RESUMEN

Siponimod (Sp) is a Sphingosine 1-phosphate (S1P) receptor modulator, and it suppresses S1P- mediated autoimmune lymphocyte transport and inflammation. Theiler's murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis (MS) exhibits inflammation-driven acute and chronic phases, spinal cord lesions, brain and spinal cord atrophy, and white matter injury. The objective of the study was to investigate whether Sp treatment could attenuate inflammation-induced pathology in the TMEV model by inhibiting microglial activation and preventing the atrophy of central nervous tissue associated with neurodegeneration. Clinical disability score (CDS), body weight (BW), and rotarod retention time measures were used to assess Sp's impact on neurodegeneration and disease progression in 4 study groups of 102 animals, including 44 Sp-treated (SpT), 44 vehicle-treated, 6 saline-injected, and 8 age-matched healthy controls (HC). Next, 58 (22 SpT, 22 vehicle, 6 saline injected, and 8 HC) out of the 102 animals were further evaluated to assess the effect of Sp on brain region-specific and spinal cord volume changes, as well as microglial activation. Sp increased CDS and decreased BW and rotarod retention time in TMEV mice, but did not significantly affect most brain region volumes, except for lateral ventricle volume. Sp suppressed ventricular enlargement, suggesting reduced TMEV-induced inflammation in LV. No significant differences in spine volume changes were observed between Sp- and vehicle-treated animals, but there were differences between HC and TMEV groups, indicating TMEV-induced inflammation contributed to increased spine volume. Spine histology revealed no significant microglial density differences between groups in gray matter, but HC animals had higher type 1 morphology and lower type 2 morphology percentages in gray and white matter regions. This suggests that Sp did not significantly affect microglial density but may have modulated neuroinflammation in the spinal cord. Sp may have some effects on neuroinflammation and ventricular enlargement. However, it did not demonstrate a significant impact on neurodegeneration, spinal volume, or lesion volume in the TMEV mouse model. Further investigation is required to fully understand Sp's effect on microglial activation and its relevance to the pathophysiology of MS. The differences between the current study and previous research using other MS models, such as EAE, highlight the differences in pathological processes in these two disease models.

Asunto(s)

Enfermedades Desmielinizantes , Theilovirus , Animales , Ratones , Enfermedades Neuroinflamatorias , Encéfalo/diagnóstico por imagen , Médula Espinal/diagnóstico por imagen , Atrofia , Modelos Animales de Enfermedad

RESUMEN

BACKGROUND: Several translational animal models have been described assessing intra-arterial (IA) treatments for malignant gliomas. We describe the first endovascular animal model that allows testing of IA drug delivery as a first-line treatment, which is difficult to do in actual patients. We report a unique protocol for vascular access and IA delivery in the rat model that, unlike prior reports, does not require direct puncture and opening of proximal cerebrovasculature which carries risk of ischemia in the animal brain post-delivery. METHODS: Wistar rats underwent left femoral artery catherization with a Balt Magic 1.2F catheter or Marathon Flow directed 1.5F Microcatheter with an Asahi Chikai 0.008 micro-guidewire which was navigated to the left internal carotid artery under x-ray. 25% mannitol was administered to test blood brain barrier breakdown (BBBB). Additional rats were implanted with C6 glioma cells in the left frontal lobe. C6 Glioma-Implanted Rats (C6GRs) were monitored for overall survival and tumor growth. Tumor volumes from MRI images were calculated utilizing 3D slicer. Additional rats underwent femoral artery catheterization with Bevacizumab, carboplatin, or irinotecan injected into the left internal carotid artery to test feasibility and safety. RESULTS: A successful endovascular access and BBBB protocol was established. BBBB was confirmed with positive Evans blue staining. 10 rats were successfully implanted with C6 gliomas with confirmed growths on MRI. Overall survival was 19.75 ± 2.21 days. 5 rats were utilized for the development of our femoral catheterization protocol and BBBB testing. With regards to IA chemotherapy dosage testing, control rats tolerated targeted 10 mg/kg of bevascizumab, 2.4 mg/kg of carboplatin, and 15 mg/kg of irinotecan IA ICA injections without any complications. CONCLUSIONS: We present the first endovascular IA rat glioma model that allows selective catheterization of the intracranial vasculature and assessment of IA therapies for gliomas without need for access and sacrifice of proximal cerebrovasculature.

