RESUMO
BACKGROUND: Colony stimulating factor 1 receptor (CSF1R) signaling is crucial for the maintenance and function of various myeloid subsets. CSF1R antagonism was previously shown to mitigate clinical severity in experimental autoimmune encephalomyelitis (EAE). The associated mechanisms are still not well delineated. METHODS: To assess the effect of CSF1R signaling, we employed the CSF1R antagonist PLX5622 formulated in chow (PLX5622 diet, PD) and its control chow (control diet, CD). We examined the effect of PD in steady state and EAE by analyzing cells isolated from peripheral immune organs and from the CNS via flow cytometry. We determined CNS infiltration sites and assessed the extent of demyelination using immunohistochemistry of cerebella and spinal cords. Transcripts of genes associated with neuroinflammation were also analyzed in these tissues. RESULTS: In addition to microglial depletion, PD treatment reduced dendritic cells and macrophages in peripheral immune organs, both during steady state and during EAE. Furthermore, CSF1R antagonism modulated numbers and relative frequencies of T effector cells both in the periphery and in the CNS during the early stages of the disease. Classical neurological symptoms were milder in PD compared to CD mice. Interestingly, a subset of PD mice developed atypical EAE symptoms. Unlike previous studies, we observed that the CNS of PD mice was infiltrated by increased numbers of peripheral immune cells compared to that of CD mice. Immunohistochemical analysis showed that CNS infiltrates in PD mice were mainly localized in the cerebellum while in CD mice infiltrates were primarily localized in the spinal cords during the onset of neurological deficits. Accordingly, during the same timepoint, cerebella of PD but not of CD mice had extensive demyelinating lesions, while spinal cords of CD but not of PD mice were heavily demyelinated. CONCLUSIONS: Our findings suggest that CSF1R activity modulates the cellular composition of immune cells both in the periphery and within the CNS, and affects lesion localization during the early EAE stages.
Assuntos
Encefalomielite Autoimune Experimental , Camundongos , Animais , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Compostos Orgânicos/farmacologia , Medula Espinal/patologia , Microglia , Receptores de Fator Estimulador de Colônias , Receptores Proteína Tirosina Quinases , Camundongos Endogâmicos C57BLRESUMO
After traumatic spinal cord injury (SCI), a scar may form with a fibrotic core (fibrotic scar) and surrounding reactive astrocytes (glial scar) at the lesion site. The scar tissue is considered a major obstacle preventing regeneration both as a physical barrier and as a source for secretion of inhibitors of axonal regeneration. Understanding the mechanism of scar formation and how to control it may lead to effective SCI therapies. Using a compression-SCI model on adult transgenic mice, we demonstrate that the canonical Wnt/ß-catenin signaling reporter TOPgal (TCF/Lef1-lacZ) positive cells appeared at the lesion site by 5 days, peaked on 7 days, and diminished by 14 days post injury. Using various representative cell lineage markers, we demonstrate that, these transiently TOPgal positive cells are a group of Fibronectin(+);GFAP(-) fibroblast-like cells in the core scar region. Some of them are proliferative. These results indicate that Wnt/ß-catenin signaling may play a key role in fibrotic scar formation after traumatic spinal cord injury.
