Oligodendroglial Progenitor Cells Research Articles

Build muscles and protect myelin

Abstract Multiple sclerosis (MS) is a chronic and debilitating autoimmune disease of the central nervous system (CNS) in which a CNS-driven immune response destroys myelin, leading to wide range of symptoms including numbness and tingling, vision problems, mobility impairment, etc. Oligodendrocytes are the myelinating cells in the CNS, which are generated from oligodendroglial progenitor cells (OPCs) via differentiation. However, for multiple reasons, OPCs fail to differentiate to oligodendrocytes in MS and as a result, stimulating the differentiation of OPCs to oligodendrocytes is considered beneficial for MS. The β-hydroxy β-methylbutyrate (HMB) is a widely-used muscle-building supplement in human and recently it has been shown that low-dose HMB is capable of stimulating the differentiation of cultured OPCs to oligodendrocytes for remyelination. Moreover, other causes of autoimmune demyelination are the decrease and/or suppression of Foxp3-expressing anti-autoimmune regulatory T cells (Tregs) and upregulation of autoimmune T-helper 1(Th1) and Th17 cells. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS in which the autoimmune demyelination is nicely visible. It has been reported that in EAE mice, oral HMB upregulates Tregs and decreases Th1 and Th17 responses, leading to remyelination in the CNS. Here, we analyze these newly-described features of HMB, highlighting the putative promyelinating nature of this supplement.

CNSC-03. LINKING CELL FATE DETERMINATION IN THE EMBRYONIC FOREBRAIN TO PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND Pediatric high-grade gliomas are treatment resistant, usually fatal cancers encompassing diffuse midline glioma, K27-altered (DMG), and diffuse hemispheric glioma, G34R/V (DHG). Most patients harbour canonical and non-canonical mutations in histone variants H3.1 and H3.3, leading to global epigenetic dysregulation and stalled differentiation states as oligodendroglial progenitor cells (OPC) in DMG and GABAergic interneuron precursors in DHG. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes GABAergic interneuron cell fate and migration during early neurogenesis. Methods & Results Our lab has shown DLX2 promoter occupancy of Gad1/Gad2, and early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo. Co-expression of DLX2 with these target sequences reduces reporter gene expression in vitro, and the Dlx1/Dlx2 double knockout (DKO) mouse showed increases in Olig2, Nkx2.2, and Plp-1 expression in vivo. Transient over-expression of a Dlx2-GFP construct into murine DMG cells resulted in significant increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3 and loss of H3K27 acetylation, as well as a reduction in migration, invasion, and colony formation in vitro. To validate in human pHGG, we first probed an RNA-seq database containing 62 patient-derived pHGG cell lines which demonstrated an inverse correlation between DLX2 expression and OLIG1/2. Cell fate changes in DHG cell lines were evaluated by knockout of DLX2 using CRISPR/Cas9. Concurrently, OLIG1/2-expressing DMG cell lines underwent stable transfection of a DLX2-mCherry overexpression construct in conjunction with a SOX2-BFP overexpression construct, confirmed by qPCR and immunoblot. Analysis of histone H3 marks, K27M, and OPC signature genes will be compared to OPC genes from Dlx1/Dlx2 DKO mouse ganglionic eminences. CONCLUSIONS Manipulation of DLX2 in pHGG provides a model system for assessing the contribution of a homeobox factor to cell fate decisions regulating differentiation and tumorigenicity.

Muscle-building supplement β-hydroxy β-methylbutyrate stimulates the maturation of oligodendroglial progenitor cells to oligodendrocytes.