RESUMEN

Chronic demyelination in the human CNS is characterized by an inhibitory microenvironment that impairs recruitment and differentiation of oligodendrocyte progenitor cells (OPCs) leading to failed remyelination and axonal atrophy. By network-based transcriptomics, we identified sulfatase 2 (Sulf2) mRNA in activated human primary OPCs. Sulf2, an extracellular endosulfatase, modulates the signaling microenvironment by editing the pattern of sulfation on heparan sulfate proteoglycans. We found that Sulf2 was increased in demyelinating lesions in multiple sclerosis and was actively secreted by human OPCs. In experimental demyelination, elevated OPC Sulf1/2 expression directly impaired progenitor recruitment and subsequent generation of oligodendrocytes thereby limiting remyelination. Sulf1/2 potentiates the inhibitory microenvironment by promoting BMP and WNT signaling in OPCs. Importantly, pharmacological sulfatase inhibition using PI-88 accelerated oligodendrocyte recruitment and remyelination by blocking OPC-expressed sulfatases. Our findings define an important inhibitory role of Sulf1/2 and highlight the potential for modulation of the heparanome in the treatment of chronic demyelinating disease.

Asunto(s)

Diferenciación Celular/genética , Microambiente Celular/genética , Enfermedades Desmielinizantes/genética , Perfilación de la Expresión Génica/métodos , Células Precursoras de Oligodendrocitos/metabolismo , Remielinización/genética , Animales , Axones/metabolismo , Células Cultivadas , Enfermedades Desmielinizantes/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Células Precursoras de Oligodendrocitos/citología , Sulfatasas/genética , Sulfatasas/metabolismo , Sulfotransferasas/genética , Sulfotransferasas/metabolismo

RESUMEN

BACKGROUND: The human myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (huMOG-EAE) model, generates B-cell driven demyelination in mice, making it a suitable multiple sclerosis model to study B cell depletion. OBJECTIVES: We investigated the effect of subcutaneous anti-CD20 antibody treatment on huMOG-EAE gray matter (GM) pathology. METHODS: C57Bl/6, 8-week old mice were immunized with 200 huMOG1-125 and treated with 50 µg/mouse of anti-CD20 antibody (n = 16) or isotype control (n = 16). Serial brain volumetric 9.4 T MRI scans was performed at baseline, 1 and 5 wkPI. Disease severity was measured by clinical disability score (CDS) and performance on rotarod test. RESULTS: Anti-CD20 antibody significantly reduced brain volume loss compared with the isotype control across all timepoints longitudinally in the basal ganglia (p = 0.01), isocortex (p = 0.025) and thalamus (p = 0.023). The CDS was reduced significantly with anti-CD20 antibody vs. the isotype control at 3 (p = 0.003) and 4 (p = 0.03) wkPI, while a trend was observed at 5 (p = 0.057) and 6 (p = 0.086) wkPI. Performance on rotarod was also improved significantly at 3 (p = 0.007) and 5 (p = 0.01) wkPI compared with the isotype control. At cellular level, anti-CD20 therapy suppressed the percentage of proliferative nuclear antigen positive microglia in huMOG-EAE isocortex (p = 0.016). Flow cytometry confirmed that anti-CD20 antibody strongly depleted the CD19-expressing B cell fraction in peripheral blood mononuclear cells, reducing it from 39.7% measured in isotype control to 1.59% in anti-CD20 treated mice (p < 0.001). CONCLUSIONS: Anti-CD20 antibody treatment delayed brain tissue neurodegeneration in GM, and showed clinical benefit on measures of disease severity in huMOG-EAE mice.

