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Tumor organoids and tumors-on-chips can be built by placing patient-derived cells within an extracellular matrix (ECM) for personalized medicine. The ECM influences the tumor response, and understanding the ECM-tumor relationship is important before translating tumor-on-chips into clinics. In this work, we tuned the physical and structural characteristics of ECM in a bioprinted soft-tissue sarcoma microtissue. We formed 3D spheroids at a controlled size and encapsulated them into our gelatin methacryloyl (GelMA)-based bioink to make perfusable hydrogel-based microfluidic chips. We then demonstrated the scalability and customization flexibility of our hydrogel-based chip via engineering tools. A multiscale physical and structural data analysis suggested a relationship between cell invasion response and bioink characteristics. Tumor cell invasive behavior and focal adhesion properties were observed in response to varying polymer network densities of the GelMA-based bioink. Immunostaining assays and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) helped assess the bioactivity of the microtissue and measure the cell invasion. The RT-qPCR results showed higher expressions of HIF-1α, CD44, and MMP2 genes in a lower polymer density, highlighting the correlation between bioink structural porosity, ECM stiffness, and tumor spheroid response. In conclusion, this work is the first step in modeling STS tumor invasiveness in hydrogel-based microfluidic chips, and our tunable bioink may help reduce the variability of current tumor-on-chips. STATEMENT OF SIGNIFICANCE: We optimized an engineering protocol for making tumor spheroids at a controlled size, embedding spheroids into a gelatin-based matrix, and constructing a perfusable microfluidic device. A higher tumor invasion was observed in a low-stiffness matrix than a high-stiffness matrix. The physical characterizations revealed how the stiffness is controlled by the density of polymer chain networks and porosity. The biological assays revealed how the structural properties of the gelatin matrix and hypoxia in tumor progression impact cell invasion. The cell spheroids' responses underscore the importance of replicating physical and structural properties to mimic tumor response. This work can contribute to personalized medicine by making more effective, tailored cancer models.
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Myelination has evolved as a mechanism to ensure fast and efficient propagation of nerve impulses along axons. Within the central nervous system (CNS), myelination is carried out by highly specialized glial cells, oligodendrocytes. The formation of myelin is a prolonged aspect of CNS development that occurs well into adulthood in humans, continuing throughout life in response to injury or as a component of neuroplasticity. The timing of myelination is tightly tied to the generation of oligodendrocytes through the differentiation of their committed progenitors, oligodendrocyte precursor cells (OPCs), which reside throughout the developing and adult CNS. In this article, we summarize our current understanding of some of the signals and pathways that regulate the differentiation of OPCs, and thus the myelination of CNS axons.
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Diferenciación Celular , Vaina de Mielina , Oligodendroglía , Oligodendroglía/fisiología , Oligodendroglía/citología , Humanos , Animales , Vaina de Mielina/fisiología , Vaina de Mielina/metabolismo , Transducción de Señal , Sistema Nervioso Central/fisiología , Axones/fisiología , Axones/metabolismoRESUMEN
Myelin loss and oligodendrocyte death are well documented in patients with traumatic brain injury (TBI), as well as in experimental animal models after moderate-to-severe TBI. In comparison, mild TBI (mTBI) does not necessarily result in myelin loss or oligodendrocyte death, but causes structural alterations in the myelin. To gain more insight into the impact of mTBI on oligodendrocyte lineage in the adult brain, we subjected mice to mild lateral fluid percussion injury (mFPI) and characterized the early impact (1 and 3 days post-injury) on oligodendrocytes in the corpus callosum using multiple oligodendrocyte lineage markers (platelet-derived growth factor receptor [PDGFR]-α, glutathione S-transferase [GST]-π, CC1, breast carcinoma-amplified sequence 1 [BCAS1], myelin basic protein [MBP], myelin-associated glycoprotein [MAG], proteolipid protein [PLP], and FluoroMyelin™). Two regions of the corpus callosum in relation to the impact site were analyzed: areas near (focal) and anterior (distal) to the impact site. mFPI did not result in oligodendrocyte death in either the focal or distal corpus callosum, nor impact on oligodendrocyte precursors (PDGFR-α+) and GST-π+ oligodendrocyte numbers. In the focal but not distal corpus callosum, mFPI caused a decrease in CC1+ as well as BCAS1+ actively myelinating oligodendrocytes and reduced FluoroMyelin intensity without altering myelin protein expression (MBP, PLP, and MAG). Disruption in node-paranode organization and loss of Nav1.6+ nodes were observed in both the focal and distal regions, even in areas without obvious axonal damage. Altogether, our study shows regional differences in mature and myelinating oligodendrocyte in response to mFPI. Further, mFPI elicits a widespread impact on node-paranode organization that affects regions both close to and remotely located from the site of injury.
