RESUMO
BACKGROUND: The multi-drug resistance transporter ABCG2, a member of the ATP-binding cassette (ABC) transporter family, mediates the efflux of different immunotherapeutics used in multiple sclerosis (MS), e.g., teriflunomide (teri), cladribine, and mitoxantrone, across cell membranes and organelles. Hence, the modulation of ABCG2 activity could have potential therapeutic implications in MS. In this study, we aimed at investigating the functional impact of abcg2 modulation on teri-induced effects in vitro and in vivo. METHODS: T cells from C57BL/6 J wild-type (wt) and abcg2-knockout (KO) mice were treated with teri at different concentrations with/without specific abcg2-inhibitors (Ko143; Fumitremorgin C) and analyzed for intracellular teri concentration (HPLC; LS-MS/MS), T cell apoptosis (annexin V/PI), and proliferation (CSFE). Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6J by active immunization with MOG35-55/CFA. Teri (10 mg/kg body weight) was given orally once daily after individual disease onset. abcg2-mRNA expression (spinal cord, splenic T cells) was analyzed using qRT-PCR. RESULTS: In vitro, intracellular teri concentration in T cells was 2.5-fold higher in abcg2-KO mice than in wt mice. Teri-induced inhibition of T cell proliferation was two fold increased in abcg2-KO cells compared to wt cells. T cell apoptosis demonstrated analogous results with 3.1-fold increased apoptosis after pharmacological abcg2-inhibition in wt cells. abcg2-mRNA was differentially regulated during different phases of EAE within the central nervous system and peripheral organs. In vivo, at a dosage not efficacious in wt animals, teri treatment ameliorated clinical EAE in abcg2-KO mice which was accompanied by higher spinal cord tissue concentrations of teri. CONCLUSION: Functional relevance of abcg2 modulation on teri effects in vitro and in vivo warrants further investigation as a potential determinant of interindividual treatment response in MS, with potential implications for other immunotherapies.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/fisiologia , Crotonatos/uso terapêutico , Modelos Animais de Doenças , Imunoterapia/métodos , Esclerose Múltipla/imunologia , Linfócitos T/imunologia , Toluidinas/uso terapêutico , Animais , Crotonatos/farmacologia , Feminino , Humanos , Hidroxibutiratos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/tratamento farmacológico , Nitrilas , Ratos , Linfócitos T/efeitos dos fármacos , Toluidinas/farmacologiaRESUMO
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/farmacologiaRESUMO
Glatiramer acetate (GA) is a FDA-approved drug which is licensed for the treatment of relapsing-remitting multiple sclerosis and which may exert neuroprotective effects via brain-derived neurotrophic factor (BDNF). In this study, we investigate effects of GA on BDNF expression especially in astrocytes in vitro and in vivo in brains of R6/2 and YAC128 transgenic mouse models of Huntington's disease (HD) where a pathogenic role of astroglial cells has recently been shown. We show that GA increases the expression of functionally active BDNF in astrocyte culture and in astrocytes of GA treated HD mice. In the brains of these mice, GA decreases neurodegeneration and restores BDNF levels. The beneficial effect of GA in R6/2 mice also comprises reduced weight loss and prolonged life span and, for both models, also improved motor performance. Further studies with this safe and effective drug in HD are warranted.
Assuntos
Astrócitos/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Acetato de Glatiramer/uso terapêutico , Doença de Huntington/tratamento farmacológico , Doença de Huntington/patologia , Imunossupressores/uso terapêutico , Animais , Animais Recém-Nascidos , Astrócitos/química , Fator Neurotrófico Derivado do Encéfalo/genética , Caspase 3/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Acetato de Glatiramer/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Proteína Huntingtina/genética , Doença de Huntington/complicações , Doença de Huntington/genética , Imunossupressores/farmacologia , Mesencéfalo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Degeneração Neural/tratamento farmacológico , Degeneração Neural/etiologia , RNA Mensageiro/metabolismoRESUMO
The development of the spinal cord represents one of the most complex structure developments of the central nervous system (CNS) as it has to unfold along the longitudinal axis and within segmental cues. There it has to cope with on the one hand connection to the periphery (skeletal muscle, dermomyotome, smooth muscles) and connect it to the higher midbrain and cortical regions of the CNS. Major studies have been performed to analyze the specific subset of transcription factors of the different types of cells within the different segments of the spinal cord. But transcription factor expression is always a result of cellular positioning as the environment defines the intracellular changes during differentiation and in adulthood. The surrounding composed of mainly extracellular matrix does not only provide a "glue" to attach cells to each other but also provides signals with special domains docking to cell surface receptors and presents soluble molecules such as basic fibroblast growth factors (bFGFs) or Wnt-proteins. The availability of these molecules depends on the matrix composition and influences the transcription factor code of each cell. Recent research has also provided strong evidence that depletion of single matrix molecules like Tenascin C (TnC) can lead to developmental changes within the progenitor pools. Therefore beyond the transcription factor code that defines cellular properties we want to focus on the role of the extracellular matrix in the development of the spinal cord.