Asunto(s)

Anticuerpos/uso terapéutico , Antígenos CD20/inmunología , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Sustancia Gris/patología , Glicoproteína Mielina-Oligodendrócito , Animales , Atrofia , Linfocitos B/inmunología , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/diagnóstico por imagen , Femenino , Sustancia Gris/diagnóstico por imagen , Humanos , Macrófagos/inmunología , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/inmunología , Equilibrio Postural/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos

RESUMEN

BACKGROUND AND PURPOSE: Blood-derived monocytes/macrophages can be labeled with ultrasmall superparamagnetic iron oxides (USPIO) at periphery and subsequently migrate into areas of inflammation in the brain. We investigated temporal pattern of migration of peripheral immune cells in Theiler's murine encephalomyelitis virus (TMEV) model of chronic demyelination by USPIO-enhanced imaging. METHODS: Fifteen SJL mice (Envigo, Indianapolis, IN) were injected with TMEV (n = 12) or saline (n = 3) at 7 weeks of age. Brain MRI of 9.4 T was performed at 3 months postinfection (mpi) (the peak of inflammatory phase), at 4, 5, and 7 mpi (throughout neurodegenerative phase) using T2*-weighted gradient echo MRI, and performed 24 hours after USPIO injection. Contrast enhancing lesion (CEL) number and volume were measured and development of brain atrophy was assessed across serial time points. Clinical disability scale and rotarod score assessed disease progression. RESULTS: CEL was detected in a total of eight (66.7%) TMEV-infected animals and none of the Controls. The CEL was present in four (33.3%) TMEV-infected animals at 3 mpi, two (16.7%) at 4 mpi, six (54.5%) at 5 mpi, and four (44.4%) at 7 mpi, respectively. In TMEV-infected animals, the CEL number and volume increased significantly from 3 to 7 mpi (P < .01 for both). The correlation between total CEL number and volume with clinical and MRI outcomes was trending (P < .05). On histopathology analysis, CEL showed increased density of Iba1 staining for microglia activity. CONCLUSIONS: Serial USPIO imaging is a promising biomarker for investigating the effect of therapeutic interventions on monocytes/macrophages and microglia activation and neurodegeneration in TMEV-infected animals.

Asunto(s)

Encéfalo/diagnóstico por imagen , Enfermedades Desmielinizantes/diagnóstico por imagen , Inflamación/diagnóstico por imagen , Macrófagos/patología , Microglía/patología , Monocitos/patología , Animales , Encéfalo/inmunología , Encéfalo/patología , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/patología , Dextranos , Inflamación/inmunología , Inflamación/patología , Nanopartículas de Magnetita , Ratones , Ratones Endogámicos , Theilovirus/inmunología

RESUMEN

BACKGROUND: Leptomeningeal inflammation, as evidenced by leptomeningeal contrast enhancement (LMCE), is associated to cortical pathology in multiple sclerosis. The temporal pattern of LMCE in experimental autoimmune encephalomyelitis (EAE) myelin oligodendrocyte glycoprotein (MOG) is unknown. OBJECTIVE: To investigate LMCE using serial MRI in the EAE model of MS, and its association with clinical disease progression. To characterize the relationship between LMCE and underlying histological correlates. DESIGN: Thirteen C57BL/6J mice, MOG-immunized (35-55 amino acid) and 8 saline injected animals were assessed at pre-induction and at 3, 6, 10, 20, 27, 32, 45 and 63 days post induction (dPI). LMCE scan was obtained using FLAIR-RARE sequence after post-contrast gadolinium administration on 9.4 T scanner. Brain cryo-sections were assessed for measuring cellular density of Iba1 positive macrophage/microglia at 10 dPI and 32 dPI, and for the presence of T, B and macrophage cells in the meningeal layer at 10 dPI and 63 dPI. RESULTS: All EAE-MOG animals showed presence of LMCE and none of the control mice. The peak signal intensity of LMCE was evidenced at 10dPI in the meninges and decreased through 10-63 dPI. The peak of LMCE was associated with a weight loss starting at 1 week PI and with clinical symptoms starting at 2 weeks PI. Histological analysis of the brain tissue showed a higher density of Iba1 positive microglial cells in the EAE-MOG animals, corresponding to the areas of LMCE. Meninges of EAE mice showed higher density of Iba1 stained macrophage cells relative to saline animals. EAE animals also showed the presence of T and B cells in the meninges which were absent in the saline animals. CONCLUSIONS: LMCE peak intensity in the meninges corresponds to the acute inflammatory phase of EAE-MOG disease progression, and is associated with clinical symptoms and higher inflammatory cell density.