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Despite decades of research presenting insulin-like growth factor-1 receptor (IGF1R) as an attractive target for cancer therapy, IGF1R inhibitors ultimately failed in clinical trials. This was surprising due to the known cancer-promoting functions of IGF1R, including stimulation of cell invasion, proliferation, and survival. Discourse in the literature has acknowledged that a lack of patient stratification may have impacted the success of IGF1R-inhibitor trials. This argument alludes to the possibility that IGF1R function may be contingent on tumor type and cellular composition. Looking into the known roles of IGF1R, it becomes clear that this receptor interacts with a multitude of different proteins and even has tumor-suppressing functions. IGF1R is implicated in both cell-cell and cell-surface adhesion dynamics, and the effects of either IGF1R downregulation or pharmacological inhibition on cellular adhesion remain poorly understood. In turn, adhesion receptors modulate IGF1R signaling. In addition, our understanding of IGF1R function in tumor-associated immune and stromal cells is lacking, which could contribute to the overwhelming failure of IGF1R inhibitors in the clinic. In this review, we re-investigate clinical trial data to make connections between the failure of these drugs in human cancer patients and the understudied facets of IGF1R function. We describe lesser-known and potentially tumor-suppressive functions of IGF1R that include promoting cell-cell adhesion through E-cadherin, augmenting a pro-inflammatory macrophage phenotype, and stimulating B cells to produce immunoglobulins. We also highlight the important role of adhesion receptors in regulating IGF1R function, and we use this information to infer stratification criteria for selecting patients that might benefit from IGF1R inhibitors.
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Factor I del Crecimiento Similar a la Insulina , Neoplasias , Receptor IGF Tipo 1 , Humanos , Cadherinas/genética , Cadherinas/metabolismo , Cadherinas/farmacología , Línea Celular Tumoral , Proliferación Celular , Factor I del Crecimiento Similar a la Insulina/metabolismo , Integrinas , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transducción de SeñalRESUMEN
In the CNS, oligodendrocyte progenitor cells (OPCs) differentiate into mature oligodendrocytes to generate myelin, an essential component for normal nervous system function. OPC differentiation is driven by signaling pathways, such as mTOR, which functions in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), containing Raptor or Rictor, respectively. In the current studies, mTORC2 signaling was selectively deleted from OPCs in PDGFRα-Cre X Rictorfl/fl mice. This study examined developmental myelination in male and female mice, comparing the impact of mTORC2 deletion in the corpus callosum and spinal cord. In both regions, Rictor loss in OPCs resulted in early reduction in myelin RNAs and proteins. However, these deficits rapidly recovered in spinal cord, where normal myelin was noted at P21 and P45. By contrast, the losses in corpus callosum resulted in severe hypomyelination and increased unmyelinated axons. The hypomyelination may result from decreased oligodendrocytes in the corpus callosum, which persisted in animals as old as postnatal day 350. The current studies focus on uniquely altered signaling pathways following mTORC2 loss in developing oligodendrocytes. A major mTORC2 substrate is phospho-Akt-S473, which was significantly reduced throughout development in both corpus callosum and spinal cord at all ages measured, yet this had little impact in spinal cord. Loss of mTORC2 signaling resulted in decreased expression of actin regulators, such as gelsolin in corpus callosum, but only minimal loss in spinal cord. The current study establishes a regionally specific role for mTORC2 signaling in OPCs, particularly in the corpus callosum.SIGNIFICANCE STATEMENT mTORC1 and mTORC2 signaling has differential impact on myelination in the CNS. Numerous studies identify a role for mTORC1, but deletion of Rictor (mTORC2 signaling) in late-stage oligodendrocytes had little impact on myelination in the CNS. However, the current studies establish that deletion of mTORC2 signaling from oligodendrocyte progenitor cells results in reduced myelination of brain axons. These studies also establish a regional impact of mTORC2, with little change in spinal cord in these conditional Rictor deletion mice. Importantly, in both brain and spinal cord, mTORC2 downstream signaling targets were impacted by Rictor deletion. Yet, these signaling changes had little impact on myelination in spinal cord, while they resulted in long-term alterations in myelination in brain.