Oligodendrocytes are the myelinating cells in the CNS and multiple sclerosis (MS) is a demyelinating disorder that is characterized by progressive loss of myelin. Although oligodendroglial progenitor cells (OPCs) should be differentiated into oligodendrocytes, for multiple reasons, OPCs fail to differentiate into oligodendrocytes in MS. Therefore, increasing the maturation of OPCs to oligodendrocytes may be of therapeutic benefit for MS. The β-hydroxy β-methylbutyrate (HMB) is a muscle-building supplement in humans and this study underlines the importance of HMB in stimulating the maturation of OPCs to oligodendrocytes. HMB treatment upregulated the expression of different maturation markers including PLP, MBP, and MOG in cultured OPCs. Double-label immunofluorescence followed by immunoblot analyses confirmed the upregulation of OPC maturation by HMB. While investigating mechanisms, we found that HMB increased the maturation of OPCs isolated from peroxisome proliferator-activated receptor β-/- (PPARβ-/-) mice, but not PPARα-/- mice. Similarly, GW6471 (an antagonist of PPARα), but not GSK0660 (an antagonist of PPARβ), inhibited HMB-induced maturation of OPCs. GW9662, a specific inhibitor of PPARγ, also could not inhibit HMB-mediated stimulation of OPC maturation. Furthermore, PPARα agonist GW7647, but neither PPARβ agonist GW0742 nor PPARγ agonist GW1929, alone increased the maturation of OPCs. Finally, HMB treatment of OPCs led to the recruitment of PPARα, but neither PPARβ nor PPARγ, to the PLP gene promoter. These results suggest that HMB stimulates the maturation of OPCs via PPARα and that HMB may have therapeutic prospects in remyelination.

Oligodeoxynucleotide IMT504: Effects on Central Nervous System Repair Following Demyelination.

Multiple sclerosis (MS) is an immune-mediated central nervous system (CNS) disease characterized by demyelination resulting from oligodendrocyte loss and inflammation. Cuprizone (CPZ) administration experimentally replicates MS pattern-III lesions, generating an inflammatory response through microgliosis and astrogliosis. Potentially remyelinating agents include oligodeoxynucleotides (ODN) with a specific immunomodulatory sequence consisting of the active motif PyNTTTTGT. In this work, the remyelinating effects of ODN IMT504 were evaluated through immunohistochemistry and qPCR analyses in a rat CPZ-induced demyelination model. Subcutaneous IMT504 administration exacerbated the pro-inflammatory response to demyelination and accelerated the transition to an anti-inflammatory state. IMT504 reduced microgliosis in general and the number of phagocytic microglia in particular and expanded the population of oligodendroglial progenitor cells (OPCs), later reflected in an increase in mature oligodendrocytes. The intracranial injection of IMT504 and intravenous inoculation of IMT504-treated B lymphocytes rendered comparable results. Altogether, these findings unveil potentially beneficial properties of IMT504 in the regulation of neuroinflammation and oligodendrogenesis, which may aid the development of therapies for demyelinating diseases such as MS.

MODL-12. REGULATION OF OLIGODENDROGLIAL PROGENITOR CELL (OPC) CELL FATE IN DIFFUSE MIDLINE GLIOMA (DMG) BY THE HOMEOBOX TRANSCRIPTION FACTOR DLX2

Abstract Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. 80% of patients with DMG harbor K27M mutations in canonical histone variants H3.3 and H3.1, leading to global epigenetic dysregulation and an oligodendroglial progenitor cell (OPC) transcriptional profile. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes a GABAergic interneuron cell fate during neurogenesis. Our lab has previously identified DLX2 transcription factor occupancy of genes required for OPC differentiation in vivo (Olig2, Nkx2.2, Myt1, Plp1), and shown that co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. Likewise, increased expression of Olig2, Nkx2.2, and Plp-1 are observed in a Dlx1/Dlx2 double knockout (DKO) mouse model. Transient overexpression of a Dlx2-GFP construct into cells of a genetically engineered mouse model (GEMM) of DMG resulted in increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, and a reduction in migration, invasion, and colony formation in vitro. Inverse expression of DLX2 and OLIG1/2 was observed in a bulk RNAseq database of 45 human DMG cell lines. We have established a pipeline to evaluate cell fate changes following changes in DLX2 expression levels. OLIG1/2high/DLX2low DMG expressing cell lines underwent stable transfection of a DLX2-mCherry overexpression construct. Successful alteration of DLX2 expression was confirmed by qPCR and immunoblot in knockout and overexpressing human DMG cell lines. Analysis of histone marks (H3K27me3, H3K27ac), K27M, and OPC signature genes will be presented with comparison of altered OPC genes from the DKO mouse as determined by RNA-seq. The manipulation of DLX2 expression in human DMG cells in vitro and in vivo provides a model system for assessing the contribution of a homeobox transcription factor required for neurogenesis to cell fate regulation and effects on tumorigenicity in DMG.

Differential activity of transcription factor Sox9 in early and adult oligodendroglial progenitor cells.

