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1.
Proc Natl Acad Sci U S A ; 113(43): 12304-12309, 2016 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-27791020

RESUMO

Despite the recognized role of tumor necrosis factor (TNF) in inflammation and neuronal degeneration, anti-TNF therapeutics failed to treat neurodegenerative diseases. Animal disease models had revealed the antithetic effects of the two TNF receptors (TNFR) in the central nervous system, whereby TNFR1 has been associated with inflammatory degeneration and TNFR2 with neuroprotection. We here show the therapeutic potential of selective inhibition of TNFR1 and activation of TNFR2 by ATROSAB, a TNFR1-selective antagonistic antibody, and EHD2-scTNFR2, an agonistic TNFR2-selective TNF, respectively, in a mouse model of NMDA-induced acute neurodegeneration. Coadministration of either ATROSAB or EHD2-scTNFR2 into the magnocellular nucleus basalis significantly protected cholinergic neurons and their cortical projections against cell death, and reverted the neurodegeneration-associated memory impairment in a passive avoidance paradigm. Simultaneous blocking of TNFR1 and TNFR2 signaling, however, abrogated the therapeutic effect. Our results uncover an essential role of TNFR2 in neuroprotection. Accordingly, the therapeutic activity of ATROSAB is mediated by shifting the balance of the antithetic activity of endogenous TNF toward TNFR2, which appears essential for neuroprotection. Our data also explain earlier results showing that complete blocking of TNF activity by anti-TNF drugs was detrimental rather than protective and argue for the use of next-generation TNFR-selective TNF therapeutics as an effective approach in treating neurodegenerative diseases.


Assuntos
Inflamação/tratamento farmacológico , Degeneração Neural/tratamento farmacológico , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/genética , Animais , Anticorpos/farmacologia , Núcleo Basal de Meynert/metabolismo , Núcleo Basal de Meynert/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Morte Celular/efeitos dos fármacos , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/patologia , Células HEK293 , Humanos , Inflamação/genética , Inflamação/patologia , Camundongos , N-Metilaspartato/genética , Degeneração Neural/induzido quimicamente , Degeneração Neural/genética , Degeneração Neural/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo II do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores
2.
Glia ; 62(2): 272-83, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24310780

RESUMO

Tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2 have pleiotropic effects in neurodegenerative disorders. For example, while TNFR1 mediates neurodegenerative effects in multiple sclerosis, TNFR2 is protective and contributes to remyelination. The exact mode of TNFR2 action, however, is poorly understood. Here, we show that TNFR2-mediated activation of the PI3K-PKB/Akt pathway in primary astrocytes increased the expression of neuroprotective genes, including that encoding the neurotrophic cytokine leukemia inhibitory factor (LIF). To investigate whether intercellular signaling between TNFR2-stimulated astrocytes and oligodendrocytes plays a role in oligodendrocyte maturation, we established an astrocyte-oligodendrocyte coculture model, composed of primary astrocytes from huTNFR2-transgenic (tgE1335) mice and oligodendrocyte progenitor cells (OPCs) from wild-type mice, capable of differentiating into mature myelinating oligodendrocytes. In this model, selective stimulation of human TNFR2 on astrocytes, promoted differentiation of cocultured OPCs to myelin basic protein-positive mature oligodendrocytes. Addition of LIF neutralizing antibodies inhibited oligodendrocyte differentiation, indicating a crucial role of TNFR2-induced astrocyte derived LIF for oligodendrocyte maturation.


Assuntos
Astrócitos/metabolismo , Fator Inibidor de Leucemia/metabolismo , Oligodendroglia/citologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oligodendroglia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Fator de Necrose Tumoral alfa/metabolismo
3.
Brain ; 136(Pt 1): 116-31, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23365094