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Células Precursoras de Oligodendrocitos , Animales , Femenino , Masculino , Ratones , Diferenciación Celular/fisiología , Sistema Nervioso Central/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratones Noqueados , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Introduction: The acquisition of a metastatic phenotype is the critical event that determines patient survival from breast cancer. Several receptor tyrosine kinases have functions both in promoting and inhibiting metastasis in breast tumors. Although the insulin-like growth factor 1 receptor (IGF1R) has been considered a target for inhibition in breast cancer, low levels of IGF1R expression are associated with worse overall patient survival. Methods: To determine how reduced IGF1R impacts tumor phenotype in human breast cancers, we used weighted gene co-expression network analysis (WGCNA) of Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) patient data to identify gene modules associated with low IGF1R expression. We then compared these modules to single cell gene expression analyses and phenotypes of mouse mammary tumors with reduced IGF1R signaling or expression in a tumor model of triple negative breast cancer. Results: WGCNA from METABRIC data revealed gene modules specific to cell cycle, adhesion, and immune cell signaling that were inversely correlated with IGF1R expression in human breast cancers. Integration of human patient data with single cell sequencing data from mouse tumors revealed similar pathways necessary for promoting metastasis in basal-like mammary tumors with reduced signaling or expression of IGF1R. Functional analyses revealed the basis for the enhanced metastatic phenotype including alterations in E- and P-cadherins. Discussion: Human breast and mouse mammary tumors with reduced IGF1R are associated with upregulation of several pathways necessary for promoting metastasis supporting the conclusion that IGF1R normally helps maintain a metastasis suppressive tumor microenvironment. We further found that reduced IGF1R signaling in tumor epithelial cells dysregulates cadherin expression resulting in reduced cell adhesion.
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This protocol describes isolation and live-cell metabolic analysis of O4+ oligodendroglia from brain and spinal cord of postnatal mice. We have optimized existing protocols for O4+ isolation from neonatal brain and expanded the protocol to include isolation of highly viable oligodendroglia from spinal cords of postnatal mice up to 18 days of age. Isolated oligodendroglia can be used in multiple downstream analyses, and here we describe an optimized real-time metabolic assay using Agilent Seahorse Analyzer to measure mitochondrial respiration. For complete details on the use and execution of this protocol, please refer to Khandker et al. (2022).
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Oligodendroglía , Médula Espinal , Animales , Encéfalo/metabolismo , Ratones , Mitocondrias/metabolismo , Oligodendroglía/metabolismo , Consumo de Oxígeno , Médula Espinal/metabolismoRESUMEN
Historically, the body of literature surrounding the insulin-like growth factor type 1 receptor (IGF1R) has described a largely pro-tumorigenic role in breast cancer cells and in several transgenic or xenograft mouse models of breast cancer. Interestingly, however, more recent evidence has emerged that suggests an additional, previously undescribed, tumor and metastasis suppressive function for IGF1R in both human breast tumors and mammary oncogenesis in mice. These seemingly conflicting reports can be reconciled when considering what is currently known about IGF1R function in the context of tissue development and cancer as it relates to cellular growth, proliferation, and differentiation. In this mini review, we will summarize the currently existing data with a particular focus on mouse models that have been developed to study IGF1R function in mammary development, tumorigenesis, and metastasis in vivo and propose hypotheses for how both the tumor-promoting and tumor-suppressing schools of thought regarding IGF1R in these histological contexts are compatible.
