20% also caused cell loss and the remaining oligodendrocytes appeared retracted with decreased myelin protein expression. Finally, high-magnitude tensile strain applied to 158N oligodendroglial cells altered myelin protein expression, dampened MAPK-ERK1/2 and MAPK-p38 signaling, and enhanced the production of reactive oxygen species. The latter was accompanied by increased protein oxidation and an alteration of anti-oxidant defense that was strain magnitude-dependent. In conclusion, mechanical stretch of high magnitude provokes axonal injury with significant alterations in oligodendrocyte biology that could initiate demyelination.


Assuntos
Axônios/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Transdução de Sinais , Estresse Mecânico , Animais , Antioxidantes/metabolismo , Adesão Celular , Linhagem Celular , Forma Celular , Cerebelo/patologia , Regulação da Expressão Gênica , Glutationa/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Resistência à Tração
16.
Glia ; 67(4): 668-687, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30585359

RESUMO

The lack of endogenous repair following spinal cord injury (SCI) accounts for the frequent permanent deficits for which effective treatments are absent. Previously, we demonstrated that low sulfated modified heparin mimetics (LS-mHeps) attenuate astrocytosis, suggesting they may represent a novel therapeutic approach. mHeps are glycomolecules with structural similarities to resident heparan sulfates (HS), which modulate cell signaling by both sequestering ligands, and acting as cofactors in the formation of ligand-receptor complexes. To explore whether mHeps can affect the myelination and neurite outgrowth necessary for repair after SCI, we created lesioned or demyelinated neural cell co-cultures and exposed them with a panel of mHeps with varying degrees and positions of their sulfate moieties. LS-mHep7 enhanced neurite outgrowth and myelination, whereas highly sulfated mHeps (HS-mHeps) had attenuating effects. LS-mHeps had no effects on myelination or neurite extension in developing, uninjured myelinating cultures, suggesting they might exert their proregenerating effects by modulating or sequestering inhibitory factors secreted after injury. To investigate this, we examined conditioned media from cultures using chemokine arrays and conducted an unbiased proteomics approach by applying TMT-LC/MS to mHep7 affinity purified conditioned media from these cultures. Multiple protein factors reported to play a role in damage or repair mechanisms were identified, including amyloid betaA4. Amyloid beta peptide (1-42) was validated as an important candidate by treating myelination cultures and shown to inhibit myelination. Thus, we propose that LS-mHeps exert multiple beneficial effects on mechanisms supporting enhanced repair, and represent novel candidates as therapeutics for CNS damage.


Assuntos
Doenças do Sistema Nervoso Central/tratamento farmacológico , Doenças do Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes/tratamento farmacológico , Heparitina Sulfato/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Recém-Nascidos , Antimetabólitos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Desoxiuridina/farmacologia , Embrião de Mamíferos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas da Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Neuritos/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia
17.
Acta Neurobiol Exp (Wars) ; 78(3): 198-209, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30295677

RESUMO

Charcot­Marie­Tooth type 1A (CMT1A) is a dysmyelinating disease of the peripheral nervous system that results in a slow progressive weakening and wasting of the distal muscles of the upper and lower limbs. Despite extensive research and clinical trials there is still no treatment for CMT1A that results in complete neurological improvement. Recent studies investigating various pharmacological modulators of adenylyl cyclase activity, including ascorbic acid and ligands of G protein­coupled receptors (GPCRs), provide hope for future treatments of this type of hereditary motor and sensory neuropathy. A review of mechanisms of action of several compounds tested for CMT1A in pre­clinical and clinical studies ascorbic acid, onapristone, PXT3003 (baclofen, naltrexone, and sorbitol), and ADX71441, very clearly indicates an important role for adenylyl cyclase activity and GPCRs in the pathomechanism of the disease. Metabotropic γ­aminobutyric acid receptors (GABABR), subtype mu (µ) opioid receptors (MOR), and muscarinic acetylcholine receptors (mACh) appear to be particularly significant in both pathogenesis and treatment, and their activation may exert a similar and synergistic effect on the physiology of Schwann cells as well as neurons. These receptors participate in proliferation and differentiation of Schwann cells and influence excitatory transmission in neurons. We also hypothesize that onapristone might act through a non­classical mechanism via membrane progesterone receptor (mPR) and cAMP signaling. This review endeavors to outline a pathway leading inversely from therapy to an indispensable role for adenylyl cyclase activity and GPCRs in the modulation of dosage sensitive peripheral myelin protein (PMP22) gene expression.


Assuntos
Adenilil Ciclases/metabolismo , Doença de Charcot-Marie-Tooth/tratamento farmacológico , Proteínas da Mielina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Doença de Charcot-Marie-Tooth/metabolismo , Neuropatia Hereditária Motora e Sensorial/tratamento farmacológico , Humanos , Receptores Acoplados a Proteínas-G/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos
18.
PLoS One ; 13(9): e0202590, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30231069

RESUMO

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication and as possible therapeutic agents in inflammation-mediated demyelinating diseases, including multiple sclerosis (MS). In the present study, we investigated whether intravenously administered EVs derived from mesenchymal stem cells (MSCs) from human adipose tissue might mediate recovery in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a progressive model of MS. SJL/J mice were subjected to EV treatment once the disease was established. We found that intravenous EV administration improved motor deficits, reduced brain atrophy, increased cell proliferation in the subventricular zone and decreased inflammatory infiltrates in the spinal cord in mice infected with TMEV. EV treatment was also capable of modulating neuroinflammation, given glial fibrillary acidic protein and Iba-1 staining were reduced in the brain, whereas myelin protein expression was increased. Changes in the morphology of microglial cells in the spinal cord suggest that EVs also modulate the activation state of microglia. The clear reduction in plasma cytokine levels, mainly in the Th1 and Th17 phenotypes, in TMEV mice treated with EVs confirms the immunomodulatory ability of intravenous EVs. According to our results, EV administration attenuates motor deficits through immunomodulatory actions, diminishing brain atrophy and promoting remyelination. Further studies are necessary to establish EV delivery as a possible therapy for the neurodegenerative phase of MS.