Asunto(s)

Encefalomielitis Autoinmune Experimental/patología , Meninges/patología , Esclerosis Múltiple/patología , Animales , Linfocitos B/patología , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Corteza Cerebral/patología , Encefalomielitis Autoinmune Experimental/diagnóstico por imagen , Femenino , Inflamación/patología , Macrófagos/metabolismo , Macrófagos/patología , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/genética , Microglía/metabolismo , Microglía/patología , Esclerosis Múltiple/diagnóstico por imagen , Glicoproteína Mielina-Oligodendrócito/biosíntesis , Glicoproteína Mielina-Oligodendrócito/genética , Linfocitos T/patología , Pérdida de Peso

RESUMEN

BACKGROUND AND PURPOSE: Teriflunomide reduces disability progression and brain atrophy in multiple sclerosis patients. The exact mechanism of action by which teriflunomide exerts these effects is currently unknown. We assessed the effect of teriflunomide on brain glial cells in the Theiler's murine encephalomyelitis virus (TMEV) by using a histological approach in combination with neuroimaging. METHODS: Forty-eight SJL female mice received an intracerebral injection of TMEV at 6-8 weeks of age and were then treated with teriflunomide (n = 24) or placebo (n = 24) for 9 months. They were examined with MRI and behavioral testing at 2, 6, and 9 months postinduction (mPI). Of those, 18 teriflunomide-treated and 17 controls mice were analyzed histologically at 9 mPI to sample from different brain regions for myelination status, microglial density, and oligodendroglial lineage. The histological and MRI outcomes were correlated. RESULTS: Corpus callosum microglial density was numerically lower in the teriflunomide-treated mice compared to the control group (141.1 ± 21.7 SEM vs. 214.74 ± 34.79 SEM, Iba1+ cells/mm2 , P = .087). Basal ganglia (BG) microglial density in the teriflunomide group exhibited a negative correlation with fractional anisotropy (P = .021) and a positive correlation with mean diffusivity (P = .034), indicating less inflammation and axonal damage. Oligodendroglial lineage cell and myelin density were not significantly different between treatment groups. However, a significant positive correlation between BG oligodendrocytes and BG volume (P = .027), and with N-acetyl aspartate concentration (P = .008), was found in the teriflunomide group, indicating less axonal loss. CONCLUSION: Teriflunomide altered microglia density and oligodendrocytes differentiation, which was associated with less evident microstructural damage on MRI.

Asunto(s)

Cuerpo Calloso/diagnóstico por imagen , Crotonatos/farmacología , Enfermedades Desmielinizantes/diagnóstico por imagen , Neuroglía/efectos de los fármacos , Toluidinas/farmacología , Animales , Cuerpo Calloso/patología , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Femenino , Hidroxibutiratos , Ratones , Neuroglía/patología , Neuroimagen , Nitrilos , Theilovirus

RESUMEN

Human oligodendrocyte progenitor cells (hOPCs) persist into adulthood as an abundant precursor population capable of division and differentiation. The transcriptional mechanisms that regulate hOPC homeostasis remain poorly defined. Herein, we identify paired related homeobox protein 1 (PRRX1) in primary PDGFαR+ hOPCs. We show that enforced PRRX1 expression results in reversible G1/0 arrest. While both PRRX1 splice variants reduce hOPC proliferation, only PRRX1a abrogates migration. hOPC engraftment into hypomyelinated shiverer/rag2 mouse brain is severely impaired by PRRX1a, characterized by reduced cell proliferation and migration. PRRX1 induces a gene expression signature characteristic of stem cell quiescence. Both IFN-γ and BMP signaling upregulate PRRX1 and induce quiescence. PRRX1 knockdown modulates IFN-γ-induced quiescence. In mouse brain, PRRX1 mRNA was detected in non-dividing OPCs and is upregulated in OPCs following demyelination. Together, these data identify PRRX1 as a regulator of quiescence in hOPCs and as a potential regulator of pathological quiescence.