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Neoplasias de la Mama , Transformación Celular Neoplásica , Animales , Neoplasias de la Mama/patología , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Receptor IGF Tipo 1RESUMEN
The insulin receptor (INSR) is an evolutionarily conserved signaling protein that regulates development and cellular metabolism. INSR signaling promotes neurogenesis in Drosophila; however, a specific role for the INSR in maintaining adult neural stem cells (NSCs) in mammals has not been investigated. We show that conditionally deleting the Insr gene in adult mouse NSCs reduces subventricular zone NSCs by â¼70% accompanied by a corresponding increase in progenitors. Insr deletion also produced hyposmia caused by aberrant olfactory bulb neurogenesis. Interestingly, hippocampal neurogenesis and hippocampal-dependent behaviors were unperturbed. Highly aggressive proneural and mesenchymal glioblastomas had high INSR/insulin-like growth factor (IGF) pathway gene expression, and isolated glioma stem cells had an aberrantly high ratio of INSR:IGF type 1 receptor. Moreover, INSR knockdown inhibited GBM tumorsphere growth. Altogether, these data demonstrate that the INSR is essential for a subset of normal NSCs, as well as for brain tumor stem cell self-renewal.
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Células Madre Adultas , Ventrículos Laterales/metabolismo , Células-Madre Neurales , Receptor de Insulina/metabolismo , Somatomedinas , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Ventrículos Laterales/citología , Ratones , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis , Somatomedinas/metabolismoRESUMEN
Brain and spinal cord oligodendroglia have distinct functional characteristics, and cell-autonomous loss of individual genes can result in different regional phenotypes. However, a molecular basis for these distinctions is unknown. Using single-cell analysis of oligodendroglia during developmental myelination, we demonstrate that brain and spinal cord precursors are transcriptionally distinct, defined predominantly by cholesterol biosynthesis. We further identify the mechanistic target of rapamycin (mTOR) as a major regulator promoting cholesterol biosynthesis in oligodendroglia. Oligodendroglia-specific loss of mTOR decreases cholesterol biosynthesis in both the brain and the spinal cord, but mTOR loss in spinal cord oligodendroglia has a greater impact on cholesterol biosynthesis, consistent with more pronounced deficits in developmental myelination. In the brain, mTOR loss results in a later adult myelin deficit, including oligodendrocyte death, spontaneous demyelination, and impaired axonal function, demonstrating that mTOR is required for myelin maintenance in the adult brain.
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Células Precursoras de Oligodendrocitos , Encéfalo/metabolismo , Diferenciación Celular/genética , Colesterol , Vaina de Mielina/metabolismo , Células Precursoras de Oligodendrocitos/metabolismo , Oligodendroglía/metabolismo , Médula Espinal/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Despite evidence for prominent metabolic dysfunction within multiple sclerosis (MS) lesions, the mechanisms controlling metabolic shifts in oligodendroglia are poorly understood. The cuprizone model of demyelination and remyelination is a valuable tool for assessing metabolic insult during oligodendrocyte death and myelin degradation, closely resembling the distal oligodendrogliopathy seen in Pattern III MS lesions. In this review we discuss how metabolic processes in oligodendrocytes are disrupted in both MS and the cuprizone model, as well as the evidence for mechanistic target of rapamycin (mTOR) signaling as a key regulator of oligodendroglial metabolic function and efficient remyelination.
