Characterization of a murine mixed neuron-glia model and cellular responses to regulatory T cell-derived factors.
ABSTRACT: One of the unmet clinical needs in demyelinating diseases such as Multiple Sclerosis (MS) is to provide therapies that actively enhance the process of myelin regeneration (remyelination) in the central nervous system (CNS). Oligodendrocytes, the myelinating cells of the CNS, play a central role in remyelination and originate from oligodendrocyte progenitor cells (OPCs). We recently showed that depletion of regulatory T cells (Treg) impairs remyelination in vivo, and that Treg-secreted factors directly enhance oligodendrocyte differentiation. Here we aim to further characterize the dynamics of Treg-enhanced oligodendrocyte differentiation as well as elucidate the cellular components of a murine mixed neuron-glia model. Murine mixed neuron-glia cultures were generated from P2-7 C57BL/6 mice and characterized for percentage of neuronal and glial cell populations prior to treatment at 7 days in vitro (div) as well as after treatment with Treg-conditioned media at multiple timepoints up to 12 div. Mixed neuron-glia cultures consisted of approximately 30% oligodendroglial lineage cells, 20% neurons and 10% microglia. Furthermore, a full layer of astrocytes, that could not be quantified, was present. Treatment with Treg-conditioned media enhanced the proportion of MBP+ oligodendrocytes and decreased the proportion of PDGFR?+ OPCs, but did not affect OPC proliferation or survival. Treg-enhanced oligodendrocyte differentiation was not caused by Treg polarizing factors, was dependent on the number of activation cycles Treg underwent and was robustly achieved by using 5% conditioned media. These studies provide in-depth characterization of a murine mixed neuron-glia model as well as further insights into the dynamics of Treg-enhanced oligodendrocyte differentiation.
Project description:The differentiation and maturation of oligodendrocyte precursor cells (OPCs) is essential for myelination and remyelination in the CNS. The failure of OPCs to achieve terminal differentiation in demyelinating lesions often results in unsuccessful remyelination in a variety of human demyelinating diseases. However, the molecular mechanisms controlling OPC differentiation under pathological conditions remain largely unknown. Myt1L (myelin transcription factor 1-like), mainly expressed in neurons, has been associated with intellectual disability, schizophrenia, and depression. In the present study, we found that Myt1L was expressed in oligodendrocyte lineage cells during myelination and remyelination. The expression level of Myt1L in neuron/glia antigen 2-positive (NG2+) OPCs was significantly higher than that in mature CC1+ oligodendrocytes. In primary cultured OPCs, overexpression of Myt1L promoted, while knockdown inhibited OPC differentiation. Moreover, Myt1L was potently involved in promoting remyelination after lysolecithin-induced demyelination in vivo. ChIP assays showed that Myt1L bound to the promoter of Olig1 and transcriptionally regulated Olig1 expression. Taken together, our findings demonstrate that Myt1L is an essential regulator of OPC differentiation, thereby supporting Myt1L as a potential therapeutic target for demyelinating diseases.
Project description:Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.
Project description:Promotion of remyelination is a major goal in treating demyelinating diseases such as multiple sclerosis (MS). The recombinant human monoclonal IgM, rHIgM22, targets myelin and oligodendrocytes (OLs) and promotes remyelination in animal models of MS. It is unclear whether rHIgM22-mediated stimulation of lesion repair is due to promotion of oligodendrocyte progenitor cell (OPC) proliferation and survival, OPC differentiation into myelinating OLs or protection of mature OLs. It is also unknown whether astrocytes or microglia play a functional role in IgM-mediated lesion repair.We assessed the effect of rHIgM22 on cell proliferation in mixed CNS glial and OPC cultures by tritiated-thymidine uptake and by double-label immunocytochemistry using the proliferation marker, Ki-67. Antibody-mediated signaling events, OPC differentiation and OPC survival were investigated and quantified by Western blots.rHIgM22 stimulates OPC proliferation in mixed glial cultures but not in purified OPCs. There is no proliferative response in astrocytes or microglia. rHIgM22 activates PDGF?R in OPCs in mixed glial cultures. Blocking PDGFR-kinase inhibits rHIgM22-mediated OPC proliferation in mixed glia. We confirm in isolated OPCs that rHIgM22-mediated anti-apoptotic signaling and inhibition of OPC differentiation requires PDGF and FGF-2. We observed no IgM-mediated effect in mature OLs in the absence of PDGF and FGF-2.Stimulation of OPC proliferation by rHIgM22 depends on co-stimulatory astrocytic and/or microglial factors. We demonstrate that rHIgM22-mediated activation of PDGF?R is required for stimulation of OPC proliferation. We propose that rHIgM22 lowers the PDGF threshold required for OPC proliferation and protection, which can result in remyelination of CNS lesions.