The high-mobility-group domain-containing transcription factor Sox9 confers glial competence to neuroepithelial precursors in the developing central nervous system and is an important determinant of astroglial and oligodendroglial specification. In oligodendroglial cells, it remains expressed in oligodendrocyte progenitor cells (OPCs) of the developing nervous system, but is shut off in differentiating oligodendrocytes as well as in OPCs that persist in the adult nervous system. To better understand the role of Sox9 in OPCs, we generated mouse models that allowed oligodendroglial expression of a Sox9 transgene during development or in the adult. With transgene expression beginning in the last trimester of pregnancy, the number of OPCs increased dramatically, followed by comparable gains in the number of pre-myelinating and myelinating oligodendrocytes as assessed by marker gene expression. This argues that Sox9 boosts oligodendrogenesis during ontogenetic development at all stages, including terminal oligodendrocyte differentiation. When Sox9 transgene expression started in the adult, many transgene-expressing OPCs failed to maintain their progenitor cell identity and instead converted into myelinating oligodendrocytes. As infrequent and inefficient differentiation of adult OPCs is one of the main obstacles to effective remyelination in demyelinating diseases such as Multiple Sclerosis, increased Sox9 levels in adult OPCs may substantially increase their remyelination capacity.

The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.

Divergence between Neuronal and Oligodendroglial Cell Fate, in Postnatal Brain Neural Stem Cells, Leads to Divergent Properties in Polymorphic In Vitro Assays.

Two main stem cell pools exist in the postnatal mammalian brain that, although they share some “stemness” properties, also exhibit significant differences. Multipotent neural stem cells survive within specialized microenvironments, called niches, and they are vulnerable to ageing. Oligodendroglial lineage-restricted progenitor cells are widely distributed in the brain parenchyma and are more resistant to the effects of ageing. Here, we create polymorphic neural stem cell cultures and allow cells to progress towards the neuronal and the oligodendroglial lineage. We show that the divergence of cell fate is accompanied by a divergence in the properties of progenitors, which reflects their adaptation to life in the niche or the parenchyma. Neurogenesis shows significant spatial restrictions and a dependence on laminin, a major niche component, while oligodendrogenesis shows none of these constraints. Furthermore, the blocking of integrin-β1 leads to opposing effects, reducing neurogenesis and enhancing oligodendrogenesis. Therefore, polymorphic neural stem cell assays can be used to investigate the divergence of postnatal brain stem cells and also to predict the in vivo effects of potential therapeutic molecules targeting stem and progenitor cells, as we do for the microneurotrophin BNN-20.

Temporal and spatial evolution of various functional neurons during demyelination induced by cuprizone.

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS). Here we report the temporal and spatial evolution of various functional neurons during demyelination in a cuprizone (CPZ)-induced mouse model. CPZ did not significantly induce the damage of axons and neurons after 2 wk of feeding. However, after 4-6 wk of CPZ feeding, axons and neurons were markedly reduced in the cortex, posterior thalamic nuclear group, and hippocampus. Simultaneously, the expression of TPH+ tryptophan neurons and VGLUT1+ glutamate neurons was obviously decreased, and the expression of TH+ dopaminergic neurons was slightly decreased in the tail part of the substantia nigra striatum, whereas the number of ChAT+ cholinergic neurons was not significantly different in the brain. In the second week of feeding, CPZ caused a higher level of glutamate secretion and upregulated the expression of EAAT2 on astrocytes, which should contribute to rapid and sufficient glutamate uptake and removal. This finding reveals that astrocyte-driven glutamate reuptake protected the CNS from excitotoxicity by rapid reuptake of glutamate in 4-6 wk of CPZ feeding. At this stage, although NG2+ oligodendroglia progenitor cells (OPCs) were enhanced in the demyelination foci, the myelin sheath was still absent. In conclusion, we comprehensively observed the temporal and spatial evolution of various functional neurons. Our results will assist with understanding how demyelination affects neurons during CPZ-induced demyelination and provide novel information for neuroprotection in myelin regeneration and demyelinating diseases.NEW & NOTEWORTHY Our results further indicate temporal and spatial evolution of various functional neurons during the demyelination in a cuprizone (CPZ)-induced mouse model, which mainly occur 4-6 wk after CPZ feeding. At the same time, the axonal compartment is damaged and, consequently, neuronal death occurs, while glutamate neurons are lost obviously. The astrocyte-mediated glutamate reuptake could protect the neurons from the excitatory effects of glutamate.