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Enfermedades Desmielinizantes , Esclerosis Múltiple , Remielinización , Animales , Cuprizona/metabolismo , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/patología , Sirolimus/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The p70 ribosomal S6 kinases (p70 ribosomal S6 kinase 1 and p70 ribosomal S6 kinase 2) are downstream targets of the mechanistic target of rapamycin signalling pathway. p70 ribosomal S6 kinase 1 specifically has demonstrated functions in regulating cell size in Drosophila and in insulin-sensitive cell populations in mammals. Prior studies demonstrated that the mechanistic target of the rapamycin pathway promotes oligodendrocyte differentiation and developmental myelination; however, how the immediate downstream targets of mechanistic target of rapamycin regulate these processes has not been elucidated. Here, we tested the hypothesis that p70 ribosomal S6 kinase 1 regulates oligodendrocyte differentiation during developmental myelination and remyelination processes in the CNS. We demonstrate that p70 ribosomal S6 kinase activity peaks in oligodendrocyte lineage cells at the time when they transition to myelinating oligodendrocytes during developmental myelination in the mouse spinal cord. We further show p70 ribosomal S6 kinase activity in differentiating oligodendrocytes in acute demyelinating lesions induced by lysophosphatidylcholine injection or by experimental autoimmune encephalomyelitis in mice. In demyelinated lesions, the expression of the p70 ribosomal S6 kinase target, phosphorylated S6 ribosomal protein, was transient and highest in maturing oligodendrocytes. Interestingly, we also identified p70 ribosomal S6 kinase activity in oligodendrocyte lineage cells in active multiple sclerosis lesions. Consistent with its predicted function in promoting oligodendrocyte differentiation, we demonstrate that specifically inhibiting p70 ribosomal S6 kinase 1 in cultured oligodendrocyte precursor cells significantly impairs cell lineage progression and expression of myelin basic protein. Finally, we used zebrafish to show in vivo that inhibiting p70 ribosomal S6 kinase 1 function in oligodendroglial cells reduces their differentiation and the number of myelin internodes produced. These data reveal an essential function of p70 ribosomal S6 kinase 1 in promoting oligodendrocyte differentiation during development and remyelination across multiple species.
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In demyelinating diseases, such as multiple sclerosis, primary loss of myelin and subsequent neuronal degeneration throughout the CNS impair patient functionality. While the importance of mechanistic target of rapamycin (mTOR) signaling during developmental myelination is known, no studies have yet directly examined the function of mTOR signaling specifically in the oligodendrocyte (OL) lineage during remyelination. Here, we conditionally deleted Mtor from adult oligodendrocyte precursor cells (OPCs) using Ng2-CreERT in male adult mice to test its function in new OLs responsible for remyelination. During early remyelination after cuprizone-induced demyelination, mice lacking mTOR in adult OPCs had unchanged OL numbers but thinner myelin. Myelin thickness recovered by late-stage repair, suggesting a delay in myelin production when Mtor is deleted from adult OPCs. Surprisingly, loss of mTOR in OPCs had no effect on efficiency of remyelination after lysophosphatidylcholine lesions in either the spinal cord or corpus callosum, suggesting that mTOR signaling functions specifically in a pathway dysregulated by cuprizone to promote remyelination efficiency. We further determined that cuprizone and inhibition of mTOR cooperatively compromise metabolic function in primary rat OLs undergoing differentiation. Together, our results support the conclusion that mTOR signaling in OPCs is required to overcome the metabolic dysfunction in the cuprizone-demyelinated adult brain.SIGNIFICANCE STATEMENT Impaired remyelination by oligodendrocytes contributes to the progressive pathology in multiple sclerosis, so it is critical to identify mechanisms of improving remyelination. The goal of this study was to examine mechanistic target of rapamycin (mTOR) signaling in remyelination. Here, we provide evidence that mTOR signaling promotes efficient remyelination of the brain after cuprizone-mediated demyelination but has no effect on remyelination after lysophosphatidylcholine demyelination in the spinal cord or brain. We also present novel data revealing that mTOR inhibition and cuprizone treatment additively affect the metabolic profile of differentiating oligodendrocytes, supporting a mechanism for the observed remyelination delay. These data suggest that altered metabolic function may underlie failure of remyelination in multiple sclerosis lesions and that mTOR signaling may be of therapeutic potential for promoting remyelination.