Project description:Muscarinic receptor antagonists act as potent inducers of oligodendrocyte differentiation and accelerate remyelination. However, the use of muscarinic antagonists in the clinic is limited by poor understanding of the operant receptor subtype, and questions regarding possible species differences between rodents and humans. Based on high selective expression in human oligodendrocyte progenitor cells (OPCs), we hypothesized that M3R is the functionally relevant receptor. Lentiviral M3R knockdown in human primary CD140a/PDGF?R+ OPCs resulted in enhanced differentiation in vitro and substantially reduced the calcium response following muscarinic agonist treatment. Importantly, following transplantation in hypomyelinating shiverer/rag2 mice, M3R knockdown improved remyelination by human OPCs. Furthermore, conditional M3R ablation in adult NG2-expressing OPCs increased oligodendrocyte differentiation and led to improved spontaneous remyelination in mice. Together, we demonstrate that M3R receptor mediates muscarinic signaling in human OPCs that act to delay differentiation and remyelination, suggesting that M3 receptors are viable targets for human demyelinating disease.SIGNIFICANCE STATEMENT The identification of drug targets aimed at improving remyelination in patients with demyelination disease is a key step in development of effective regenerative therapies to treat diseases, such as multiple sclerosis. Muscarinic receptor antagonists have been identified as effective potentiators of remyelination, but the receptor subtypes that mediate these receptors are unclear. In this study, we show that genetic M3R ablation in both mouse and human cells results in improved remyelination and is mediated by acceleration of oligodendrocyte commitment from oligodendrocyte progenitor cells. Therefore, M3R represents an attractive target for induced remyelination in human disease.
Project description:Regeneration of CNS myelin involves differentiation of oligodendrocytes from oligodendrocyte progenitor cells. In multiple sclerosis, remyelination can fail despite abundant oligodendrocyte progenitor cells, suggesting impairment of oligodendrocyte differentiation. T cells infiltrate the CNS in multiple sclerosis, yet little is known about T cell functions in remyelination. We report that regulatory T cells (Treg) promote oligodendrocyte differentiation and (re)myelination. Treg-deficient mice exhibited substantially impaired remyelination and oligodendrocyte differentiation, which was rescued by adoptive transfer of Treg. In brain slice cultures, Treg accelerated developmental myelination and remyelination, even in the absence of overt inflammation. Treg directly promoted oligodendrocyte progenitor cell differentiation and myelination in vitro. We identified CCN3 as a Treg-derived mediator of oligodendrocyte differentiation and myelination in vitro. These findings reveal a new regenerative function of Treg in the CNS, distinct from immunomodulation. Although the cells were originally named 'Treg' to reflect immunoregulatory roles, this also captures emerging, regenerative Treg functions.
Project description:Demyelinated lesions of the central nervous system are characteristic for multiple sclerosis (MS). Remyelination is not very effective, particular at later stages of the disease, which results in a chronic neurodegenerative character with worsening of symptoms. Previously, we have shown that the enzyme Tissue Transglutaminase (TG2) is downregulated upon differentiation of oligodendrocyte progenitor cells (OPCs) into myelin-forming oligodendrocytes and that TG2 knock-out mice lag behind in remyelination after cuprizone-induced demyelination. Here, we examined whether astrocytic or oligodendroglial TG2 affects OPCs in a cell-specific manner to modulate their differentiation, and therefore myelination. Our findings indicate that human TG2-expressing astrocytes did not modulate OPC differentiation and myelination. In contrast, persistent TG2 expression upon OPC maturation or exogenously added recombinant TG2 accelerated OPC differentiation and myelin membrane formation. Continuous exposure of recombinant TG2 to OPCs at different consecutive developmental stages, however, decreased OPC differentiation and myelin membrane formation, while it enhanced myelination in dorsal root ganglion neuron-OPC co-cultures. In MS lesions, TG2 is absent in OPCs, while human OPCs show TG2 immunoreactivity during brain development. Exposure to the MS-relevant pro-inflammatory cytokine IFN-? increased TG2 expression in OPCs and prolonged expression of endogenous TG2 upon differentiation. However, despite the increased TG2 levels, OPC maturation was not accelerated, indicating that TG2-mediated OPC differentiation may be counteracted by other pathways. Together, our data show that TG2, either endogenously expressed, or exogenously supplied to OPCs, accelerates early OPC differentiation. A better understanding of the role of TG2 in the OPC differentiation process during MS is of therapeutic interest to overcome remyelination failure.
Project description:Oligodendrocyte precursor cells (OPCs) have the ability to repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. Recent evidence suggests that miR-219 helps regulate the differentiation of OPCs into oligodendrocytes. We performed oligodendrocyte differentiation studies using miR-219-overexpressing mouse embryonic stem cells (miR219-mESCs). The self-renewal and multiple differentiation properties of miR219-mESCs were analyzed by the expression of the stage-specific cell markers Nanog, Oct4, nestin, musashi1, GFAP, Tuj1 and O4. MiR-219 accelerated the differentiation of mESC-derived neural precursor cells (NPCs) into OPCs. We further transplanted OPCs derived from miR219-mESCs (miR219-OPCs) into cuprizone-induced chronically demyelinated mice to observe remyelination, which resulted in well-contained oligodendrocyte grafts that migrated along the corpus callosum and matured to express myelin basic protein (MBP). Ultrastructural studies further confirmed the presence of new myelin sheaths. Improved cognitive function in these mice was confirmed by behavioral tests. Importantly, the transplanted miR219-OPCs induced the proliferation of endogenous NPCs. In conclusion, these data demonstrate that miR-219 rapidly transforms mESCs into oligodendrocyte lineage cells and that the transplantation of miR219-OPCs not only promotes remyelination and improves cognitive function but also enhances the proliferation of host endogenous NPCs following chronic demyelination. These results support the potential of a therapeutic role for miR-219 in demyelinating diseases.