RESUMO

Remyelination following central nervous system demyelination is essential to prevent axon degeneration. However, remyelination ultimately fails in demyelinating diseases such as multiple sclerosis. This failure of remyelination is likely mediated by many factors, including changes in the extracellular signalling environment. Here, we examined the expression of the extracellular matrix molecule fibronectin on demyelinating injury and how this affects remyelination by oligodendrocytes progenitors. In toxin-induced lesions undergoing efficient remyelination, fibronectin expression was transiently increased within demyelinated areas and declined as remyelination proceeded. Fibronectin levels increased both by leakage from the blood circulation and by production from central nervous system resident cells. In chronically demyelinated multiple sclerosis lesions, fibronectin expression persisted in the form of aggregates, which may render fibronectin resistant to degradation. Aggregation of fibronectin was similarly observed at the relapse phase of chronic experimental autoimmune encephalitis, but not on toxin-induced demyelination, suggesting that fibronectin aggregation is mediated by inflammation-induced demyelination. Indeed, the inflammatory mediator lipopolysaccharide induced fibronectin aggregation by astrocytes. Most intriguingly, injection of astrocyte-derived fibronectin aggregates in toxin-induced demyelinated lesions inhibited oligodendrocyte differentiation and remyelination, and fibronectin aggregates are barely expressed in remyelinated multiple sclerosis lesions. Therefore, these findings suggest that fibronectin aggregates within multiple sclerosis lesions contribute to remyelination failure. Hence, the inhibitory signals induced by fibronectin aggregates or factors that affect fibronectin aggregation could be potential therapeutic targets for promoting remyelination.


Assuntos
Doenças Desmielinizantes/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Fibronectinas/metabolismo , Esclerose Múltipla/metabolismo , Bainha de Mielina/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Axônios/metabolismo , Axônios/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Células Cultivadas , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/fisiopatologia , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Humanos , Esclerose Múltipla/patologia , Esclerose Múltipla/fisiopatologia , Bainha de Mielina/patologia , Regeneração Nervosa/fisiologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Medula Espinal/patologia , Medula Espinal/fisiopatologia
4.
Biochem Biophys Res Commun ; 440(2): 336-41, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-24076392

RESUMO

The neuroprotective role of TNF receptor 2 (TNFR2) has been shown in various studies. However, a direct role of TNFR2 in oligodendrocyte function has not yet been demonstrated. Using primary oligodendrocytes of transgenic mice expressing human TNFR2, we show here that TNFR2 is primarily expressed on oligodendrocyte progenitor cells. Interestingly, preconditioning with a TNFR2 agonist protects these cells from oxidative stress, presumably by increasing the gene expression of distinct anti-apoptotic and detoxifying proteins, thereby providing a potential mechanism for the neuroprotective role of TNFR2 in oligodendrocyte progenitor cells.


Assuntos
Oligodendroglia/efeitos dos fármacos , Receptores Tipo II do Fator de Necrose Tumoral/fisiologia , Células-Tronco/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Humanos , Camundongos , Camundongos Transgênicos , Oligodendroglia/fisiologia , Estresse Oxidativo , Receptores Tipo II do Fator de Necrose Tumoral/agonistas , Receptores Tipo II do Fator de Necrose Tumoral/biossíntese
5.
J Neurosci Res ; 88(15): 3361-75, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20857509

RESUMO

Statins have attracted interest as a treatment option for multiple sclerosis (MS) because of their pleiotropic antiinflammatory and immunomodulatory effects. However, contradictory results have been described when they are applied to oligodendrocytes (OLGs), the cell type predominantly affected in MS. In this study we focus on the in vitro effect of statins on process outgrowth in OLN-93 cells, a well-characterized OLG-derived cell line, and primary cultures of neonatal rat OLGs. Application of the lipophilic simvastatin, as low as 0.1-1 µM, disturbs process formation of both cell types, leading to less ramified cells. We show that both protein isoprenylation and cholesterol synthesis are required for the normal differentiation of OLGs. It is further demonstrated that the expression of 2',3'-cyclic-nucleotide-3' phosphodiesterase (CNP) and tubulin is lowered, concomitant with a reduction of membrane-bound CNP as well as tubulin. Therefore, we propose that lack of isoprenylation of CNP could help to explain the altered morphological and biochemical differentiation state of treated OLGs. Moreover, expression of specific myelin markers, such as myelin basic protein, myelin-associated glycoprotein, and myelin oligodendrocyte glycoprotein, was compromised after treatment. We conclude that simvastatin treatment has detrimental effects on OLG process outgrowth, the prior step in (re)myelination, thereby mortgaging long-term healing of MS lesions.