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Encéfalo/metabolismo , Cuprizona/toxicidad , Células Precursoras de Oligodendrocitos/metabolismo , Remielinización/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Encéfalo/efectos de los fármacos , Quelantes/toxicidad , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas Sprague-Dawley , Remielinización/efectos de los fármacos , Serina-Treonina Quinasas TOR/genéticaRESUMEN
The actin cytoskeleton is crucial for oligodendrocyte differentiation and myelination. Here we show that p21-activated kinase 1 (PAK1), a well-known actin regulator, promotes oligodendrocyte morphologic change and myelin production in the CNS. A combination of in vitro and in vivo models demonstrated that PAK1 is expressed throughout the oligodendrocyte lineage with highest expression in differentiated oligodendrocytes. Inhibiting PAK1 early in oligodendrocyte development decreased oligodendrocyte morphologic complexity and altered F-actin spreading at the tips of oligodendrocyte progenitor cell processes. Constitutively activating AKT in oligodendrocytes in male and female mice, which leads to excessive myelin wrapping, increased PAK1 expression, suggesting an impact of PAK1 during active myelin wrapping. Furthermore, constitutively activating PAK1 in oligodendrocytes in zebrafish led to an increase in myelin internode length while inhibiting PAK1 during active myelination decreased internode length. As myelin parameters influence conduction velocity, these data suggest that PAK1 may influence communication within the CNS. These data support a model in which PAK1 is a positive regulator of CNS myelination.SIGNIFICANCE STATEMENT Myelin is a critical component of the CNS that provides metabolic support to neurons and also facilitates communication between cells in the CNS. Recent data demonstrate that actin dynamics drives myelin wrapping, but how actin is regulated during myelin wrapping is unknown. The authors investigate the role of the cytoskeletal modulator PAK1 during differentiation and myelination by oligodendrocytes, the myelinating cells of the CNS. They demonstrate that PAK1 promotes oligodendrocyte differentiation and myelination by modulating the cytoskeleton and thereby internode length, thus playing a critical role in the function of the CNS.
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Vaina de Mielina/metabolismo , Neurogénesis/fisiología , Oligodendroglía/citología , Oligodendroglía/metabolismo , Quinasas p21 Activadas/metabolismo , Animales , Diferenciación Celular/fisiología , Femenino , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Pez CebraRESUMEN
BACKGROUND: Musculoskeletal traumas are on the rise in the United States; however, limited studies are available to help trauma providers assess and treat concerns beyond the physical impact. Little is understood about the psychological, social, and spiritual factors that protect patients from adverse effects after a physical trauma or their experiences with each factor afterward. OBJECTIVE: This systematic review was conducted to investigate and review advancements in research related to risk and resiliency factors experienced by survivors of traumatic musculoskeletal injuries. The use of biopsychosocial-spiritual (BPS-S) framework and resiliency theory guided the analysis. METHODS: Researchers reviewed 1003 articles, but only seven met the search criteria. Due to the complexity and uniqueness of traumatic brain injuries, studies on that target population were excluded. RESULTS: Of the seven articles reviewed, three identified psychological protective factors that protect against negative health outcomes; three identified negative psychological, social, or spiritual outcomes; and none investigated social or spiritual health. CONCLUSIONS: There are significant gaps in the literature surrounding risk and resiliency factors related to the BPS-S health of musculoskeletal injury survivors.