Asunto(s)

Ciclo Celular , Proteínas de Homeodominio/metabolismo , Células Precursoras de Oligodendrocitos/citología , Células Precursoras de Oligodendrocitos/metabolismo , Animales , Proteínas Morfogenéticas Óseas/farmacología , Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Antígeno Ki-67/metabolismo , Ratones , Vaina de Mielina/metabolismo , Células Precursoras de Oligodendrocitos/trasplante , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Sustancia Blanca/metabolismo , Sustancia Blanca/patología

RESUMEN

Impaired human oligodendrocyte progenitor cell (hOPC) differentiation likely contributes to failed remyelination in multiple sclerosis. The characterization of molecular pathways that regulate hOPC differentiation will provide means to induce remyelination. In this study, we determined the gene expression profile of PDGFαR+ hOPCs during initial oligodendrocyte commitment. Weighted gene coexpression network analysis was used to define progenitor and differentiation-specific gene expression modules and functionally important hub genes. These modules were compared with rodent OPC and oligodendrocyte data to determine the extent of species conservation. These analyses identified G-protein ß4 (GNB4), which was associated with hOPC commitment. Lentiviral GNB4 overexpression rapidly induced human oligodendrocyte differentiation. Following xenograft in hypomyelinating shiverer/rag2 mice, GNB4 overexpression augmented myelin synthesis and the ability of hOPCs to ensheath host axons, establishing GNB4 as functionally important in human myelination. As such, network analysis of hOPC gene expression accurately predicts genes that influence human oligodendrocyte differentiation in vivo.

Asunto(s)

Diferenciación Celular/genética , Biología Computacional/métodos , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genómica , Células Precursoras de Oligodendrocitos/citología , Células Precursoras de Oligodendrocitos/metabolismo , Animales , Axones/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Perfilación de la Expresión Génica , Genómica/métodos , Humanos , Oligodendroglía/citología , Oligodendroglía/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Roedores , Transducción de Señal , Transcriptoma

RESUMEN

BACKGROUND: Pathology of gray matter is associated with development of physical and cognitive disability in patients with multiple sclerosis. In particular, glutamatergic dysregulation in the cortex-basal ganglia-thalamus (CxBGTh) circuit could be associated with decline in these behaviors. OBJECTIVES: To investigate the effect of an immunomodulatory therapy (teriflunomide, Aubagio®) on changes of the CxBGTh loop in the Theiler's Murine Encephalomyelitis Virus, (TMEV) mouse model of MS. METHODS: Forty-eight (48) mice were infected with TMEV, treated with teriflunomide (24) or control vehicle (24) and followed for 39 weeks. Mice were examined with MRS and volumetric MRI scans (0, 8, 26, and 39 weeks) in the cortex, basal ganglia and thalamus, using a 9.4T scanner, and with behavioral tests (0, 4, 8, 12, 17, 26, and 39 weeks). Within conditions, MRI measures were compared between two time points by paired samples t-test and across multiple time points by repeated measures ANOVA (rmANOVA), and between conditions by independent samples t-test and rmANOVA, respectively. Data were considered as significant at the p<0.01 level and as a trend at p<0.05 level. RESULTS: In the thalamus, the teriflunomide arm exhibited trends toward decreased glutamate levels at 8 and 26 weeks compared to the control arm (p = 0.039 and p = 0.026), while the control arm exhibited a trend toward increased glutamate between 0 to 8 weeks (p = 0.045). In the basal ganglia, the teriflunomide arm exhibited a trend toward decreased glutamate earlier than the control arm, from 0 to 8 weeks (p = 0.011), resulting in decreased glutamate compared to the control arm at 8 weeks (p = 0.016). CONCLUSIONS: Teriflunomide may reduce possible excitotoxicity in the thalamus and basal ganglia by lowering glutamate levels.

Asunto(s)

Ganglios Basales/efectos de los fármacos , Crotonatos/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Tálamo/efectos de los fármacos , Toluidinas/farmacología , Animales , Ganglios Basales/metabolismo , Línea Celular , Cuerpo Calloso/efectos de los fármacos , Cuerpo Calloso/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Femenino , Ácido Glutámico/metabolismo , Hidroxibutiratos , Mesocricetus , Ratones , Mielitis/tratamiento farmacológico , Nitrilos , Tálamo/metabolismo , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

Oligodendrocyte development has been studied for several decades, and has served as a model system for both neurodevelopmental and stem/progenitor cell biology. Until recently, the vast majority of studies have been conducted in lower species, especially those focused on rodent development and remyelination. In humans, the process of myelination requires the generation of vastly more myelinating glia, occurring over a period of years rather than weeks. Furthermore, as evidenced by the presence of chronic demyelination in a variety of human neurologic diseases, it appears likely that the mechanisms that regulate development and become dysfunctional in disease may be, in key ways, divergent across species. Improvements in isolation techniques, applied to primary human neural and oligodendrocyte progenitors from both fetal and adult brain, as well as advancements in the derivation of defined progenitors from human pluripotent stem cells, have begun to reveal the extent of both species-conserved signaling pathways and potential key differences at cellular and molecular levels. In this article, we will review the commonalities and differences in myelin development between rodents and man, describing the approaches used to study human oligodendrocyte differentiation and myelination, as well as heterogeneity within targetable progenitor pools, and discuss the advances made in determining which conserved pathways may be both modeled in rodents and translate into viable therapeutic strategies to promote myelin repair.