Assuntos
Fatores Imunológicos/farmacologia , Oligodendroglia/efeitos dos fármacos , Sinvastatina/farmacologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Ratos
6.
Cell Death Dis ; 11(3): 212, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32235829

RESUMO

Non-alcoholic fatty liver disease (NAFLD) shows an increasing prevalence and is associated with the development of liver fibrosis and cirrhosis as the major risk factors of liver-related mortality in this disease. The therapeutic possibilities are limited and restricted to life style intervention, since specific drugs for NAFLD are unavailable so far. TNFα has been implicated as a major pathogenic driver of NAFLD. TNFα-mediated liver injury occurs mainly via TNF-receptor-1 (TNFR1) signaling, whereas TNFR2 mediates protective pathways. In this study, we analyzed the therapeutic effects of a novel antibody, which selectively inhibits TNFR1 while retaining protective TNFR2 signaling in a high-fat diet (HFD) mouse model of NAFLD. Mice were fed with HFD for 32 weeks and treated with anti-TNFR1-antibody or control-antibody for the last 8 weeks. We then investigated the mechanisms of TNFR1 inhibition on liver steatosis, inflammatory liver injury, insulin resistance and fibrosis. Compared to control-antibody treatment, TNFR1 inhibition significantly reduced liver steatosis and triglyceride content, which was accompanied by reduced expression and activation of the transcription factor SREBP1 and downstream target genes of lipogenesis. Furthermore, inhibition of TNFR1 resulted in reduced activation of the MAP kinase MKK7 and its downstream target JNK, which was associated with significant improvement of insulin resistance. Apoptotic liver injury, NAFLD activity and alanine aminotransferase (ALT) levels, as well as liver fibrosis significantly decreased by anti-TNFR1 compared to control-antibody treatment. Thus, our results suggest selective TNFR1 inhibition as a promising approach for NAFLD treatment.


Assuntos
Fígado Gorduroso/terapia , Cirrose Hepática/terapia , Hepatopatia Gordurosa não Alcoólica/terapia , Fator 1 Associado a Receptor de TNF/antagonistas & inibidores , Animais , Fígado Gorduroso/genética , Humanos , Cirrose Hepática/genética , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética
7.
Glia ; 57(4): 402-13, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18814266

RESUMO

Statins, well-known inhibitors of cholesterol synthesis and protein isoprenylation, have been proposed as therapeutic drugs for multiple sclerosis (MS). As lovastatin and simvastatin, which are currently tested for their use in MS, can cross the blood-brain barrier, they may affect cellular processes in the central nervous system. This is especially relevant with respect to remyelination as a proposed additional treatment for MS, because cholesterol is a major component of myelin. Here, we show that primary oligodendrocytes, treated with lovastatin, form extensive membrane sheets, which contain galactosphingolipids. However, these membrane sheets are devoid of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP). Reduced MBP protein expression was confirmed by SDS-PAGE and Western blotting, and in situ hybridization experiments revealed that lovastatin blocks MBP mRNA transport into oligodendrocyte processes. In contrast, PLP expression was only mildly affected by lovastatin. However, lovastatin treatment resulted in intracellular accumulation of PLP and prevented its translocation to the cell surface. Interestingly, another inhibitor of cholesterol synthesis (ro48-8071), which does not interfere with isoprenylation, had a similar effect on the localization of PLP, but it did not affect MBP expression and localization. These results suggest that lovastatin affects PLP transport predominantly by the inhibition of cholesterol synthesis, whereas reduced MBP expression is caused by impaired isoprenylation. Based on these results we recommend to carefully monitor the effect of statins on myelination prior to their use in demyelinating diseases.