SKAP2 as a new regulator of oligodendroglial migration and myelin sheath formation.

Oligodendroglial progenitor cells (OPCs) are highly proliferative and migratory cells, which differentiate into complex myelin forming and axon ensheathing mature oligodendrocytes during myelination. Recent studies indicate that the oligodendroglial cell population is heterogeneous on transcriptional and functional level depending on the location in the central nervous system. Here, we compared intrinsic properties of OPC from spinal cord and brain on functional and transcriptional level. Spinal cord OPC demonstrated increased migration as well as differentiation capacity. Moreover, transcriptome analysis revealed differential expression of several genes between both OPC populations. In spinal cord OPC, we confirmed upregulation of SKAP2, a cytoplasmatic adaptor protein known for its implication in cytoskeletal remodeling and migration in other cell types. Recent findings suggest that actin dynamics determine not only oligodendroglial migration, but also differentiation: Whereas actin polymerization is important for process extension, actin destabilization and depolymerization is required for myelin sheath formation. Downregulation or complete lack of SKAP2 in OPC resulted in reduced migration and impaired morphological maturation in oligodendrocytes. In contrast, overexpression of SKAP2 as well as constitutively active SKAP2 increased OPC migration suggesting that SKAP2 function is dependent on activation by phosphorylation. Furthermore, lack of SKAP2 enhanced the positive effect on OPC migration after integrin activation suggesting that SKAP2 acts as modulator of integrin dependent migration. In summary, we demonstrate the presence of intrinsic differences between spinal cord and brain OPC and identified SKAP2 as a new regulator of oligodendroglial migration and sheath formation.

The remyelination effect of DNA framework nucleic acids on demyelinating diseases

Multiple sclerosis (MS) is one of the most famous and common demyelinating diseases in young adults, characterized by motor, sensory, and cognitive impairments. In MS patients, multifocal demyelinating lesions, inflammatory infiltrates, axonal damage, and deletion of oligodendrocytes are observed. For the solely immunomodulatory focus has exhibited unsatisfactory ability to combat the progression of MS disability. There is an absence of first-line drugs against MS to delay the progression and obtain clinical improvement. Tetrahedral framework nucleic acids (tFNAs), novel self-assembled nucleic-acid nanomaterials, have been found to exhibit broad application value. Previous studies have reported positive reactions between tFNAs and various cell types, and tFNAs are able to act the advantages of neuroprotective, anti-apoptosis and anti-inflammatory. To investigate a new research direction for remyelination and potential mechanism for MS therapies, we attempted to characterize the effects of tFNAs on remyelination. The results in vitro demonstrated that tFNAs were able to rescue oligodendroglial progenitor cells (OPCs) from interferon-γ (IFN-γ) induced cell death. In in vivo study, functional remyelination was assessed by behavior test and histological examination in cuprizone-induced mice treated with tFNAs. The tFNAs-treated groups exhibited excellent remyelination. Proteins related to the PI3K-AKT-mTOR and mitochondrial apoptotic pathways were examined to further support this finding. In conclusion, our study suggested the therapeutic potential of tFNAs for MS patients by enhancing the remyelination.

The Wnt Effector TCF7l2 Promotes Oligodendroglial Differentiation by Repressing Autocrine BMP4-Mediated Signaling.