Assuntos
Matriz Extracelular/fisiologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Animais , Humanos , Medula Espinal/citologiaRESUMO
Diabetic neuropathy (DNP), afflicting sensory and motor nerve fibers, is a major complication in diabetes. The underlying cellular mechanisms of axon degeneration are poorly understood. IGFBP5, an inhibitory binding protein for insulin-like growth factor 1 (IGF1) is highly up-regulated in nerve biopsies of patients with DNP. We investigated the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP5 in motor axons and sensory nerve fibers. These mice develop motor axonopathy and sensory deficits similar to those seen in DNP. Motor axon degeneration was also observed in mice in which the IGF1 receptor (IGF1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF1 on IGF1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP5 in diabetic nerves reduces the availability of IGF1 for IGF1R on motor axons, thus leading to progressive neurodegeneration. Inhibition of IGFBP5 could thus offer novel treatment strategies for DNP.
Assuntos
Axônios/fisiologia , Proteínas de Transporte/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Neuropatias Diabéticas/fisiopatologia , Neurônios Motores/fisiologia , Degeneração Neural/fisiopatologia , Animais , Axônios/patologia , Crescimento Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Neuropatias Diabéticas/patologia , Humanos , Camundongos Transgênicos , Atividade Motora/fisiologia , Neurônios Motores/patologia , Degeneração Neural/patologia , Nervo Frênico/patologia , Nervo Frênico/fisiopatologia , Receptor IGF Tipo 1/metabolismo , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , Sensação/fisiologiaRESUMO
Bias-free, three-dimensional imaging of entire living cellular specimen is required for investigating shape and volume changes that occur during cellular growth or migration. Here we present fifty consecutive recordings of a living cultured neuron from a mouse dorsal root ganglion obtained by Scanning ion conductance microscopy (SICM). We observed a saltatory migration of the neuron with a mean velocity of approximately 20 µm/h. These results demonstrate the non-invasiveness of SICM, which makes it unique among the scanning probe microscopes. In contrast to SICM, most scanning probe techniques require a usually denaturating preparation of the cells, or they exert a non-negligible force on the cellular membrane, impeding passive observation. Moreover, the present series of recordings demonstrates the potential use of SICM for the detailed investigation of cellular migration and membrane surface dynamics even of such delicate samples as living neurons.
Assuntos
Movimento Celular , Microscopia/métodos , Neurônios/fisiologia , Análise de Célula Única/métodos , Animais , Células Cultivadas , Imageamento Tridimensional , CamundongosRESUMO
The development of the spinal cord needs a concerted interaction of transcription factors activating diverse genes and signals from outside acting on the specification of the different cells. Signals have to act on the segments of the embryo as well as on the cranial-caudal axis and the dorso-ventral axis. Additionally the axons of the motoneurons have to cross the central nervous system barrier to connect to the periphery. Intensive anatomical studies have been followed by molecular characterization of the different subsets of transcription factors that are expressed by cells of the developing spinal cord. Here, intensive studies for the most important appearing cells, the motoneurons, have resulted in a good knowledge on the expression patterns of these proteins. Nonetheless motoneurons are by far not the only important cells and the concert activity of all cells besides them is necessary for the correct function and integrity of motoneurons within the spinal cord. This article will briefly summarize the different aspects on spinal cord development and focuses on the differentiation as well as the functionalization of motoneurons.
RESUMO
The 30-amino acid peptide Y-P30, generated from the N-terminus of the human dermcidin precursor protein, has been found to promote neuronal survival, cell migration and neurite outgrowth by enhancing the interaction of pleiotrophin and syndecan-3. We now show that Y-P30 activates Src kinase and extracellular signal-regulated kinase (ERK). Y-P30 promotes axonal growth of mouse embryonic stem cell-derived neurons, embryonic mouse spinal cord motoneurons, perinatal rat retinal neurons, and rat cortical neurons. Y-P30-mediated axon growth was dependent on heparan sulfate chains. Y-P30 decreased the proportion of collapsing/degenerating growth cones of cortical axons in an Src and ERK-dependent manner. Y-P30 increased for 90 min in axonal growth cones the level of Tyr418-phosphorylated Src kinase and the amount of F-actin, and transiently the level of Tyr-phosphorylated ERK. Levels of total Src kinase, actin, GAP-43, cortactin and the glutamate receptor subunit GluN2B were not altered. When exposed to semaphorin-3a, Y-P30 protected a significant fraction of growth cones of cortical neurons from collapse. These results suggest that Y-P30 promotes axonal growth via Src- and ERK-dependent mechanisms which stabilize growth cones and confer resistance to collapsing factors.