Assuntos
Membrana Celular/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lovastatina/farmacologia , Bainha de Mielina/metabolismo , Oligodendroglia/citologia , Animais , Animais Recém-Nascidos , Benzofenonas/farmacologia , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Oligodendroglia/efeitos dos fármacos , Fosfatos/metabolismo , Fosfatos de Poli-Isoprenil/farmacologia , Prosencéfalo/citologia , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar
8.
Antimicrob Agents Chemother ; 53(3): 1027-38, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19075071

RESUMO

LL-37 is a cationic host defense peptide that is highly expressed during acute inflammation and that kills bacteria by poorly defined mechanisms, resulting in permeabilization of microbial membranes. High concentrations of LL-37 have also been reported to have cytotoxic effects against eukaryotic cells, but the peptide is clearly capable of differentiating between membranes with different compositions (eukaryotic versus bacterial membranes). Eukaryotic cells such as leukocytes change their membrane composition during apoptotic cell death, when they are turned into nonfunctional but structurally intact entities. We tested whether LL-37 exerted specific activity on apoptotic cells and found that the peptide selectively permeabilized the membranes of apoptotic human leukocytes, leaving viable cells unaffected. This activity was seemingly analogous to the direct microbicidal effect of LL-37, in that it was rapid, independent of known surface receptors and/or active cell signaling, and inhibitable by serum components such as high-density lipoprotein. A similar selective permeabilization of apoptotic cells was recorded for both NK cells and neutrophils. In the latter cell type, LL-37 permeabilized both the plasma and granule membranes, resulting in the release of both lactate dehydrogenase and myeloperoxidase. Apoptosis is a way for inflammatory cells to die silently and minimize collateral tissue damage by retaining tissue-damaging and proinflammatory substances within intact membranes. Permeabilization of apoptotic leukocytes by LL-37, accompanied by the leakage of cytoplasmic as well as intragranular molecules, may thus shift the balance between pro- and anti-inflammatory signals and in this way be of importance for the termination of acute inflammation.


Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/fisiologia , Apoptose/imunologia , Leucócitos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Humanos , Peróxido de Hidrogênio/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , L-Lactato Desidrogenase/análise , L-Lactato Desidrogenase/metabolismo , Leucócitos/metabolismo , Testes de Sensibilidade Microbiana , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Oxidantes/farmacologia , Permeabilidade , Peroxidase/análise , Peroxidase/metabolismo , Catelicidinas
9.
J Mol Neurosci ; 35(1): 35-53, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18172773

RESUMO

In vertebrates, myelination is required for the saltatory signal conductance along the axon. At the onset of myelination, the myelinating cells, i.e., oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system, are heavily engaged in the biogenesis of membranes that are wrapped around the axon to form the myelin sheath. Although the membrane of the myelin sheath is continuous with the plasma membrane surrounding the cell body, the composition of both membrane domains is clearly distinct implying that myelinating cells are polarized cells. The coordinated manner of myelin sheath formation requires the existence of sorting and trafficking pathways to establish and maintain this highly polarized phenotype. Although in vitro data show that the formation of myelin-like membranes is an intrinsic property of oligodendrocytes, exogenous factors modulate myelination and are required for the subcompartmentation and compaction of the myelin sheath in vivo. In this paper, we discuss the sorting and trafficking of myelin proteins and lipids in oligodendrocytes in relation to polarity development and maintenance, including the role of exogenous factors, and give examples how the perturbation of trafficking pathways may contribute to the development of demyelinating diseases of the central nervous system.


Assuntos
Polaridade Celular , Bainha de Mielina , Oligodendroglia , Transporte Proteico/fisiologia , Animais , Axônios/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/fisiopatologia , Humanos , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas da Mielina/metabolismo , Bainha de Mielina/química , Bainha de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Vesículas Transportadoras/metabolismo
10.
Sci Rep ; 8(1): 13628, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206422