Assuntos
Cicatriz/metabolismo , Cicatriz/patologia , Compressão da Medula Espinal/metabolismo , Compressão da Medula Espinal/patologia , Medula Espinal/patologia , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Cicatriz/etiologia , Fibrose , Proteína Glial Fibrilar Ácida , Camundongos , Camundongos Transgênicos , Medula Espinal/metabolismo , Compressão da Medula Espinal/complicaçõesRESUMO
Non-canonical Wnt/planar cell polarity (PCP) signaling plays a primary role in the convergent extension that drives neural tube closure and body axis elongation. PCP signaling gene mutations cause severe neural tube defects (NTDs). However, the role of canonical Wnt/ß-catenin signaling in neural tube closure and NTDs remains poorly understood. This study shows that conditional gene targeting of ß-catenin in the dorsal neural folds of mouse embryos represses the expression of the homeobox-containing genes Pax3 and Cdx2 at the dorsal posterior neuropore (PNP), and subsequently diminishes the expression of the Wnt/ß-catenin signaling target genes T, Tbx6 and Fgf8 at the tail bud, leading to spina bifida aperta, caudal axis bending and tail truncation. We demonstrate that Pax3 and Cdx2 are novel downstream targets of Wnt/ß-catenin signaling. Transgenic activation of Pax3 cDNA can rescue the closure defect in the ß-catenin mutants, suggesting that Pax3 is a key downstream effector of ß-catenin signaling in the PNP closure process. Cdx2 is known to be crucial in posterior axis elongation and in neural tube closure. We found that Cdx2 expression is also repressed in the dorsal PNPs of Pax3-null embryos. However, the ectopically activated Pax3 in the ß-catenin mutants cannot restore Cdx2 mRNA in the dorsal PNP, suggesting that the presence of both ß-catenin and Pax3 is required for regional Cdx2 expression. Thus, ß-catenin signaling is required for caudal neural tube closure and elongation, acting through the transcriptional regulation of key target genes in the PNP.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Homeodomínio/metabolismo , Tubo Neural/embriologia , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Animais , Padronização Corporal/genética , Fator de Transcrição CDX2 , Adesão Celular/genética , Polaridade Celular/fisiologia , Fator 8 de Crescimento de Fibroblasto/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Fator de Transcrição MSX1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/metabolismo , Defeitos do Tubo Neural/genética , Neurulação , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/genética , Disrafismo Espinal/genética , Proteínas com Domínio T , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genéticaRESUMO
BACKGROUND: Periventricular leukomalacia (PVL) is the leading cause of neurological disabilities including motor and cognitive deficits in premature infants. Periventricular leukomalacia is characterized by damage to the white matter in the immature brain, but the mechanisms by which damage to immature white matter results in widespread deficits of cognitive and motor function are unclear. The thalamocortical system is crucial for human consciousness and cognitive functions, and impaired development of the cortico-thalamic projections in the neonatal period is implicated to contribute importantly to abnormalities of cognitive function in children with PVL. RESULTS: In this study, using a mouse model of PVL, we sought to test the hypothesis that PVL-like injury affects the different components of the thalamocortical circuitry that can be defined by vesicular glutamate transporters 1 and 2 (vGluT1 and vGluT2), both of which are required for glutamatergic synaptic transmission in the central nervous system. We combined immunocytochemistry and immuno-electron microscopy to investigate changes in cortico-thalamic synapses which were specifically identified by vGluT1 immunolabeling. We found that a drastic reduction in the density of vGluT1 labeled profiles in the somatosensory thalamus, with a reduction of 72-74 % in ventroposterior (VP) nucleus and a reduction of 42-82 % in thalamic reticular nucleus (RTN) in the ipsilateral side of PVL mice. We further examined these terminals at the electron microscopic level and revealed onefold-twofold decrease in the sizes of vGluT1 labeled corticothalamic terminals in VP and RTN. The present study provides anatomical and ultrastructural evidence to elucidate the cellular mechanisms underlying alteration of thalamic circuitry in a mouse model of PVL, and reveals that PVL-like injury has a direct impact on the corticothalamic projection system. CONCLUSIONS: Our findings provide the first set of evidence showing that the thalamocortical circuitry is affected and vulnerable in PVL mice, supporting a working model in which vGluT1 defined corticothalamic synapses are altered in PVL mice, and vGluT2 defined thalamocortical synapses are associated with such changes, leading to the compromised thalamocortical circuitry in the PVL mice. Our study demonstrates that the thalamocortical circuitry is highly vulnerable to hypoxia-ischemia in the PVL model, thus identifying a novel target site in PVL pathology.