Assuntos
Axônios/efeitos dos fármacos , Cones de Crescimento/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/citologia , Peptídeos/farmacologia , Actinas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Proteína GAP-43/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Imagem Molecular , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Ratos Long-Evans , Retina/citologia , Retina/efeitos dos fármacos , Semaforina-3A/metabolismoRESUMO
Ionotropic glutamate receptors (iGluRs) do not only mediate the majority of excitatory neurotransmission in the vertebrate CNS, but also modulate pre- and postnatal neurogenesis. Most of the studies on the developmental role of iGluRs are performed on neural progenitors and neural stem cells (NSCs). We took a step back in our study by examining the role of iGluRs in the earliest possible cell type, embryonic stem cells (ESCs), by looking at the mRNA expression of the major iGluR subfamilies in undifferentiated mouse ESCs. For that, we used two distinct murine ES cell lines, 46C ESCs and J1 ESCs. Regarding 46C ESCs, we found transcripts of kainate receptors (KARs) (GluK2 to GluK5), AMPA receptors (AMPARs) (GluA1, GluA3, and GluA4), and NMDA receptors (NMDARs) (GluN1, and GluN2A to GluN2D). Analysis of 46C-derived cells of later developmental stages, namely neuroepithelial precursor cells (NEPs) and NSCs, revealed that the mRNA expression of KARs is significantly upregulated in NEPs and, subsequently, downregulated in NSCs. However, we could not detect any protein expression of any of the KAR subunits present on the mRNA level either in ESCs, NEPs, or NSCs. Regarding AMPARs and NMDARs, GluN2A is weakly expressed at the protein level only in NSCs. Matching our findings for iGluRs, all three cell types were found to weakly express pre- and postsynaptic markers of glutamatergic synapses only at the mRNA level. Finally, we performed patch-clamp recordings of 46C ESCs and could not detect any current upon iGluR agonist application. Similar to 46C ESCs, J1 ESCs express KARs (GluK2 to GluK5), AMPARs (GluA3), and NMDARs (GluN1, and GluN2A to GluN2D) at the mRNA level, but these transcripts are not translated into receptor proteins either. Thus, we conclude that ESCs do not contain functional iGluRs, although they do express an almost complete set of iGluR subunit mRNAs.
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/biossínteseRESUMO
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/embriologiaRESUMO
A series of Ni-based electrocatalysts, [Ni(7P(Ph)2N(C6H4X))2](BF4)2, featuring seven-membered cyclic diphosphine ligands incorporating a single amine base, 1-para-X-phenyl-3,6-triphenyl-1-aza-3,6-diphosphacycloheptane (7P(Ph)2N(C6H4X), where X = OMe, Me, Br, Cl, or CF3), have been synthesized and characterized. X-ray diffraction studies have established that the [Ni(7P(Ph)2N(C6H4X))2](2+) complexes have a square planar geometry, with bonds to four phosphorus atoms of the two bidentate diphosphine ligands. Each of the complexes is an efficient electrocatalyst for hydrogen production at the potential of the Ni(II/I) couple, with turnover frequencies ranging from 2400 to 27,000 s(-1) with [(DMF)H](+) in acetonitrile. Addition of water (up to 1.0 M) accelerates the catalysis, giving turnover frequencies ranging from 4100 to 96,000 s(-1). Computational studies carried out on the [Ni(7P(Ph)2N(C6H4X))2](2+) family indicate the catalytic rates reach a maximum when the electron-donating character of X results in the pKa of the Ni(I) protonated pendant amine matching that of the acid used for proton delivery. Additionally, the fast catalytic rates for hydrogen production by the [Ni(7P(Ph)2N(C6H4X))2](2+) family relative to the analogous [Ni(P(Ph)2N(C6H4X)2)2](2+) family are attributed to preferred formation of endo protonated isomers with respect to the metal center in the former, which is essential to attain suitable proximity to the reduced metal center to generate H2. The results of this work highlight the importance of precise pKa matching with the acid for proton delivery to obtain optimal rates of catalysis.