RESUMO

Tumour necrosis factor (TNF) signalling is mediated via two receptors, TNF-receptor 1 (TNFR1) and TNF-receptor 2 (TNFR2), which work antithetically to balance CNS immune responses involved in autoimmune diseases such as multiple sclerosis. To determine the therapeutic potential of selectively inhibiting TNFR1 in mice with experimental autoimmune encephalomyelitis, we used chimeric human/mouse TNFR1 knock-in mice allowing the evaluation of antagonistic anti-human TNFR1 antibody efficacy. Treatment of mice after onset of disease with ATROSAB resulted in a robust amelioration of disease severity, correlating with reduced central nervous system immune cell infiltration. Long-term efficacy of treatment was achieved by treatment with the parental mouse anti-human TNFR1 antibody, H398, and extended by subsequent re-treatment of mice following relapse. Our data support the hypothesis that anti-TNFR1 therapy restricts immune cell infiltration across the blood-brain barrier through the down-regulation of TNF-induced adhesion molecules, rather than altering immune cell composition or activity. Collectively, we demonstrate the potential for anti-human TNFR1 therapies to effectively modulate immune responses in autoimmune disease.


Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo
11.
PLoS One ; 11(10): e0163314, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27755542

RESUMO

Chronic inflammatory conditions during peritoneal dialysis (PD)-treatment lead to the impairment of peritoneal tissue integrity. The resulting structural and functional reorganization of the peritoneal membrane diminishes ultrafiltration rate and thereby enhances mortality by limiting dialysis effectiveness over time. Tumour necrosis factor (TNF) and its receptors TNFR1 and TNFR2 are key players during inflammatory processes. To date, the role of TNFR1 in peritoneal tissue damage during PD-treatment is completely undefined. In this study, we used an acute PD-mouse model to investigate the role of TNFR1 on structural and morphological changes of the peritoneal membrane. TNFR1-mediated TNF signalling in transgenic mice expressing human TNFR1 was specifically blocked by applying a monoclonal antibody (H398) highly selective for human TNFR1 prior to PD-treatment. Cancer antigen-125 (CA125) plasma concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Western blot analyses were applied to determine TNFR2 protein concentrations. Histological staining of peritoneal tissue sections was performed to assess granulocytes within the peritoneal membrane as well as the content of hyaluronic acid and collagen. We show for the first time that the number of granulocytes within the peritoneal membrane is significantly reduced in mice pre-treated with H398. Moreover, we demonstrate that blocking of TNFR1 not only influences CA125 values but also hyaluronic acid and collagen contents of the peritoneal tissue in these mice. These results strongly suggest that TNFR1 inhibition attenuates peritoneal damage caused by peritoneal dialysis fluid (PDF) and therefore may represent a new therapeutic approach in the treatment of PD-related side effects.


Assuntos
Inflamação/prevenção & controle , Peritônio/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Antígeno Ca-125/sangue , Colágeno/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Granulócitos/citologia , Granulócitos/metabolismo , Ácido Hialurônico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Diálise Peritoneal , Peritônio/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo
12.
Int Rev Cytol ; 226: 127-64, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12921237

RESUMO

Plasma membranes of epithelial cells consist of two domains, an apical and a basolateral domain, the surfaces of which differ in composition. The separation of these domains by a tight junction and the fact that specific transport pathways exist for intracellular communication between these domains and distinct intracellular compartments relevant to cell polarity development, have triggered extensive research on issues that focus on how the polarity is generated and maintained. Apart from proper assembly of tight junctions, their potential functioning as landmark for the transport machinery, cell-cell adhesion is obviously instrumental in barrier formation. In recent years, distinct endocytic compartments, defined as subapical compartment or common endosome, were shown to play a prominent role in regulating membrane trafficking to and from polarized membrane domains. Sorting devices remain to be determined but likely include distinct rab proteins, and evidence is accumulating to indicate that signaling events may direct intracellular membrane transport, intimately involved in the biogenesis and maintenance of polarized membrane domains and hence the development of cell polarity.