Assuntos
Córtex Cerebral/ultraestrutura , Modelos Animais de Doenças , Hipóxia-Isquemia Encefálica/complicações , Leucomalácia Periventricular/patologia , Sinapses/ultraestrutura , Tálamo/ultraestrutura , Animais , Córtex Cerebral/metabolismo , Leucomalácia Periventricular/etiologia , Leucomalácia Periventricular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Vias Neurais/metabolismo , Vias Neurais/ultraestrutura , Sinapses/metabolismo , Tálamo/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
Pluripotent stem cells (PSCs) have been differentiated into oligodendroglial progenitor cells (OPCs), providing promising cell replacement therapies for many central nervous system disorders. Studies from rodents have shown that brain OPCs express a variety of ion channels, and that a subset of brain OPCs express voltage-gated sodium channel (NaV ), mediating the spiking properties of OPCs. However, it is unclear whether PSC-derived OPCs exhibit electrophysiological properties similar to brain OPCs and the role of NaV in the functional maturation of OPCs is unknown. Here, using a mouse embryonic stem cell (mESC) green fluorescent protein (GFP)-Olig2 knockin reporter line, we demonstrated that unlike brain OPCs, all the GFP(+) /Olig2(+) mESC-derived OPCs (mESC-OPCs) did not express functional NaV and failed to generate spikes (hence termed "nonspiking mESC-OPCs"), while expressing the delayed rectifier and inactivating potassium currents. By ectopically expressing NaV 1.2 α subunit via viral transduction, we successfully generated mESC-OPCs with spiking properties (termed "spiking mESC-OPCs"). After transplantation into the spinal cord and brain of myelin-deficient shiverer mice, the spiking mESC-OPCs demonstrated better capability in differentiating into myelin basic protein expressing oligodendrocytes and in myelinating axons in vivo than the nonspiking mESC-OPCs. Thus, by generating spiking and nonspiking mESC-OPCs, this study reveals a novel function of NaV in OPCs in their functional maturation and myelination, and sheds new light on ways to effectively develop PSC-derived OPCs for future clinical applications.
Assuntos
Células-Tronco Embrionárias/citologia , Oligodendroglia/citologia , Células-Tronco Pluripotentes/citologia , Potenciais de Ação , Animais , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Embrionárias/transplante , Hipocampo/citologia , Hipocampo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Neurônios/citologia , Neurônios/fisiologia , Oligodendroglia/fisiologia , Células-Tronco Pluripotentes/fisiologia , Células-Tronco Pluripotentes/transplante , Transmissão SinápticaRESUMO
Neither the mechanisms that govern lip morphogenesis nor the cause of cleft lip are well understood. We report that genetic inactivation of Lrp6, a co-receptor of the Wnt/beta-catenin signaling pathway, leads to cleft lip with cleft palate. The activity of a Wnt signaling reporter is blocked in the orofacial primordia by Lrp6 deletion in mice. The morphological dynamic that is required for normal lip formation and fusion is disrupted in these mutants. The expression of the homeobox genes Msx1 and Msx2 is dramatically reduced in the mutants, which prevents the outgrowth of orofacial primordia, especially in the fusion site. We further demonstrate that Msx1 and Msx2 (but not their potential regulator Bmp4) are the downstream targets of the Wnt/beta-catenin signaling pathway during lip formation and fusion. By contrast, a ;fusion-resistant' gene, Raldh3 (also known as Aldh1a3), that encodes a retinoic acid-synthesizing enzyme is ectopically expressed in the upper lip primordia of Lrp6-deficient embryos, indicating a region-specific role of the Wnt/beta-catenin signaling pathway in repressing retinoic acid signaling. Thus, the Lrp6-mediated Wnt signaling pathway is required for lip development by orchestrating two distinctively different morphogenetic movements.