RESUMO
Cells of the central nervous system are notoriously difficult to transfect. This is not only true for neurons and glial cells but also for dividing neural stem and progenitor cells (NSCs). About ten years ago a major advance was provided by introduction of the nucleofection technology that allowed for transfection of approximately half of the exposed NSCs. However, limitations were encountered with the need for large numbers of NSCs for a single transfection and compromised survival rates with typically only one-third of the cells surviving the pulse conditions. Here, we report the establishment of a pulse protocol that targets NSCs with high efficiency and twofold higher NSC survival rates using the 4D Nucleofector device. We demonstrate that the established protocol not only provides a clear and significant improvement over existing protocols with transfection rates above 80% and two-thirds of the NSCs surviving for at least 48h, but also their unaltered differentiation along neuronal and glial lineages. This improved protocol for the transfection of sensitive mouse central nervous system derived cells will provide an important step forward for studies of gene function by overexpression or knock-down of genes in cultured NSCs.
Assuntos
Células-Tronco Adultas/metabolismo , Eletroporação/métodos , Células-Tronco Embrionárias/metabolismo , Animais , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Proteínas de Fluorescência Verde/genética , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , NestinaRESUMO
Research of the past 25 years has shown that astrocytes do more than participating and building up the blood-brain barrier and detoxify the active synapse by reuptake of neurotransmitters and ions. Indeed, astrocytes express neurotransmitter receptors and, as a consequence, respond to stimuli. Within the tripartite synapse, the astrocytes owe more and more importance. Besides the functional aspects the differentiation of astrocytes has gained a more intensive focus. Deeper knowledge of the differentiation processes during development of the central nervous system might help explaining and even help treating neurological diseases like Alzheimer's disease, Amyotrophic lateral sclerosis, Parkinsons disease, and psychiatric disorders in which astrocytes have been shown to play a role. Specific differentiation of neural stem cells toward the astroglial lineage is performed as a multi-step process. Astrocytes and oligodendrocytes develop from a multipotent stem cell that prior to this has produced primarily neuronal precursor cells. This switch toward the more astroglial differentiation is regulated by a change in receptor composition on the cell surface and responsiveness to Fibroblast growth factor and Epidermal growth factor (EGF). The glial precursor cell is driven into the astroglial direction by signaling molecules like Ciliary neurotrophic factor, Bone Morphogenetic Proteins, and EGF. However, the early astrocytes influence their environment not only by releasing and responding to diverse soluble factors but also express a wide range of extracellular matrix (ECM) molecules, in particular proteoglycans of the lectican family and tenascins. Lately these ECM molecules have been shown to participate in glial development. In this regard, especially the matrix protein Tenascin C (Tnc) proved to be an important regulator of astrocyte precursor cell proliferation and migration during spinal cord development. Nevertheless, ECM molecules expressed by reactive astrocytes are also known to act mostly in an inhibitory fashion under pathophysiological conditions. Thus, we further summarize resent data concerning the role of chondroitin sulfate proteoglycans and Tnc under pathological conditions.
RESUMO
BACKGROUND: Sulfated glycosaminoglycan chains are known for their regulatory functions during neural development and regeneration. However, it is still unknown whether the sulfate residues alone influence, for example, neural precursor cell behavior or whether they act in concert with the sugar backbone. Here, we provide evidence that the unique 473HD-epitope, a representative chondroitin sulfate, is expressed by spinal cord neural precursor cells in vivo and in vitro, suggesting a potential function of sulfated glycosaminoglycans for spinal cord development. RESULTS: Thus, we applied the widely used sulfation inhibitor sodium chlorate to analyze the importance of normal sulfation levels for spinal cord neural precursor cell biology in vitro. Addition of sodium chlorate to spinal cord neural precursor cell cultures affected cell cycle progression accompanied by changed extracellular signal-regulated kinase 1 or 2 activation levels. This resulted in a higher percentage of neurons already under proliferative conditions. In contrast, the relative number of glial cells was largely unaffected. Strikingly, both morphological and electrophysiological characterization of neural precursor cell-derived neurons demonstrated an attenuated neuronal maturation in the presence of sodium chlorate, including a disturbed neuronal polarization. CONCLUSIONS: In summary, our data suggest that sulfation is an important regulator of both neural precursor cell proliferation and maturation of the neural precursor cell progeny in the developing mouse spinal cord.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Sulfatos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sulfatos de Condroitina/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Camundongos , Camundongos Endogâmicos , Cultura Primária de Células , Medula Espinal/fisiologiaRESUMO
We report bifunctional reactivity of the ß-diketiminato Ni(III)-imide [Me(3)NN]NiâNAd (1), which undergoes H-atom abstraction (HAA) reactions with benzylic substrates R-H (indane, ethylbenzene, toluene). Nickel-imide 1 competes with the nickel-amide HAA product [Me(3)NN]Ni-NHAd (2) for the resulting hydrocarbyl radical R(â¢) to give the nickel-amide [Me(3)NN]Ni-N(CHMePh)Ad (3) (R-H = ethylbenzene) or aminoalkyl tautomer [Me(3)NN]Ni(η(2)-CH(Ph)NHAd) (4) (R-H = toluene). A significant amount of functionalized amine R-NHAd is observed in the reaction of 1 with indane along with the dinickel imide {[Me(3)NN]Ni}(2)(µ-NAd) (5). Kinetic and DFT analyses point to rate-limiting HAA from R-H by 1 to give R(â¢), which may add to either imide 1 or amide 2, each featuring significant N-based radical character. Thus, these studies illustrate a fundamental competition possible in C-H amination systems that proceed via a HAA/radical rebound mechanism.