Assuntos
Membrana Celular/fisiologia , Polaridade Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Animais , Humanos , Junções Íntimas/fisiologia
13.
Oxid Med Cell Longev ; 2015: 610813, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25834699

RESUMO

Neuroinflammation and mitochondrial dysfunction are common features of chronic neurodegenerative diseases of the central nervous system. Both conditions can lead to increased oxidative stress by excessive release of harmful reactive oxygen and nitrogen species (ROS and RNS), which further promote neuronal damage and subsequent inflammation resulting in a feed-forward loop of neurodegeneration. The cytokine tumor necrosis factor (TNF), a master regulator of the immune system, plays an important role in the propagation of inflammation due to the activation and recruitment of immune cells via its receptor TNF receptor 1 (TNFR1). Moreover, TNFR1 can directly induce oxidative stress by the activation of ROS and RNS producing enzymes. Both TNF-induced oxidative stress and inflammation interact and cooperate to promote neurodegeneration. However, TNF plays a dual role in neurodegenerative disease, since stimulation via its second receptor, TNFR2, is neuroprotective and promotes tissue regeneration. Here we review the interrelation of oxidative stress and inflammation in the two major chronic neurodegenerative diseases, Alzheimer's and Parkinson's disease, and discuss the dual role of TNF in promoting neurodegeneration and tissue regeneration via its two receptors.


Assuntos
Doenças Neurodegenerativas/patologia , Estresse Oxidativo , Fator de Necrose Tumoral alfa/metabolismo , Humanos , Inflamação , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo
14.
Chem Phys Lipids ; 116(1-2): 3-18, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12093532

RESUMO

Odd as it may seem, experimental challenges in lipid research are often hampered by the simplicity of the lipid structure. Since, as in protein research, mutants or overexpression of lipids are not realistic, a considerable amount of lipid research relies on the use of tagged lipid analogues. However, given the size of an average lipid molecule, special care is needed for the selection of probes, since if the size and intramolecular localization of the probe is not specifically taken into account, it may dramatically affect the properties of the lipids. The latter is particularly important in cell biological studies of lipid trafficking and sorting, where the probed lipid should resemble its natural counterpart as closely as possible. On the other hand, for biophysical applications, these considerations may be less critical. Here we provide a brief overview of the application of several lipid probes in cell biological and biophysical research, and critically analyze their validity in the various fields.


Assuntos
Corantes Fluorescentes , Lipídeos/química , Animais , Transporte Biológico , Técnicas Citológicas/métodos , Endocitose , Transferência de Energia , Corantes Fluorescentes/química , Humanos , Fusão de Membrana
15.
PLoS One ; 9(2): e90117, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24587232

RESUMO

Tumour necrosis factor (TNF) is a proinflammatory cytokine that is known to regulate inflammation in a number of autoimmune diseases, including multiple sclerosis (MS). Although targeting of TNF in models of MS has been successful, the pathological role of TNF in MS remains unclear due to clinical trials where the non-selective inhibition of TNF resulted in exacerbated disease. Subsequent experiments have indicated that this may have resulted from the divergent effects of the two TNF receptors, TNFR1 and TNFR2. Here we show that the selective targeting of TNFR1 with an antagonistic antibody ameliorates symptoms of the most common animal model of MS, experimental autoimmune encephalomyelitis (EAE), when given following both a prophylactic and therapeutic treatment regime. Our results demonstrate that antagonistic TNFR1-specific antibodies may represent a therapeutic approach for the treatment of MS in the future.


Assuntos
Anticorpos/farmacologia , Encefalomielite Autoimune Experimental/terapia , Imunoterapia , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/genética
16.
ALTEX ; 29(3): 261-74, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22847254

RESUMO

Astrocytes are activated in most chronic neurodegenerative diseases associated with inflammatory events such as Parkinson's disease or Alzheimer's disease, but also in stroke. Due to an aging population worldwide, research efforts in these areas are likely to expand in the future. This will entail an increased demand for appropriate experimental models. We introduce here the new immortalized mouse astrocyte cell line IMA 2.1 as an alternative to currently used primary astrocyte cultures. IMA 2.1 were directly compared with primary mouse astrocytes with respect to their response to proinflammatory stimuli, expression of typical astrocyte markers, and to the cell line's capacity to metabolize the parkinsonian toxin MPTP to its toxic metabolite MPP+. Under inflammatory conditions, mimicked with the addition of a cytokine mix, IMA 2.1 responded similarly to primary astrocytes with mRNA upregulation, expression of iNOS and COX-2, and the release of various inflammatory mediators. Analysis of astrocytic markers indicated that IMA 2.1 represent a relatively early, GFAP-negative stage of astrocyte development. Moreover, conversion of MPTP by monoamine oxidase-B proceeded in IMA at least as quickly as in primary cells. For all endpoints investigated, the cell line IMA 2.1, derived from a single clone, delivered reproducible results over a period of several years and allowed upscaling of experiments due to its easy handling compared with primary cells.