Assuntos
Proteínas Relacionadas a Receptor de LDL/metabolismo , Lábio/embriologia , Morfogênese/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Animais , Apoptose/fisiologia , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Proliferação de Células , Fenda Labial/metabolismo , Fenda Labial/patologia , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Lábio/anatomia & histologia , Lábio/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Retinal Desidrogenase , Proteínas Wnt/genéticaRESUMO
The biology of cerebral white matter injury has been woefully understudied, in part because of the difficulty of reliably modeling this type of injury in rodents. Periventricular leukomalacia (PVL) is the predominant form of brain injury and the most common cause of cerebral palsy in premature infants. PVL is characterized by predominant white matter injury. No specific therapy for PVL is presently available, because the pathogenesis is not well understood. Here we report that two types of mouse PVL models have been created by hypoxia-ischemia with or without systemic coadministration of lipopolysaccharide (LPS). LPS coadministration exacerbated hypoxic-ischemic white matter injury and led to enhanced microglial activation and astrogliosis. Drug trials with the antiinflammatory agent minocycline, the antiexcitotoxic agent NBQX, and the antioxidant agent edaravone showed various degrees of protection in the two models, indicating that excitotoxic, oxidative, and inflammatory forms of injury are involved in the pathogenesis of injury to immature white matter. We then applied immunoelectron microscopy to reveal fine structural changes in the injured white matter and found that synapses between axons and oligodendroglial precursor cells (OPCs) are quickly and profoundly damaged. Hypoxia-ischemia caused a drastic decrease in the number of postsynaptic densities associated with the glutamatergic axon-OPC synapses defined by the expression of vesicular glutamate transporters, vGluT1 and vGluT2, on axon terminals that formed contacts with OPCs in the periventricular white matter, resulted in selective shrinkage of the postsynaptic OPCs contacted by vGluT2 labeled synapses, and led to excitotoxicity mediated by GluR2-lacking, Ca(2+) -permeable AMPA receptors. Overall, the present study provides novel mechanistic insights into the pathogenesis of PVL and reveals that axon-glia synapses are highly vulnerable to white matter injury in the developing brain. More broadly, the study of white matter development and injury has general implications for a variety of neurological diseases, including PVL, stroke, spinal cord injury, and multiple sclerosis.
Assuntos
Lesões Encefálicas/complicações , Leucoencefalopatias/etiologia , Leucoencefalopatias/patologia , Fibras Nervosas Mielinizadas/patologia , Neuroglia/patologia , Sinapses/patologia , Animais , Animais Recém-Nascidos , Antígenos/genética , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/etiologia , Lesões Encefálicas/patologia , Doenças das Artérias Carótidas/complicações , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios , Lateralidade Funcional , Proteína Glial Fibrilar Ácida/metabolismo , Hipóxia-Isquemia Encefálica/complicações , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Minociclina/uso terapêutico , Proteína Básica da Mielina/metabolismo , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/ultraestrutura , Neuroglia/efeitos dos fármacos , Polissacarídeos/toxicidade , Proteoglicanas/genética , Quinoxalinas , Receptores de AMPA/metabolismo , Sinapses/ultraestrutura , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
Alteration in mitochondrial dynamics has been implicated in many neurodegenerative diseases. Mitochondrial apoptosis inducing factor (AIF) plays a key role in multiple cellular and disease processes. Using immunoblotting and flow cytometry analysis with Harlequin mutant mice that have a proviral insertion in the AIF gene, we first revealed that mitofusion 1 (Mfn1), a key mitochondrial fusion protein, is significantly diminished in Purkinje cells of the Harlequin cerebellum. Next, we investigated the cerebellar pathology of Harlequin mice in an age-dependent fashion, and identified a striking process of progressive and patterned Purkinje cell degeneration. Using immunohistochemistry with zebrin II, the most studied compartmentalization marker in the cerebellum, we found that zebrin II-negative Purkinje cells first started to degenerate at 7 months of age. By 11 months of age, almost half of the Purkinje cells were degenerated. Subsequently, most of the Purkinje cells disappeared in the Harlequin cerebellum. The surviving Purkinje cells were concentrated in cerebellar lobules IX and X, where these cells were positive for heat shock protein 25 and resistant to degeneration. We further showed that the patterned Purkinje cell degeneration was dependent on caspase but not poly(ADP-ribose) polymerase-1 (PARP-1) activation, and confirmed the marked decrease of Mfn1 in the Harlequin cerebellum. Our results identified a previously unrecognized role of AIF in Purkinje cell degeneration, and revealed that AIF deficiency leads to altered mitochondrial fusion and caspase-dependent cerebellar Purkinje cell loss in Harlequin mice. This study is the first to link AIF and mitochondrial fusion, both of which might play important roles in neurodegeneration.