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Lesiones Traumáticas del Encéfalo , Personas con Discapacidad , Necesidades y Demandas de Servicios de Salud , Humanos , Sobrevivientes , Estados UnidosRESUMEN
The ERK1/2 signaling pathway promotes myelin wrapping during development and remyelination, and sustained ERK1/2 activation in the oligodendrocyte (OL) lineage results in hypermyelination of the CNS. We therefore hypothesized that increased ERK1/2 signaling in the OL lineage would 1) protect against immune-mediated demyelination due to increased baseline myelin thickness and/or 2) promote enhanced remyelination and thus functional recovery after experimental autoimmune encephalomyelitis (EAE) induction. Cnp-Cre;Mek1DD-eGFP/+ mice that express a constitutively active form of MEK1 (the upstream activator of ERK1/2) in the OL lineage, exhibited a significant decrease in EAE clinical severity compared to controls. However, experiments using tamoxifen-inducible Plp-CreERT;Mek1DD-eGFP/+ or Pdgfrα-CreERT;Mek1DD-eGFP mice revealed this was not solely due to a protective or reparative effect resulting from MEK1DD expression specifically in the OL lineage. Because EAE is an immune-mediated disease, we examined Cnp-Cre;Mek1DD-eGFP/+ splenic immune cells for recombination. Surprisingly, GFP+ recombined CD19+ B-cells, CD11b+ monocytes, and CD3+ T-cells were noted when Cre expression was driven by the Cnp promoter. While ERK1/2 signaling in monocytes and T-cells is associated with proinflammatory activation, fewer studies have examined ERK1/2 signaling in B-cell populations. After in vitro stimulation, MEK1DD-expressing B-cells exhibited a 3-fold increase in CD138+ plasmablasts and a 5-fold increase in CD5+CD1dhi B-cells compared to controls. Stimulated MEK1DD-expressing B-cells also exhibited an upregulation of IL-10, known to suppress the initiation of EAE when produced by CD5+CD1dhi regulatory B-cells. Taken together, our data support the conclusion that sustained ERK1/2 activation in B-cells suppresses immune-mediated demyelination via increasing activation of regulatory B10 cells.
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2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/biosíntesis , Linfocitos B/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/prevención & control , Sistema de Señalización de MAP Quinasas/fisiología , Regiones Promotoras Genéticas/fisiología , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/inmunología , Animales , Linfocitos B/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
During differentiation, oligodendrocyte precursor cells (OPCs) extend a network of processes that make contact with axons and initiate myelination. Recent studies revealed that actin polymerization is required for initiation of myelination whereas actin depolymerization promotes myelin wrapping. Here, we used primary OPCs in culture isolated from neonatal rat cortices of both sexes and young male and female mice with oligodendrocyte-specific deletion of mechanistic target of rapamycin (mTOR) to demonstrate that mTOR regulates expression of specific cytoskeletal targets and actin reorganization in oligodendrocytes during developmental myelination. Loss or inhibition of mTOR reduced expression of profilin2 and ARPC3, actin polymerizing factors, and elevated levels of active cofilin, which mediates actin depolymerization. The deficits in actin polymerization were revealed in reduced phalloidin and deficits in oligodendrocyte cellular branching complexity at the peak of morphologic differentiation and a delay in initiation of myelination. We further show a critical role for mTOR in expression and localization of myelin basic protein (Mbp) mRNA and MBP protein to the cellular processes where it is necessary at the myelin membrane for axon wrapping. Mbp mRNA transport deficits were confirmed by single molecule RNA FISH. Moreover, expression of the kinesin family member 1B, an Mbp mRNA transport protein, was reduced in CC1+ cells in the mTOR cKO and in mTOR inhibited oligodendrocytes undergoing differentiation in vitro These data support the conclusion that mTOR regulates both initiation of myelination and axon wrapping by targeting cytoskeletal reorganization and MBP localization to oligodendrocyte processes.SIGNIFICANCE STATEMENT Myelination is essential for normal CNS development and adult axon preservation and function. The mechanistic target of rapamycin (mTOR) signaling pathway has been implicated in promoting CNS myelination; however, there is a gap in our understanding of the mechanisms by which mTOR promotes developmental myelination through regulating specific downstream targets. Here, we present evidence that mTOR promotes the initiation of myelination through regulating specific cytoskeletal targets and cellular process expansion by oligodendrocyte precursor cells as well as expression and cellular localization of myelin basic protein.