Assuntos
Vesículas Extracelulares/transplante , Células-Tronco Mesenquimais/citologia , Esclerose Múltipla/terapia , Theilovirus/patogenicidade , Tecido Adiposo/citologia , Administração Intravenosa , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Camundongos , Proteínas dos Microfilamentos/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/virologia , Proteínas da Mielina/metabolismo , Resultado do Tratamento
19.
Int J Biol Macromol ; 119: 1195-1203, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30110602

RESUMO

Schwann cell (SC) is the primary structural and functional part of the peripheral nervous system, and it plays a key role in the repair and regeneration of peripheral nerve. In order to develop a suitable scaffold for SC nerve tissue engineering, three kinds of scaffolds, including pristine collagen, pure oxidized regenerated cellulose-Ca (ORCCa) and collagen/ORC-Ca composite scaffolds, have been fabricated for carrying SC in this study. SC is then seeded on the scaffolds to form SC-scaffold nerve tissue engineering composites and evaluate their biocompatibility. The chemical and physical structure of the scaffolds are investigated by FTIR, NMR and SEM. The wettability of the collagen/ORC-Ca composite scaffold is close to that of pristine collagen, and the tensile strength of the composite scaffold (0.58 MPa) is better than that of pristine collagen (0.36 MPa). Cytotoxicity, cell proliferation, cell adhesion and western blotting assays are conducted to evaluate the biocompatibility and properties of different scaffolds. The results show that the three scaffolds exhibit no toxicity, and the proliferation rate of SC on the collagen/ORC-Ca composite scaffold is significantly higher than that of the other scaffolds (p < 0.05). The number of the adhesion cells on the composite scaffold (244.67 ±â€¯13.02) is much more than that in the pure ORC-Ca group (p < 0.01). Furthermore, the expression of N-Cadheri and PMP22 proteins in the collagen/ORC-Ca composite scaffold is significantly superior to the other two scaffolds (both p < 0.01). Therefore, it could be concluded that the collagen/ORC-Ca composite is a promising candidate as a scaffold for carrying SC to form nerve tissue engineering composites in order to assist the peripheral nervous in the repair and regeneration.


Assuntos
Materiais Biocompatíveis/química , Cálcio/química , Celulose Oxidada/química , Colágeno/química , Portadores de Fármacos/química , Células de Schwann/citologia , Materiais Biocompatíveis/farmacologia , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Celulose Oxidada/farmacologia , Portadores de Fármacos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Teste de Materiais , Fenômenos Mecânicos , Proteínas da Mielina/metabolismo , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/fisiologia , Regeneração , Células de Schwann/química , Molhabilidade
20.
Glia ; 66(11): 2324-2339, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30151840

RESUMO

To study the role of L-type voltage-gated Ca++ channels in oligodendrocyte development, we used a mouse model of Timothy syndrome (TS) in which a gain-of-function mutation in the α1 subunit of the L-type Ca++ channel Cav1.2 gives rise to an autism spectrum disorder (ASD). Oligodendrocyte progenitor cells (OPCs) isolated from the cortex of TS mice showed greater L-type Ca++ influx and displayed characteristics suggestive of advanced maturation compared to control OPCs, including a more complex morphology and higher levels of myelin protein expression. Consistent with this, expression of Cav1.2 channels bearing the TS mutation in wild-type OPCs triggered process formation and promoted oligodendrocyte-neuron interaction via the activation of Ca++ /calmodulin-dependent protein kinase II. To ascertain whether accelerated OPC maturation correlated with functional enhancements, we examined myelination in the TS brain at different postnatal time points. The expression of myelin proteins was significantly higher in the corpus callosum, cortex and striatum of TS animals, and immunohistochemical analysis for oligodendrocyte stage-specific markers revealed an increase in the density of myelinating oligodendrocytes in several areas of the TS brain. Along the same line, electron microscopy studies in the corpus callosum of TS animals showed significant increases both in the percentage of myelinated axons and in the thickness of myelin sheaths. In summary, these data indicate that OPC development and oligodendrocyte myelination is enhanced in the brain of TS mice, and suggest that this mouse model of a syndromic ASD is a useful tool to explore the role of L-type Ca++ channels in myelination.


Assuntos
Transtorno Autístico/complicações , Transtorno Autístico/patologia , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Síndrome do QT Longo/complicações , Síndrome do QT Longo/patologia , Proteínas da Mielina/metabolismo , Oligodendroglia/fisiologia , Sindactilia/complicações , Sindactilia/patologia , Animais , Animais Recém-Nascidos , Transtorno Autístico/genética , Proteínas Relacionadas à Autofagia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Síndrome do QT Longo/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Células Precursoras de Oligodendrócitos/metabolismo , Células Precursoras de Oligodendrócitos/patologia , Oligodendroglia/patologia , Oligodendroglia/ultraestrutura , Potássio/farmacologia , Sindactilia/genética
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