Project description:Cells of the oligodendrocyte lineage, which form the myelinating glia of the vertebrate central nervous system, undergo a stepwise developmental progression entailing specification from neuroepithelial precursors, proliferation, migration to expand and distribute the population, and differentiation to ensheath axons with myelin. Understanding the genetic mechanisms that regulate each of these steps during development is important, because this might lead to therapies to promote remyelination following neural injury or disease. Genetic studies in mice indicated that the Sox10 transcription factor is required during the differentiation stage to promote myelin gene expression. However, whether Sox10 also promotes other features of oligodendroctye differentiation remained unknown. In this study, we used time-lapse imaging to investigate the behavior and fates of oligodendrocyte lineage cells in zebrafish embryos and larvae that lacked Sox10 function. This revealed that the myelinating subset of oligodendrocyte progenitor cells (OPCs) migrates, divides, and wraps axons normally, but then dies. Nonmyelinating oligodendrocyte progenitors divided more frequently, maintaining a normal population size. New oligodendrocytes produced by these progenitors wrapped axons and survived, but did not express myelin genes at high levels. We conclude that, in addition to promoting myelin gene expression, Sox10 function is necessary for the survival of myelinating oligodedrocytes subsequent to axon wrapping but is not required for the survival of nonmyelinating OPCs.
Project description:Although the mammalian target of rapamycin (mTOR) is an essential regulator of developmental oligodendrocyte differentiation and myelination, oligodendrocyte-specific deletion of tuberous sclerosis complex (TSC), a major upstream inhibitor of mTOR, surprisingly also leads to hypomyelination during CNS development. However, the function of TSC has not been studied in the context of remyelination. Here, we used the inducible Cre-lox system to study the function of TSC in the remyelination of a focal, lysolecithin-demyelinated lesion in adult male mice. Using two different mouse models in which Tsc1 is deleted by Cre expression in oligodendrocyte progenitor cells (OPCs) or in premyelinating oligodendrocytes, we reveal that deletion of Tsc1 affects oligodendroglia differently depending on the stage of the oligodendrocyte lineage. Tsc1 deletion from NG2+ OPCs accelerated remyelination. Conversely, Tsc1 deletion from proteolipid protein (PLP)-positive oligodendrocytes slowed remyelination. Contrary to developmental myelination, there were no changes in OPC or oligodendrocyte numbers in either model. Our findings reveal a complex role for TSC in oligodendrocytes during remyelination in which the timing of Tsc1 deletion is a critical determinant of its effect on remyelination. Moreover, our findings suggest that TSC has different functions in developmental myelination and remyelination.SIGNIFICANCE STATEMENT Myelin loss in demyelinating disorders such as multiple sclerosis results in disability due to loss of axon conductance and axon damage. Encouragingly, the nervous system is capable of spontaneous remyelination, but this regenerative process often fails. Many chronically demyelinated lesions have oligodendrocyte progenitor cells (OPCs) within their borders. It is thus of great interest to elucidate mechanisms by which we might enhance endogenous remyelination. Here, we provide evidence that deletion of Tsc1 from OPCs, but not differentiating oligodendrocytes, is beneficial to remyelination. This finding contrasts with the loss of oligodendroglia and hypomyelination seen with Tsc1 or Tsc2 deletion in the oligodendrocyte lineage during CNS development and points to important differences in the regulation of developmental myelination and remyelination.
Project description:Chondroitin sulfate proteoglycans (CSPGs), which are enriched in demyelinating plaques in neurodegenerative diseases, such as multiple sclerosis (MS), impair remyelination by inhibiting the migration and differentiation of oligodendrocyte precursor cells (OPCs) in the central nervous system (CNS). We herein show that protamine (PRM, also known as a heparin antagonist) effectively neutralizes the inhibitory activities of CSPGs, thereby enhancing OPC differentiation and (re)myelination in mice. Cell-based assays using mouse OPC-like OL1 cells revealed that the PRM treatment exerted masking effects on extracellular CSPGs and improved oligodendrocyte differentiation on inhibitory CSPG-coated substrates. PRM also bound to the extracellular region of protein tyrosine phosphatase receptor type Z (PTPRZ), a membrane-spanning CSPG predominantly expressed in OPCs, and functioned as a ligand mimetic of PTPRZ, thereby suppressing its negative regulatory activity on oligodendrocyte differentiation. In primary cultures, the differentiation of OPCs from wild-type and Ptprz-deficient mice was equally enhanced by PRM. Moreover, the intranasal administration of PRM accelerated myelination in the developing mouse brain, and its intracerebroventricular administration stimulated remyelination after cuprizone-induced demyelination. These results indicate that PRM has CSPG-neutralizing activity which promotes oligodendrocyte differentiation under developmental and morbid conditions.