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Citoesqueleto/genética , Vaina de Mielina/genética , Oligodendroglía , Serina-Treonina Quinasas TOR/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Axones , Diferenciación Celular/genética , Cinesinas/genética , Cinesinas/metabolismo , Ratones , Ratones Noqueados , Proteína Básica de Mielina/genética , Proteína Proteolipídica de la Mielina/genética , Proteína Proteolipídica de la Mielina/metabolismo , Oligodendroglía/ultraestructura , Ratas , Ratas Sprague-Dawley , Células Madre , Serina-Treonina Quinasas TOR/genética , Pez CebraRESUMEN
Oligodendrocyte precursor cells (OPCs) differentiate and mature into oligodendrocytes, which produce myelin in the central nervous system. Prior studies have shown that the mechanistic target of rapamycin (mTOR) is necessary for proper myelination of the mouse spinal cord and that bone morphogenetic protein (BMP) signaling inhibits oligodendrocyte differentiation, in part by promoting expression of inhibitor of DNA binding 2 (Id2). Here we provide evidence that mTOR functions specifically in the transition from early stage OPC to immature oligodendrocyte by downregulating BMP signaling during postnatal spinal cord development. When mTOR is deleted from the oligodendrocyte lineage, expression of the FK506 binding protein 1A (FKBP12), a suppressor of BMP receptor activity, is reduced, downstream Smad activity is increased and Id2 expression is elevated. Additionally, mTOR inhibition with rapamycin in differentiating OPCs alters the transcriptional complex present at the Id2 promoter. Deletion of mTOR in oligodendroglia in vivo resulted in fewer late stage OPCs and fewer newly formed oligodendrocytes in the spinal cord with no effect on OPC proliferation or cell cycle exit. Finally, we demonstrate that inhibiting BMP signaling rescues the rapamycin-induced deficit in myelin protein expression. We conclude that mTOR promotes early oligodendrocyte differentiation by suppressing BMP signaling in OPCs.
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Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/fisiología , Oligodendroglía/metabolismo , Sirolimus/metabolismo , Médula Espinal/metabolismo , Animales , Ciclo Celular/fisiología , Sistema Nervioso Central/metabolismo , Ratones , Proteínas de la Mielina/metabolismo , Neurogénesis , Transducción de Señal/fisiología , Células Madre/citología , Células Madre/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The insulin receptor gene (INSR) undergoes alternative splicing to give rise to two functionally related, but also distinct, isoforms IR-A and IR-B, which dictate proliferative and metabolic regulations, respectively. Previous studies identified the RNA-binding protein CUGBP1 as a key regulator of INSR splicing. In this study, we show that the differential splicing of INSR occurs more frequently in breast cancer than in non-tumor breast tissues. In breast cancer cell lines, the IR-A:IR-B ratio varies in different molecular subtypes, knockdown or overexpression of CUGBP1 gene in breast cancer cells altered IR-A:IR-B ratio through modulation of IR-A expression, thereby reversed or enhanced the insulin-induced oncogenic behavior of breast cancer cells, respectively. Our data revealed the predominant mitogenic role of IR-A isoform in breast cancer and depicted a novel interplay between INSR and CUGBP1, implicating CUGBP1 and IR-A isoform as the potential therapeutic targets and biomarkers for breast cancer.
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Antígenos CD/genética , Neoplasias de la Mama/patología , Proteínas CELF1/metabolismo , Empalme del ARN , Receptor ErbB-2/metabolismo , Receptor de Insulina/genética , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas CELF1/genética , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Pronóstico , Isoformas de ProteínasRESUMEN
Tissue-specific stem cells have unique properties and growth requirements, but a small set of juxtacrine and paracrine signals have been identified that are required across multiple niches. Whereas insulin-like growth factor II (IGF-II) is necessary for prenatal growth, its role in adult stem cell physiology is largely unknown. We show that loss of Igf2 in adult mice resulted in a â¼50% reduction in slowly dividing, label-retaining cells in the two regions of the brain that harbor neural stem cells. Concordantly, induced Igf2 deletion increased newly generated neurons in the olfactory bulb accompanied by hyposmia, and caused impairments in learning and memory and increased anxiety. Induced Igf2 deletion also resulted in rapid loss of stem and progenitor cells in the crypts of Lieberkühn, leading to body-weight loss and lethality and the inability to produce organoids in vitro. These data demonstrate that IGF-II is critical for multiple adult stem cell niches.