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1.
PLoS One ; 12(3): e0171552, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28273090

RESUMO

Fingolimod is an oral sphingosine-1-phosphate-receptor modulator which reduces the recirculation of immune cells and may also directly target glial cells. Here we investigate effects of fingolimod on expression of astroglial glutamate transporters under pro-inflammatory conditions. In astrocyte cell culture, the addition of pro-inflammatory cytokines led to a significant downregulation of glutamate transporters glutamate transporter-1 (slc1a2/SLC1A2) and glutamate aspartate transporter (slc1a3/SLC1A3) expression on the mRNA or protein level. In this setting, the direct application of fingolimod-1 phosphate (F1P) on astrocytes did not change expression levels of slc1a2 and slc1a3 mRNA. The analysis of both transporters on the protein level by Western Blot and immunocytochemistry did also not reveal any effect of F1P. On a functional level, the addition of conditioned supernatants from F1P treated astrocytes to neuronal cell culture did not result in increased neurite growth. In experimental autoimmune encephalomyelitis as a model of multiple sclerosis, fingolimod treatment reduced T cell and macrophages/microglia mediated inflammation and also diminished astrocyte activation. At the same time, fingolimod restored the reduced expression of slc1a2 and slc1a3 in the inflamed spinal cord on the mRNA level and of SLC1A2 and SLC1A3 on the protein level, presumably via indirect, anti-inflammatory mechanisms. These findings provide further evidence for a predominantly peripheral effect of the compound in neuroinflammation.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Cloridrato de Fingolimode/farmacologia , Imunossupressores/farmacologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Células Cultivadas , Transportador 1 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/genética , Feminino , Cloridrato de Fingolimode/uso terapêutico , Imunossupressores/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/prevenção & controle , Interleucina-1beta/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células PC12 , RNA Mensageiro/metabolismo , Ratos , Fator de Necrose Tumoral alfa/farmacologia
2.
Curr Protoc Cell Biol ; 66: 1.9.1-1.9.10, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25727328

RESUMO

Isolated spinal motoneurons are a powerful tool for studying basic mechanisms of neurite growth and survival. Since motoneurons are a minor population of developing spinal cord cells, they need to be purified and enriched to separate them from non-neuronal cells. Therefore, the particular feature of embryonic motoneurons to express the low affinity neurotrophin receptor p75(NTR) is used to separate the motoneurons from other contaminating cells. Two ways are described to isolate embryonic motoneurons: the basic protocol taking advantage of the ability of p75(NTR) to bind lectin, and an alternative method using an antibody against p75(NTR) for a panning procedure. These protocols comprise suggestions for the cultivation of the isolated motoneurons for experiments regarding neural outgrowth and survival as well as instruction for the preparation of proteins of the cells.


Assuntos
Separação Celular/métodos , Neurônios Motores/citologia , Medula Espinal/citologia , Animais , Anticorpos/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Lectinas/metabolismo , Camundongos , Receptores de Fator de Crescimento Neural/metabolismo
3.
Neurochem Res ; 38(6): 1285-94, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23624942

RESUMO

Complex glycan structures and their respective carrier molecules are often expressed in a cell type specific manner. Thus, glycans can be used for the enrichment of specific cell types such as neural precursor cells (NPCs). We have recently shown that the monoclonal antibodies 487(LeX) and 5750(LeX) differentially detect the LewisX (LeX) glycan on NPCs in the developing mouse forebrain. Here, we analysed the staining pattern of both antibodies during late embryonic mouse spinal cord development. At E13.5 both antibodies strongly label the central canal region. Along these lines they detect the LeX glycan primarily on Nestin-positive NPCs at that age. Moreover, we show that spinal cord NPCs cultured as free floating neurospheres display a high immunoreactivity to both antibodies. In that context, we also demonstrate that the 487(LeX) antibody can be used to deplete a subpopulation of neurosphere forming NPCs from a mixed E13.5 spinal cord cell suspension. Towards the end of embryogenesis the overall immunoreactivity to both antibodies increases and the staining appears very diffuse. However, the 5750(LeX) antibody still labels the central canal region. The increase in immunoreactivity correlates with an expression increase of the extracellular matrix molecules Tenascin C and Receptor Protein Tyrosine Phosphatase ß/ζ, two potential LeX carrier proteins. In line with this, immunoprecipitation analyses confirmed Tenascin C as a LeX carrier protein in the embryonic mouse spinal cord. However, the immunoreactivity to both antibodies appears only to be marginally affected in the absence of Tenascin C, arguing against Tenascin C being the major LeX carrier. In conclusion our study gives some novel insights into the complex expression of LeX glycans and potential carrier proteins during the development of the mouse spinal cord.