Assuntos
Astrócitos/citologia , Astrócitos/fisiologia , Animais , Linhagem Celular , Técnicas de Cocultura , Citometria de Fluxo , Regulação da Expressão Gênica/fisiologia , Humanos , Inflamação , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos
17.
Cell Signal ; 23(1): 161-70, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20807567

RESUMO

Endocytosis is an important mechanism to regulate tumor necrosis factor (TNF) signaling. In contrast to TNF receptor 1 (TNFR1; CD120a), the relevance of receptor internalization for signaling as well as the fate and route of internalized TNF receptor 2 (TNFR2; CD120b) is poorly understood. To analyze the dynamics of TNFR2 signaling and turnover at the plasma membrane we established a human TNFR2 expressing mouse embryonic fibroblast cell line in a TNFR1(-/-)/TNFR2(-/-) background. TNF stimulation resulted in a decrease of constitutive TNFR2 ectodomain shedding. We hypothesized that reduced ectodomain release is a result of TNF/TNFR2 complex internalization. Indeed, we could demonstrate that TNFR2 was internalized together with its ligand and cytoplasmic binding partners. Upon endocytosis the TNFR2 signaling complex colocalized with late endosome/lysosome marker Rab7 and entered the lysosomal degradation pathway. Furthermore, we identified a di-leucin motif in the cytoplasmic part of TNFR2 suggesting clathrin-dependent internalization of TNFR2. Internalization defective TNFR2 mutants are capable to signal, i.e. activate NFκB, demonstrating that the di-leucin motif dependent internalization is dispensable for this response. We therefore propose that receptor internalization primarily serves as a negative feed-back to limit TNF responses via TNFR2.


Assuntos
Leucina/química , NF-kappa B/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Clatrina/metabolismo , Endocitose , Fibroblastos/metabolismo , Humanos , Ligantes , Camundongos , Receptores Tipo II do Fator de Necrose Tumoral/química , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
18.
PLoS One ; 6(11): e27621, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22110694

RESUMO

Tumor necrosis factor (TNF) plays a dual role in neurodegenerative diseases. Whereas TNF receptor (TNFR) 1 is predominantly associated with neurodegeneration, TNFR2 is involved in tissue regeneration and neuroprotection. Accordingly, the availability of TNFR2-selective agonists could allow the development of new therapeutic treatments of neurodegenerative diseases. We constructed a soluble, human TNFR2 agonist (TNC-scTNF(R2)) by genetic fusion of the trimerization domain of tenascin C to a TNFR2-selective single-chain TNF molecule, which is comprised of three TNF domains connected by short peptide linkers. TNC-scTNF(R2) specifically activated TNFR2 and possessed membrane-TNF mimetic activity, resulting in TNFR2 signaling complex formation and activation of downstream signaling pathways. Protection from neurodegeneration was assessed using the human dopaminergic neuronal cell line LUHMES. First we show that TNC-scTNF(R2) interfered with cell death pathways subsequent to H(2)O(2) exposure. Protection from cell death was dependent on TNFR2 activation of the PI3K-PKB/Akt pathway, evident from restoration of H(2)O(2) sensitivity in the presence of PI3K inhibitor LY294002. Second, in an in vitro model of Parkinson disease, TNC-scTNF(R2) rescues neurons after induction of cell death by 6-OHDA. Since TNFR2 is not only promoting anti-apoptotic responses but also plays an important role in tissue regeneration, activation of TNFR2 signaling by TNC-scTNF(R2) appears a promising strategy to ameliorate neurodegenerative processes.