Promoting oligodendrocyte (OL) differentiation represents a promising option for remyelination therapy for treating the demyelinating disease multiple sclerosis (MS). The Wnt effector transcription factor 7-like 2 (TCF7l2) was upregulated in MS lesions and had been proposed to inhibit OL differentiation. Recent data suggest the opposite yet underlying mechanisms remain elusive. Here, we unravel a previously unappreciated function of TCF7l2 in controlling autocrine bone morphogenetic protein (BMP)4-mediated signaling. Disrupting TCF7l2 in mice of both sexes results in oligodendroglial-specific BMP4 upregulation and canonical BMP4 signaling activation in vivo Mechanistically, TCF7l2 binds to Bmp4 gene regulatory element and directly represses its transcriptional activity. Functionally, enforced TCF7l2 expression promotes OL differentiation by reducing autocrine BMP4 secretion and dampening BMP4 signaling. Importantly, compound genetic disruption demonstrates that oligodendroglial-specific BMP4 deletion rescues arrested OL differentiation elicited by TCF7l2 disruption in vivo Collectively, our study reveals a novel connection between TCF7l2 and BMP4 in oligodendroglial lineage and provides new insights into augmenting TCF7l2 for promoting remyelination in demyelinating disorders such as MS.SIGNIFICANCE STATEMENT Incomplete or failed myelin repairs, primarily resulting from the arrested differentiation of myelin-forming oligodendrocytes (OLs) from oligodendroglial progenitor cells, is one of the major reasons for neurologic progression in people affected by multiple sclerosis (MS). Using in vitro culture systems and in vivo animal models, this study unraveled a previously unrecognized autocrine regulation of bone morphogenetic protein (BMP)4-mediated signaling by the Wnt effector transcription factor 7-like 2 (TCF7l2). We showed for the first time that TCF7l2 promotes oligodendroglial differentiation by repressing BMP4-mediated activity, which is dysregulated in MS lesions. Our study suggests that elevating TCF7l2 expression may be possible in overcoming arrested oligodendroglial differentiation as observed in MS patients.

DIPG-43. CAN WE REPROGRAM DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)? EXPLORING THE ROLE OF DISTALLESS/DLX HOMEOBOX GENE REGULATION OF OLIGODENDROGLIAL PROGENITOR CELLS (OPC) IN THE DEVELOPING VERTEBRATE NERVOUS SYSTEM

BACKGROUNDThe identification of H3.3/H3.1K27M in most DIPG has changed our understanding of this disease. H3K27M mutations usually demonstrate global loss of H3K27 trimethylation (me3) with gain of H3K27 acetylation (ac). Single cell RNAseq has identified the putative cell of origin as oligodendroglial progenitor cells (OPC). The distalless gene family is necessary for the differentiation and tangential migration of committed neural progenitors to become GABAergic interneurons. Dlx1/Dlx2 double knockout (DKO) cells from the ganglionic eminences (GE) transplanted into a wild-type environment become oligodendrocytes.RESULTSWe identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo and confirmed direct DLX2 protein-promoter DNA binding in vitro. Co-expression of Dlx2 with target sequences reduced reporter gene expression in vitro. There was increased expression of OLIG2, NKX2.2 and PLP-1 expression in vivo, consistent with de-repression in the absence of Dlx1/Dlx2 function. Transient over-expression of a Dlx2-GFP construct into murine DIPG cells from a GEMM that develops DIPG resulted in significant increases in expression of Gad isoforms with concomitant decreases in Olig2 and Nkx2.2. Dlx2-transfected mDIPG cells also demonstrated reduced migration, invasion and colony formation in vitro. Of significance, there was global restoration of H3K27me3 with corresponding loss of H3K27ac expression in transfected cells compared to controls.CONCLUSIONSDLX2 promotes GABAergic differentiation and migration while concomitantly repressing OPC differentiation in vivo. Developmental reprogramming of mDIPG cells by DLX2 demonstrates the potential role for directed differentiation strategies towards improving patient outcomes for this devastating pediatric cancer.

Abstinence from ethanol dependence produces concomitant cortical gray matter abnormalities, microstructural deficits and cognitive dysfunction

Previous studies demonstrate that ethanol dependence induced by repeating cycles of chronic intermittent ethanol vapor exposure (CIE) followed by protracted abstinence (CIE-PA) produces significant alterations in oligodendrogenesis in the rodent medial prefrontal cortex (mPFC). Specifically, CIE-PA produced an unprecedented increase in premyelinating oligodendroglial progenitor cells and myelin, which have been associated with persistent elevated drinking behaviors during abstinence. The current study used neuroimaging and electron microscopy to evaluate the integrity of enhanced myelin and microstructural deficits underlying enhanced myelination in the mPFC in male rats experiencing forced abstinence for 1 day (D), 7D, 21D and 42D following seven weeks of CIE. In vivo diffusion tensor imaging (DTI) detected altered microstructural integrity in the mPFC and corpus callosum (CC). Altered integrity was characterized as reduced fractional anisotropy (FA) in the CC, and enhanced mean diffusivity (MD) in the mPFC in 7D abstinent rats. Increased MD occurred concomitantly with increases in myelin associated proteins, flayed myelin and enhanced mitochondrial stress in the mPFC in 7D abstinent rats, suggesting that the increases in myelination during abstinence was aberrant. Evaluation of cognitive performance via Pavlovian conditioning in 7D abstinent rats revealed reduced retrieval and recall of fear memories dependent on the mPFC. These findings indicate that forced abstinence from moderate to severe alcohol use disorder produces gray matter damage via myelin dysfunction in the mPFC and that these microstructural changes were associated with deficits in PFC dependent behaviors.