Assuntos
Antígenos CD15/biossíntese , Células-Tronco Neurais/metabolismo , Polissacarídeos/biossíntese , Medula Espinal/embriologia , Animais , Células Cultivadas , Camundongos , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/biossíntese , Medula Espinal/metabolismo , Tenascina/biossíntese
4.
Mol Cell Neurosci ; 56: 18-28, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23500004

RESUMO

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family and a ligand for the tropomyosin-receptor kinase B (TrkB), mediates neuronal survival, differentiation, and synaptic plasticity. However, BDNF is not used to treat neurodegenerative diseases because of its poor pharmacokinetic profile, side effects, and absence of survival properties in clinical trials. Consequently, alternative approaches such as TrkB receptor agonist application are gaining importance. 7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, has been described as a robust TrkB receptor agonist in hippocampal neurons. Nevertheless, the influence of 7,8-DHF on motoneurons, one of the main targets of BDNF in vivo, is so far unknown. Therefore, we investigated the impact of 7,8-DHF treatment on primary cultured mouse motoneurons. Indeed, we found an activation of the TrkB receptor. Moreover, 7,8-DHF application promotes survival and neurite growth of cultured motoneurons and these effects appear dose-dependent. To investigate the PI3K/AKT and MAPK pathway activation in 7,8-DHF treated motoneurons, we developed a high-density culture system of primary mouse motoneurons. Analysis of both pathways demonstrated a PI3K/AKT but not MAPK pathway activation in cultured motoneurons. This is in contrast to previously published reports about BDNF-mediated activation of TrkB. The lack of MAPK pathway activation is also in contrast to what has been found for hippocampal neurons that indeed show MAPK activation after 7,8-DHF treatment. The ability of 7,8-DHF to imitate BDNF function in motoneurons by using Trk receptor signaling would provide a new approach for the treatment of motoneuron diseases, but needs a more detailed analysis of the activation profile of 7,8-DHF.


Assuntos
Flavonas/farmacologia , Sistema de Sinalização das MAP Quinases , Neurônios Motores/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Processos de Crescimento Celular , Sobrevivência Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Hipocampo/citologia , Hipocampo/embriologia , Camundongos , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neuritos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor trkB/agonistas , Receptor trkB/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia
5.
J Vis Exp ; (55)2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21946816

RESUMO

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons(1). Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract(2,3). More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)(4). Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75(NTR). The p75(NTR) can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75(NTR) is exclusively expressed by the spinal motoneurons(5). This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells(6). Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75(NTR) has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75(NTR) as well(7). The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75(NTR) antibody. The lectin is an extremely inexpensive alternative to the p75(NTR) antibody and the purification grades using lectin are comparable to that of the p75(NTR) antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.


Assuntos
Técnicas Citológicas/métodos , Neurônios Motores/citologia , Aglutininas do Germe de Trigo/química , Animais , Embrião de Mamíferos/citologia , Feminino , Camundongos , Gravidez , Medula Espinal/citologia , Medula Espinal/enzimologia
6.
J Neurosci Res ; 89(2): 127-41, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21162121

RESUMO

Mechanisms controlling neuronal survival and regeneration play an important role during development, after birth, and under lesion conditions. Isolated embryonic mouse motoneurons have been a useful tool for studying such basic mechanisms. These cultured motoneurons depend on extracellular matrix (ECM) molecules, which are potent mediators of survival and axonal growth and guidance in the CNS and in vitro, exhibiting either attractive or repellent guidance cues. Additionally, ECM proteoglycans and glycoproteins are components of the glial scar acting as a growth barrier for regenerating axons. Compared with CNS axon outgrowth, less is known about the cues that guide motoneurons toward their peripheral targets. Because we are interested in the effects of glial-derived chondroitin sulfate proteoglycans (CSPGs), we have worked out a model system for investigating the influences of glial-derived matrix molecules on motoneuron outgrowth and survival. We used cultured embryonic mouse motoneurons to investigate axon growth effects of matrix molecules produced by the glial-derived cell lines A7, Neu7, and Oli-neu primary astrocytes as well as the immortalized Schwann cell line IMS32. The results indicate that molecules of the ECM, especially chondroitin sulfates, play an important role as axon growth-promoting cues. We could demonstrate a modifying effect of the matrix components on motoneuron survival and caspase3-induced apoptosis.