Assuntos
Morte Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Receptores Tipo II do Fator de Necrose Tumoral/agonistas , Proteínas Recombinantes de Fusão/farmacologia , Tenascina/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes de Fusão/química , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Especificidade por Substrato , Superóxidos/farmacologia , Tenascina/química
19.
Glia ; 55(8): 885-95, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17405145

RESUMO

Neurofascin155 (NF155) is required for the establishment of the paranodal axo-glial junction, the predominant interaction site between myelin and axon. It has been shown that the distribution of NF155 is altered in demyelinating diseases such as multiple sclerosis (MS). However, little is known about the biochemical mechanisms underlying these changes. We therefore compared NF155 in postmortem tissue of active and chronic inactive MS lesions with white matter from healthy controls. Although NF155 showed a very similar expression in all control white matter samples, a strong individual variation was observed in MS-lesions with NF155-levels reduced in most samples. At the same time an NF155-fragment was increased in MS-lesions, suggesting that NF155 is subject to protein degradation in lesion sites. Interestingly, the association of NF155 to membrane microdomains (rafts) was reduced in all lesions, irrespective of the amount of NF155, indicating that membrane association of NF155 was generally affected. Therefore, myelin fractionation experiments were performed to analyze the fate of paranodal proteins during demyelination. Although NF155 was enriched in heavy myelin from both control white matter and active MS-lesions, association of Caspr1/paranodin with heavy myelin was abolished in MS-lesions, demonstrating that paranodal junctions are disrupted. In conclusion, the data support the hypothesis that efficient raft-association of NF155 is essential for the assembly of the paranodal junction and demonstrate that reduced association of NF155 to lipid rafts is accompanied by the disassembly of the paranodal junction and thus contributes to the demyelination process in MS.


Assuntos
Moléculas de Adesão Celular/metabolismo , Microdomínios da Membrana/metabolismo , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Fatores de Crescimento Neural/metabolismo , Nós Neurofibrosos/metabolismo , Nós Neurofibrosos/patologia , Animais , Animais Recém-Nascidos , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular Neuronais/metabolismo , Fracionamento Celular , Células Cultivadas , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Feminino , Humanos , Peso Molecular , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/patologia , Fatores de Crescimento Neural/química , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Ratos , Ratos Wistar , Esfingolipídeos/metabolismo
20.
Exp Cell Res ; 312(4): 500-11, 2006 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-16360652

RESUMO

Formation of the paranodal axo-glial junction requires the oligodendrocyte-specific 155-kDa isoform of neurofascin (NF155). Here, we report the presence of two peptides in cultured oligodendrocytes, which are recognized by distinct NF155-specific antibodies and correspond to a membrane anchor of 30 kDa and a 125 kDa peptide, which is shed from the cells, indicating that it consists of the NF155 ectodomain. Transfection of OLN-93 cells with NF155 verified that both peptides originate from NF155 cleavage, and we present evidence that metalloproteases mediate NF155 processing. Interestingly, metalloprotease activity is required for NF155 transport into oligodendrocyte processes supporting the functional significance of NF155 cleavage. To further characterize NF155 cleavage and function, we transfected MDCK cells with NF155. Although ectodomain shedding was observed in polarized and non-polarized MDCK cells, surface localization of NF155 was restricted to the lateral membrane of polarized cells consistent with a role in cell-cell adhesion. Aggregation assays performed with OLN-93 cells confirmed that NF155 accelerates cell-cell adhesion in a metalloprotease-dependent manner. The physiological relevance of NF155 processing is corroborated by the presence of NF155 cleavage products in heavy myelin, suggesting a role of NF155 ectodomain shedding for the generation and/or stabilization of the nodal/paranodal architecture.


Assuntos
Moléculas de Adesão Celular/fisiologia , Metaloproteases/metabolismo , Fatores de Crescimento Neural/fisiologia , Oligodendroglia/metabolismo , Animais , Animais Recém-Nascidos , Química Encefálica , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/fisiologia , Crescimento Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Dipeptídeos/farmacologia , Células Epiteliais/química , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Metaloproteases/antagonistas & inibidores , Bainha de Mielina/química , Fatores de Crescimento Neural/metabolismo , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional , Ratos , Ratos Wistar , Transfecção
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