Asunto(s)

Trasplante de Células/métodos , Enfermedades Desmielinizantes/cirugía , Oligodendroglía/fisiología , Células Madre/fisiología , Animales , Diferenciación Celular , Humanos , Regeneración/fisiología

RESUMEN

Therapeutic repair of myelin disorders may be limited by the relatively slow rate of human oligodendrocyte differentiation. To identify appropriate pharmacological targets with which to accelerate differentiation of human oligodendrocyte progenitors (hOPCs) directly, we used CD140a/O4-based FACS of human forebrain and microarray to hOPC-specific receptors. Among these, we identified CHRM3, a M3R muscarinic acetylcholine receptor, as being restricted to oligodendrocyte-biased CD140a(+)O4(+) cells. Muscarinic agonist treatment of hOPCs resulted in a specific and dose-dependent blockade of oligodendrocyte commitment. Conversely, when hOPCs were cocultured with human neurons, M3R antagonist treatment stimulated oligodendrocytic differentiation. Systemic treatment with solifenacin, an FDA-approved muscarinic receptor antagonist, increased oligodendrocyte differentiation of transplanted hOPCs in hypomyelinated shiverer/rag2 brain. Importantly, solifenacin treatment of engrafted animals reduced auditory brainstem response interpeak latency, indicative of increased conduction velocity and thereby enhanced functional repair. Therefore, solifenacin and other selective muscarinic antagonists represent new adjunct approaches to accelerate repair by engrafted human progenitors.

Asunto(s)

Células Madre Fetales/citología , Antagonistas Muscarínicos/farmacología , Vaina de Mielina/metabolismo , Oligodendroglía/citología , Quinuclidinas/farmacología , Regeneración , Tetrahidroisoquinolinas/farmacología , Animales , Tronco Encefálico/citología , Tronco Encefálico/fisiología , Células Cultivadas , Proteínas de Unión al ADN/genética , Potenciales Evocados Auditivos del Tronco Encefálico , Femenino , Células Madre Fetales/efectos de los fármacos , Células Madre Fetales/metabolismo , Células Madre Fetales/trasplante , Humanos , Masculino , Ratones , Agonistas Muscarínicos/farmacología , Vaina de Mielina/genética , Neurogénesis , Antígenos O/genética , Antígenos O/metabolismo , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Oligodendroglía/trasplante , Prosencéfalo/citología , Prosencéfalo/embriología , Receptor Muscarínico M3 , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Succinato de Solifenacina

RESUMEN

Human oligodendrocyte progenitor cell (OPC) specification and differentiation occurs slowly and limits the potential for cell-based treatment of demyelinating disease. In this study, using FACS-based isolation and microarray analysis, we identified a set of transcription factors expressed by human primary CD140a(+)O4(+) OPCs relative to CD133(+)CD140a(-) neural stem/progenitor cells (NPCs). Among these, lentiviral overexpression of transcription factors ASCL1, SOX10, and NKX2.2 in NPCs was sufficient to induce Sox10 enhancer activity, OPC mRNA, and protein expression consistent with OPC fate; however, unlike ASCL1 and NKX2.2, only the transcriptome of SOX10-infected NPCs was induced to a human OPC gene expression signature. Furthermore, only SOX10 promoted oligodendrocyte commitment, and did so at quantitatively equivalent levels to native OPCs. In xenografts of shiverer/rag2 animals, SOX10 increased the rate of mature oligodendrocyte differentiation and axon ensheathment. Thus, SOX10 appears to be the principle and rate-limiting regulator of myelinogenic fate from human NPCs.