RESUMO
To clarify the physiological role of Na(+)-D-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1(-/-) mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1(-/-) mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1(-/-) mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1(-/-) mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed â¼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.
Assuntos
Glucose/farmacocinética , Incretinas/metabolismo , Absorção Intestinal/genética , Transportador 1 de Glucose-Sódio/fisiologia , Animais , Feminino , Glucose/farmacologia , Glicosúria/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Intestino Delgado/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismoRESUMO
Laquinimod is a promising, orally available compound that has been successfully evaluated in placebo-controlled phase II/III studies of relapsing-remitting multiple sclerosis (MS). Studies are ongoing to further define laquinimod's modulatory mechanisms. Analyses in the animal model of experimental autoimmune encephalomyelitis (EAE) demonstrate that laquinimod reduces infiltration of leukocytes into the central nervous system, induces a Th1 to Th2/3 shift, and suppresses Th17 responses. To evaluate the potential neuroprotective capacity of laquinimod via modulation of brain-derived neurotrophic factor (BDNF), we analyzed the expression of BDNF in blood samples from 203 MS patients treated with laquinimod. Furthermore, we investigated the effect of laquinimod in EAE using a conditional BDNF knockout strain lacking BDNF expression in myeloid cells and T cells (LLF mice). Treatment with laquinimod resulted in a significant and persistent increase in BDNF serum levels of MS patients when compared to baseline and placebo-treated patients. LLF mice treated with laquinimod display a more severe EAE disease course in comparison to wild-type mice. Furthermore, laquinimod-treated wild-type monocytes secreted an anti-inflammatory cytokine pattern in comparison to untreated wild-type monocytes and treated LLF monocytes. Adoptive transfer of laquinimod stimulated monocytes into mice with EAE ameliorated the disease course. Consistent with immunomodulatory properties, laquinimod skewed monocytes toward a regulatory phenotype and also acted via modulation of BDNF, which may contribute to neuroprotection in MS patients.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Encefalomielite Autoimune Experimental/prevenção & controle , Esclerose Múltipla/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Quinolonas/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Esclerose Múltipla/metabolismoRESUMO
The generation of astrocytes during the development of the mammalian spinal cord is poorly understood. Here, we demonstrate for the first time that the extracellular matrix glycoprotein tenascin C regulates the expression of key patterning genes during late embryonic spinal cord development, leading to a timely maturation of gliogenic neural precursor cells. We first show that tenascin C is expressed by gliogenic neural precursor cells during late embryonic development. The loss of tenascin C leads to a sustained generation and delayed migration of Fgfr3-expressing immature astrocytes in vivo. Consistent with an increased generation of astroglial cells, we documented an increased number of GFAP-positive astrocytes at later stages. Mechanistically, we could demonstrate an upregulation and domain shift of the patterning genes Nkx6.1 and Nkx2.2 in vivo. In addition, sulfatase 1, a known downstream target of Nkx2.2 in the ventral spinal cord, was also upregulated. Sulfatase 1 regulates growth factor signalling by cleaving sulphate residues from heparan sulphate proteoglycans. Consistent with this function, we observed changes in both FGF2 and EGF responsiveness of spinal cord neural precursor cells. Taken together, our data implicate Tnc in the regulation of proliferation and lineage progression of astroglial progenitors in specific domains of the developing spinal cord.
Assuntos
Astrócitos/citologia , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Medula Espinal/crescimento & desenvolvimento , Tenascina/metabolismo , Animais , Movimento Celular , Células Cultivadas , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Sulfotransferases/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteínas de Peixe-ZebraRESUMO
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.