Assuntos
Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Neurônios Motores/citologia , Neuritos , Neuroglia/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Sobrevivência Celular , Embrião de Mamíferos , Imunofluorescência , Camundongos , Neurônios Motores/metabolismo , Neuritos/metabolismo
7.
Nat Protoc ; 5(1): 31-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20057379

RESUMO

Cultured spinal motoneurons are a valuable tool for studying the basic mechanisms of axon and dendrite growth and also for analyses of pathomechanisms underlying diseases like amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). As motoneurons in the developing spinal cord of mice constitute only a minor population of neurons, these cells need to be enriched in order to study them in the absence of contaminating neuronal and non-neuronal cells. Here, we describe a protocol for the isolation and in vitro cultivation of embryonic primary motoneurons from individual mouse embryos. Tissue dissection, cell isolation and a p75(NTR)-antibody-based panning technique, which highly enriches motoneurons within <8 h are described. This protocol is aimed to provide an alternative to the established FACS-based protocols describing p75(NTR)-based enrichments of neurons. This protocol will help in facilitating the research on molecular mechanisms underlying motoneuron development, survival and disease mechanisms.


Assuntos
Embrião de Mamíferos/citologia , Neurônios Motores/citologia , Medula Espinal/citologia , Medula Espinal/embriologia , Animais , Técnicas de Cultura de Células , Sobrevivência Celular , Meios de Cultura , Região Lombossacral , Camundongos
8.
J Comp Neurol ; 504(6): 659-79, 2007 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-17722031

RESUMO

The chondroitin sulfate proteoglycan DSD-1-PG/phosphacan represents one of four splice variants of receptor-protein-tyrosine-phosphatase-beta/zeta (RPTPbeta/zeta). This receptor is expressed by glial cells and occurs in two isoforms, RPTPbeta(long) and RPTPbeta(short). The secreted forms phosphacan and phosphacan short isoform (PSI) bind to extracellular matrix and adhesion molecules and might mediate astroglial effects on neuronal differentiation. Phosphacan and RPTPbeta(long) both carry the DSD-1 epitope, a glycosaminoglycan modification that is involved in stimulating neurite outgrowth of embryonic rat mesencephalic and hippocampal neurons in a polycationic environment. Additionally, phosphacan inhibits neurite outgrowth of embryonic DRG neurons in the presence of laminin. In the light of these functional properties we examined the expression patterns of the DSD-1 epitope and phosphacan isoforms in the developing mouse visual system. During retinal development the DSD-1 epitope appears around embryonic day (E)13, peaks around postnatal day (P)6, and is downregulated from P9 to adolescence. By comparison, the phosphacan core protein is first detectable at E12, reaches maximal levels around P14, and persists, although at lower levels, to adulthood. The DSD-1 epitope is restricted to the nerve fiber and the inner plexiform layers. In contrast, the phosphacan core protein immunoreactivity extends from the nerve fiber layer to the outer plexiform layer. The level of expression of the phosphacan/RPTPbeta gene was investigated by reverse-transcriptase polymerase chain reaction. These experiments suggest that there is a shift in the expression patterns of the different phosphacan/RPTPbeta isoforms during late embryonic and postnatal development. In situ hybridization experiments support the conclusion that at least one of the phosphacan/RPTPbeta isoforms in the retina is expressed by neurons.


Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Vias Visuais , Animais , Animais Recém-Nascidos , Embrião de Mamíferos , Feminino , Masculino , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Vias Visuais/embriologia , Vias Visuais/crescimento & desenvolvimento , Vias Visuais/metabolismo
9.
Cell Tissue Res ; 321(3): 323-33, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16028071

RESUMO

Phosphacan is a chondroitin sulfate proteoglycan representing the secreted extracellular part of a transmembrane receptor protein tyrosine phosphatase (RPTP-beta). These isoforms have been implicated in cell-extracellular matrix signaling events associated with myelination, axon growth, and cell migration in the developing central nervous system and may play critical roles in the context of brain pathologies. Recently, we have reported the identification of a new isoform of phosphacan, the phosphacan short isoform (PSI), the expression of which peaks in the second postnatal week. PSI interacts with the neuronal receptors L1 and F3/contactin and can promote neurite growth of cortical neurons. In this study, we have assessed, by in situ hybridization, the expression profile of PSI in the rat brain at postnatal day 7. PSI is largely expressed in the gray matter of the developing cerebral cortex in which it colocalizes with phosphacan, whereas the expression of RPTPbeta receptor forms is restricted to the ventricular area in which PSI has not been observed. Neurons from all layers of the cortex express PSI. In the cerebellum, on the other hand, no expression of PSI has been detected, although the other phosphacan/RPTP-beta isoforms show strong PSI expression here. Overall, our study suggests that PSI is expressed during the postnatal period in differentiated neurons of the cortex but is absent from structures in which proliferation and migration occur. The significance of these observations is discussed in the context of previous models of phosphacan/RPTP-beta functions.


Assuntos
Córtex Cerebral/citologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Fracionamento Celular , Cerebelo/citologia , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Proteoglicanas de Sulfatos de Condroitina/genética , Hibridização In Situ , Neurônios/citologia , Isoformas de Proteínas/genética , Ratos , Ratos Wistar , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores
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