Asunto(s)

Diferenciación Celular , Regulación de la Expresión Génica , Células-Madre Neurales/metabolismo , Oligodendroglía/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Elementos de Facilitación Genéticos , Xenoinjertos , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio , Humanos , Lentivirus , Ratones , Células-Madre Neurales/citología , Proteínas Nucleares , Oligodendroglía/citología , Trasplante de Células Madre , Factores de Transcripción/genética , Transcriptoma , Transducción Genética

RESUMEN

In this study, we sought to establish a novel method to prospectively and dynamically identify live human oligodendrocyte precursor cells (OPCs) and oligodendrocyte lineage cells from brain dissociates and pluripotent stem cell culture. We selected a highly conserved enhancer element of the Sox10 gene, known as MCS5, which directs reporter expression to oligodendrocyte lineage cells in mouse and zebrafish. We demonstrate that lentiviral Sox10-MCS5 induced expression of GFP at high levels in a subpopulation of human CD140a/PDGFαR-sorted OPCs as well as their immature oligodendrocyte progeny. Furthermore, we show that almost all Sox10-MCS5:GFP(high) cells expressed OPC antigen CD140a and human OPCs expressing SOX10, OLIG2, and PDGFRA mRNAs could be prospectively identified using GFP based fluorescence activated cells sorting alone. Additionally, we established a human induced pluripotent cell (iPSC) line transduced with the Sox10-MCS5:GFP reporter using a Rex-Neo cassette. Similar to human primary cells, GFP expression was restricted to embryoid bodies containing both oligodendrocyte progenitor and oligodendrocyte cells and co-localized with NG2 and O4-positive cells respectively. As such, we have developed a novel reporter system that can track oligodendrocyte commitment in human cells, establishing a valuable tool to improve our understanding and efficiency of human oligodendrocyte derivation.

Asunto(s)

Diferenciación Celular/genética , Células Madre Embrionarias/fisiología , Elementos de Facilitación Genéticos/genética , Oligodendroglía/metabolismo , Factores de Transcripción SOXE/metabolismo , Antígenos/metabolismo , Células Cultivadas , Feto , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Antígenos O/metabolismo , Proteoglicanos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factores de Transcripción SOXE/genética

RESUMEN

The mechanisms underlying the specification of oligodendrocyte fate from multipotent neural progenitor cells (NPCs) in developing human brain are unknown. In this study, we sought to identify antigens sufficient to distinguish NPCs free from oligodendrocyte progenitor cells (OPCs). We investigated the potential overlap of NPC and OPC antigens using multicolor fluorescence-activated cell sorting (FACS) for CD133/PROM1, A2B5, and CD140a/PDGFαR antigens. Surprisingly, we found that CD133, but not A2B5, was capable of enriching for OLIG2 expression, Sox10 enhancer activity, and oligodendrocyte potential. As a subpopulation of CD133-positive cells expressed CD140a, we asked whether CD133 enriched bone fide NPCs regardless of CD140a expression. We found that CD133(+)CD140a(-) cells were highly enriched for neurosphere initiating cells and were multipotent. Importantly, when analyzed immediately following isolation, CD133(+)CD140a(-) NPCs lacked the capacity to generate oligodendrocytes. In contrast, CD133(+)CD140a(+) cells were OLIG2-expressing OPCs capable of oligodendrocyte differentiation, but formed neurospheres with lower efficiency and were largely restricted to glial fate. Gene expression analysis further confirmed the stem cell nature of CD133(+)CD140a(-) cells. As human CD133(+) cells comprised both NPCs and OPCs, CD133 expression alone cannot be considered a specific marker of the stem cell phenotype, but rather comprises a heterogeneous mix of glial restricted as well as multipotent neural precursors. In contrast, CD133/CD140a-based FACS permits the separation of defined progenitor populations and the study of neural stem and oligodendrocyte fate specification in the human brain.