Assuntos
Fator de Indução de Apoptose/genética , Doenças Cerebelares/genética , Doenças Cerebelares/patologia , GTP Fosfo-Hidrolases/biossíntese , GTP Fosfo-Hidrolases/deficiência , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Células de Purkinje/patologia , Animais , Fator de Indução de Apoptose/deficiência , Doenças Cerebelares/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Doenças Neurodegenerativas/metabolismo , Células de Purkinje/metabolismoRESUMO
Dextromethorphan (DM) is a dextrorotary morphinan and a widely used component of cough medicine. Relatively high doses of DM in combination with quinidine are used for the treatment of mood disorders for patients with multiple sclerosis (MS). However, at lower doses, morphinans exert anti-inflammatory activities through the inhibition of NOX2-dependent superoxide production in activated microglia. Here we investigated the effects of high (10 mg/kg, i.p., "DM-10") and low (0.1 mg/kg, i.p., "DM-0.1") doses of DM on the development and progression of mouse experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We found no protection by high dose DM treatment. Interestingly, a minor late attenuation by low dose DM treatment was seen in severe EAE that was characterized by a chronic disease course and a massive spinal cord infiltration of CD45(+) cells including T-lymphocytes, macrophages and neutrophils. Furthermore, in a less severe form of EAE, where lower levels of CD4(+) and CD8(+) T-cells, Iba1(+) microglia/macrophages and no significant infiltration of neutrophils were seen in the spinal cord, the treatment with DM-0.1 was remarkably more beneficial. The effect was the most significant at the peak of disease and was associated with an inhibition of NOX2 expression and a decrease in infiltration of monocytes and lymphocytes into the spinal cord. In addition, chronic treatment with low dose DM resulted in decreased demyelination and reduced axonal loss in the lumbar spinal cord. Our study is the first report to show that low dose DM is effective in treating EAE of moderate severity. Our findings reveal that low dose morphinan DM treatment may represent a new promising protective strategy for treating MS.
Assuntos
Dextrometorfano/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/enzimologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , NADPH Oxidases/antagonistas & inibidores , Fármacos Neuroprotetores , Medula Espinal/patologia , Animais , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/efeitos dos fármacos , Doenças Desmielinizantes/patologia , Dextrometorfano/administração & dosagem , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/patologia , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Glicoproteínas/biossíntese , Imuno-Histoquímica , Contagem de Linfócitos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Glicoproteína Mielina-Oligodendrócito , NADPH Oxidase 2 , Infiltração de Neutrófilos/efeitos dos fármacos , Fragmentos de Peptídeos/biossíntese , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Superóxidos/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Poly(ADP-ribose) polymerase-1 (PARP-1) has been implicated in the pathogenesis of several central nervous system (CNS) disorders. However, the role of PARP-1 in autoimmune CNS injury remains poorly understood. Therefore, we studied experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis in mice with a targeted deletion of PARP-1. We identified inherent physiological abnormalities in the circulating and splenic immune composition between PARP-1(-/-) and wild type (WT) mice. Upon EAE induction, PARP-1(-/-) mice had an earlier onset and developed a more severe EAE compared with WT cohorts. Splenic response was significantly higher in PARP-1(-/-) mice largely because of B cell expansion. Although formation of Th1 and Th17 effector T lymphocytes was unaffected, PARP-1(-/-) mice had significantly earlier CD4+ T lymphocyte and macrophage infiltration into the CNS during EAE. However, we did not detect significant differences in cytokine profiles between PARP-1(-/-) and WT spinal cords at the peak of EAE. Expression analysis of different PARP isozymes in EAE spinal cords showed that PARP-1 was down-regulated in WT mice and that PARP-3 but not PARP-2 was dramatically up-regulated in both PARP-1(-/-) and WT mice, suggesting that these PARP isozymes could have distinct roles in different CNS pathologies. Together, our results indicate that PARP-1 plays an important role in regulating the physiological immune composition and in immune modulation during EAE; our finding identifies a new aspect of immune regulation by PARPs in autoimmune CNS pathology.
Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Esclerose Múltipla/enzimologia , Poli(ADP-Ribose) Polimerases , Medula Espinal/enzimologia , Animais , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/biossíntese , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/imunologia , Medula Espinal/imunologia , Medula Espinal/patologia , Células Th1/enzimologia , Células Th1/imunologia , Células Th1/patologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Astrocytes, once considered passive support cells, are increasingly appreciated as dynamic regulators of neuronal development and function, in part via secreted factors. The extent to which they similarly regulate oligodendrocytes or proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) is less understood. Here, we generated astrocytes from human pluripotent stem cells (hiPSC-Astros) and demonstrated that immature astrocytes, as opposed to mature ones, promote oligodendrogenesis in vitro. In the PVL mouse model of neonatal hypoxic/ischemic encephalopathy, associated with cerebral palsy in humans, transplanted immature hiPSC-Astros promoted myelinogenesis and behavioral outcome. We further identified TIMP-1 as a selectively upregulated component secreted from immature hiPSC-Astros. Accordingly, in the rat PVL model, intranasal administration of conditioned medium from immature hiPSC-Astros promoted oligodendrocyte maturation in a TIMP-1-dependent manner. Our findings suggest stage-specific developmental interactions between astroglia and oligodendroglia and have important therapeutic implications for promoting myelinogenesis.
Assuntos
Astrócitos/citologia , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Oligodendroglia/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Administração Intranasal , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Comportamento Animal , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucomalácia Periventricular/patologia , Leucomalácia Periventricular/terapia , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Fenótipo , Ratos , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
We previously established cell lines from brains of p53-deficient embryos, and have now characterized one of these lines, FBD-103a, in detail. Recloning FBD-103a yielded 3 types of subclones: 5 generated the neuronal lineage (Type 1), 3 generated the glial lineage (Type 2), and 10 gave rise to both lineages as the parental line (Type 3), indicating that FBD-103a is a multipotent neural progenitor cell line indistinguishable from true neural stem cells. RT-PCR analyses of transcription factor expression indicated that the transition of multipotent Type 3 clones to either neuronally or glially differentiated progeny was marked by down-regulation of Ascl1/Mash1 and Olig1 and up-regulation of Nrsf/Rest. As expected for neural stem cells, FBD-103a and Type 3 clones formed neurospheres when cultured on a non-adhesive substrate in a serum-free medium containing fibroblast growth factor-2 (FGF2). Interestingly, the transition between Type 3 and Type 1 neuronal- or Type 2 glial-specified cells proved to be reversible; Type 1 and Type 2 subclones could also form neurospheres, from which both neuron-generating and glia-generating progenies could be derived. Moreover, when maintained on an adherent substratum that prevented neurosphere formation, but with FGF2 and without serum, Type 2 clones could generate Type 3 multipotent cells. Thus, at least in the absence of p53, specialized cell-cell interactions within neurospheres are not essential for persistence or recapture of the capacity for self-renewal and multipotency by cells differentiating along glial pathways, a result of possible significance to the pathogenesis of malignant brain tumors.
Assuntos
Genes p53/genética , Neurônios/fisiologia , Células-Tronco/fisiologia , Animais , Northern Blotting , Western Blotting , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem da Célula , Células Clonais , Fator de Crescimento Epidérmico/fisiologia , Fator 2 de Crescimento de Fibroblastos/genética , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TranscriçãoRESUMO
Down's syndrome (DS), caused by trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons, and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally, we show that the FDA-approved antibiotic drug, minocycline, partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B, GFAP, inducible nitric oxide synthase, and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug.
Assuntos
Astrócitos/fisiologia , Síndrome de Down/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Astrócitos/efeitos dos fármacos , Estudos de Casos e Controles , Diferenciação Celular , Sobrevivência Celular , Transplante de Células/métodos , Meios de Cultivo Condicionados/farmacologia , Síndrome de Down/genética , Perfilação da Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Minociclina/farmacologia , Células-Tronco Neurais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Trombospondina 1/metabolismo , Trombospondinas/metabolismoRESUMO
Local production of neurosteroids such as progesterone and allopregnanolone confers neuroprotection in central nervous system (CNS) inflammatory diseases. The mitochondrial translocator protein (TSPO) performs a rate-limiting step in the conversion of cholesterol to pregnenolone and its steroid derivatives. Previous studies have shown that TSPO is upregulated in microglia and astroglia during neural inflammation, and radiolabelled TSPO ligands such as PK11195 have been used to image and localize injury in the CNS. Recent studies have shown that modulating TSPO activity with pharmacological ligands such as etifoxine can initiate the production of neurosteroids locally in the injured CNS. In this study, we examined the effects of etifoxine, a clinically available anxiolytic drug, in the development and progression of mouse experimental autoimmune encephalomyelitis (EAE), an experimental model for multiple sclerosis (MS). Our results showed that etifoxine attenuated EAE severity when administered before the development of clinical signs and also improved symptomatic recovery when administered at the peak of the disease. In both cases, recovery was correlated with diminished inflammatory pathology in the lumbar spinal cord. Modulation of TSPO activity by etifoxine led to less peripheral immune cell infiltration of the spinal cord, and increased oligodendroglial regeneration after inflammatory demyelination in EAE. Our results suggest that a TSPO ligand, e.g. etifoxine, could be a potential new therapeutic option for MS with benefits that could be comparable to the administration of systemic steroids but potentially avoiding the detrimental side effects of long-term direct use of steroids.
Assuntos
Ansiolíticos/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Oxazinas/uso terapêutico , Receptores de GABA/metabolismo , Animais , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Receptores de GABA/química , Medula Espinal/metabolismoRESUMO
Human pluripotent stem cells (hPSCs) have been differentiated to astroglia, but the utilization of hPSC-derived astroglia as cell therapy for neurological diseases has not been well studied. Astroglia are heterogeneous, and not all astroglia are equivalent in promoting neural repair. A prerequisite for cell therapy is to derive defined cell populations with superior therapeutic effects. Here we use an Olig2-GFP human embryonic stem cell (hESC) reporter to demonstrate that hESC-derived Olig2(+) progenitors generate a subtype of previously uncharacterized astroglia (Olig2PC-Astros). These Olig2PC-Astros differ substantially from astroglia differentiated from Olig2-negative hESC-derived neural progenitor cells (NPC-Astros), particularly in their neuroprotective properties. When grafted into brains subjected to global ischaemia, Olig2PC-Astros exhibit superior neuroprotective effects and improved behavioural outcome compared to NPC-Astros. Thus, this new paradigm of human astroglial differentiation is useful for studying the heterogeneity of human astroglia, and the unique Olig2PC-Astros may constitute a new cell therapy for treating cerebral ischaemia and other neurological diseases.