Asunto(s)

Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Células-Madre Neurales/metabolismo , Oligodendroglía/metabolismo , Péptidos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Antígeno AC133 , Biomarcadores/metabolismo , Separación Celular , Células Cultivadas , Citometría de Flujo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Esferoides Celulares/citología , Esferoides Celulares/metabolismo , Transcriptoma

RESUMEN

The molecular mechanisms controlling human oligodendrocyte development are poorly characterized. Microarray analysis of human oligodendrocyte progenitor cells (OPCs) and immature oligodendrocytes revealed that specific-class I histone deacetylase (HDAC) target genes were actively repressed during oligodendrocyte commitment. Although epigenetic regulation of oligodendrocyte differentiation has been established in rodent development, the role of HDACs in human OPCs remains undefined. We used HDAC inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate to determine the importance of HDAC activity in human primary OPC differentiation. Treatment with either drug resulted in significant dose-dependent inhibition of O4(+) oligodendrocyte cell differentiation, reduction of oligodendrocyte morphological maturation, and downregulation of myelin basic protein mRNA. High dose TSA treatment was also associated with reduction in OPC proliferation. HDACi treatment prevented downregulation of SOX2, ID4, and TCF7L2 mRNAs but did not regulate HES5, suggesting that targets of HDAC repression may differ between species. These results predict that HDACi treatment would impair proliferation and differentiation by parenchymal oligodendrocyte progenitors, and thereby degrade their potential for endogenous repair in human demyelinating disease. © 2012 Wiley Periodicals, Inc.

Asunto(s)

Diferenciación Celular/fisiología , Células Madre Embrionarias/enzimología , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Oligodendroglía/enzimología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Feto/citología , Feto/efectos de los fármacos , Feto/enzimología , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/fisiología , Humanos , Oligodendroglía/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Células Madre/efectos de los fármacos , Células Madre/enzimología

RESUMEN

BACKGROUND: Proteases play a central role in cellular homeostasis and are responsible for the spatio-temporal regulation of function. Many putative proteases have been recently identified through genomic approaches, leading to a surge in global profiling attempts to characterize their function. Through such efforts and others it has become evident that many proteases play non-traditional roles. Accordingly, the number and the variety of the substrate repertoire of proteases are expected to be much larger than previously assumed. In line with such global profiling attempts, we present here a method for the prediction of natural substrates of endo proteases (human proteases used as an example) by employing short peptide sequences as specificity determinants. METHODOLOGY/PRINCIPAL FINDINGS: Our method incorporates specificity determinants unique to individual enzymes and physiologically relevant dual filters namely, solvent accessible surface area--a parameter dependent on protein three-dimensional structure and subcellular localization. By incorporating such hitherto unused principles in prediction methods, a novel ligand docking strategy to mimic substrate binding at the active site of the enzyme, and GO functions, we identify and perform subjective validation on putative substrates of matriptase and highlight new functions of the enzyme. Using relative solvent accessibility to rank order we show how new protease regulatory networks and enzyme cascades can be created. CONCLUSION: We believe that our physiologically relevant computational approach would be a very useful complementary method in the current day attempts to profile proteases (endo proteases in particular) and their substrates. In addition, by using functional annotations, we have demonstrated how normal and unknown functions of a protease can be envisaged. We have developed a network which can be integrated to create a proteolytic world. This network can in turn be extended to integrate other regulatory networks to build a system wide knowledge of the proteome.

Asunto(s)

Modelos Moleculares , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Análisis de Secuencia de Proteína/métodos , Bases de Datos de Proteínas , Furina/química , Furina/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Proteoma/química , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato , Trombina/química , Trombina/metabolismo

RESUMEN

Development of sessile organisms requires adaptation to an ever-changing environment. In order to respond quickly to these challenges, complex signaling mechanisms have evolved to facilitate cellular modifications. The importance of phospholipid-based signaling pathways in plants, as well as animals, has recently been gaining attention. Both the PLD and PLC pathways produce the signaling molecule PA, which modulates MTs, F-actin and endomembrane trafficking. We have examined the roles of the PLD signaling pathway during development of the marine brown alga Silvetia compressa. Zygotes were treated with 1- and 2-butanol, both of which activate the PLD enzyme. However, only 1-butanol competes with water as a transphosphatidylation substrate, at the expense of PA production. Interestingly, we found that 1- and 2-butanol both disrupted MT organization and thereby cell division, with 1-butanol being more potent. These findings question whether the effects of butyl alcohol treatment are due to lowered PA levels or activation of the PLD enzyme. Additionally, preliminary results show that inhibition of DAGK results in loss of centrosomal MTs and formation of cortical MT cages that are strikingly similar to those formed following 1-butanol treatment. These data suggest that perturbation of the PLD or PLC pathway leads to cortical stabilization and/or nucleation of MT arrays.