Assuntos
Astrócitos/citologia , Astrócitos/transplante , Isquemia Encefálica/terapia , Células-Tronco Embrionárias/citologia , Animais , Astrócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Injeções Intraventriculares , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição 2 de Oligodendrócitos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Técnicas Estereotáxicas , Transplante HeterólogoAssuntos
Biópsia/efeitos adversos , Análise Mutacional de DNA/tendências , Doenças do Sistema Nervoso Periférico/diagnóstico , Polirradiculoneuropatia/diagnóstico , Nervo Sural/patologia , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/normas , Humanos , Mutação/genética , Proteínas da Mielina/análise , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Neuralgia/etiologia , Neuralgia/fisiopatologia , Neuralgia/prevenção & controle , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/fisiopatologia , Polirradiculoneuropatia/genética , Polirradiculoneuropatia/fisiopatologia , Pele/inervação , Nervo Sural/cirurgiaRESUMO
Periventricular leukomalacia (PVL) is the predominant pathology in premature infants, characterized by prominent cerebral white matter injury, and commonly caused by hypoxia-ischemia and inflammation. Activated microglia trigger white matter damage and play a major role in the development of PVL. Erythropoietin (EPO) and its derivative carbamylated erythropoietin (CEPO) have been shown to be neuroprotective in several brain disease models. Here we investigated whether EPO and CEPO could provide protection in mouse models of PVL induced by hypoxia-ischemia or hypoxia-ischemia-inflammation. We administered EPO or CEPO to mice with PVL, and found that both EPO and CEPO treatments decreased microglia activation, oligodendrocyte damage and myelin depletion. We also noted improved performance in neurological function assays. Inhibited disease progression in PVL mice by EPO or CEPO treatment was associated with decreased poly-(ADP-ribose) polymerase-1 (PARP-1) activity. PARP-1 activity was increased dramatically in activated microglia in untreated mice with PVL. Furthermore, we demonstrated that the neuroprotective properties of EPO and CEPO were diminished after PARP-1 gene depletion. The therapeutic doses of EPO and CEPO used in this study did not interfere with normal oligodendrocyte maturation and myelination. Together, our data demonstrate that EPO and CEPO are neuroprotective in cerebral white matter injury via a novel microglial PARP-1 dependent mechanism, and hold promise as a future treatment for PVL and other hypoxic-ischemic/inflammatory white matter diseases.
Assuntos
Eritropoetina/análogos & derivados , Eritropoetina/uso terapêutico , Leucomalácia Periventricular/tratamento farmacológico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Isquemia Encefálica/complicações , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Eritropoetina/farmacologia , Humanos , Hipóxia/complicações , Hipóxia/tratamento farmacológico , Hipóxia/patologia , Recém-Nascido , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/patologia , Leucomalácia Periventricular/etiologia , Leucomalácia Periventricular/patologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologiaRESUMO
Basic helix-loop-helix transcription factors Olig1 and Olig2 critically regulate oligodendrocyte development. Initially identified as a downstream effector of Olig1, an oligodendrocyte-specific zinc finger transcription repressor, Zfp488, cooperates with Olig2 function. Although Zfp488 is required for oligodendrocyte precursor formation and differentiation during embryonic development, its role in oligodendrogenesis of adult neural progenitor cells is not known. In this study, we tested whether Zfp488 could promote an oligodendrogenic fate in adult subventricular zone (SVZ) neural stem/progenitor cells (NSPCs). Using a cuprizone-induced demyelination model in mice, we examined the effect of retrovirus-mediated Zfp488 overexpression in SVZ NSPCs. Our results showed that Zfp488 efficiently promoted the differentiation of the SVZ NSPCs into mature oligodendrocytes in vivo. After cuprizone-induced demyelination injury, Zfp488-transduced mice also showed significant restoration of motor function to levels comparable to control mice. Together, these findings identify a previously unreported role for Zfp488 in adult oligodendrogenesis and functional remyelination after injury.
Assuntos
Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Camundongos , Camundongos KnockoutRESUMO
Minocycline is a clinically available antibiotic and anti-inflammatory drug that also demonstrates neuroprotective properties in a variety of experimental models of neurological diseases. There have thus far been more than 300 publications on minocycline neuroprotection, including a growing number of human studies. Our objective is to critically review the biological basis and translational potential of this action of minocycline on the nervous system.