Extrinsic immune cell-derived, but not intrinsic oligodendroglial factors contribute to oligodendroglial differentiation block in multiple sclerosis

Multiple sclerosis (MS) is the most frequent demyelinating disease in young adults and despite significant advances in immunotherapy, disease progression still cannot be prevented. Promotion of remyelination, an endogenous repair mechanism resulting in the formation of new myelin sheaths around demyelinated axons, represents a promising new treatment approach. However, remyelination frequently fails in MS lesions, which can in part be attributed to impaired differentiation of oligodendroglial progenitor cells into mature, myelinating oligodendrocytes. The reasons for impaired oligodendroglial differentiation and defective remyelination in MS are currently unknown. To determine whether intrinsic oligodendroglial factors contribute to impaired remyelination in relapsing–remitting MS (RRMS), we compared induced pluripotent stem cell-derived oligodendrocytes (hiOL) from RRMS patients and controls, among them two monozygous twin pairs discordant for MS. We found that hiOL from RRMS patients and controls were virtually indistinguishable with respect to remyelination-associated functions and proteomic composition. However, while analyzing the effect of extrinsic factors we discovered that supernatants of activated peripheral blood mononuclear cells (PBMCs) significantly inhibit oligodendroglial differentiation. In particular, we identified CD4+ T cells as mediators of impaired oligodendroglial differentiation; at least partly due to interferon-gamma secretion. Additionally, we observed that blocked oligodendroglial differentiation induced by PBMC supernatants could not be restored by application of oligodendroglial differentiation promoting drugs, whereas treatment of PBMCs with the immunomodulatory drug teriflunomide prior to supernatant collection partly rescued oligodendroglial differentiation. In summary, these data indicate that the oligodendroglial differentiation block is not due to intrinsic oligodendroglial factors but rather caused by the inflammatory environment in RRMS lesions which underlines the need for drug screening approaches taking the inflammatory environment into account. Combined, these findings may contribute to the development of new remyelination promoting strategies.

Progesterone through Progesterone Receptor B Isoform Promotes Rodent Embryonic Oligodendrogenesis.

Oligodendrocytes are the myelinating cells of the central nervous system (CNS). These cells arise during the embryonic development by the specification of the neural stem cells to oligodendroglial progenitor cells (OPC); newly formed OPC proliferate, migrate, differentiate, and mature to myelinating oligodendrocytes in the perinatal period. It is known that progesterone promotes the proliferation and differentiation of OPC in early postnatal life through the activation of the intracellular progesterone receptor (PR). Progesterone supports nerve myelination after spinal cord injury in adults. However, the role of progesterone in embryonic OPC differentiation as well as the specific PR isoform involved in progesterone actions in these cells is unknown. By using primary cultures obtained from the embryonic mouse spinal cord, we showed that embryonic OPC expresses both PR-A and PR-B isoforms. We found that progesterone increases the proliferation, differentiation, and myelination potential of embryonic OPC through its PR by upregulating the expression of oligodendroglial genes such as neuron/glia antigen 2 (NG2), sex determining region Y-box9 (SOX9), myelin basic protein (MBP), 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP1), and NK6 homeobox 1 (NKX 6.1). These effects are likely mediated by PR-B, as they are blocked by the silencing of this isoform. The results suggest that progesterone contributes to the process of oligodendrogenesis during prenatal life through specific activation of PR-B.

Temporal and Spatial Dynamics of Astroglial Reaction and Immune Response in Cuprizone-Induced Demyelination.

The cuprizone (CPZ)-induced demyelination is a relatively reproducible animal model and has been extremely useful for identifying the specific cellular and molecular signals that regulate oligodendrocyte survival and efficiency of oligodendrogenesis and remyelination. Here, we reported the temporal and spatial dynamics of astroglial reaction and immune response in CPZ-induced demyelinating model. CPZ did not induce significant microglia and astrocyte reaction after 2 weeks of feeding. After 4-6 weeks of CPZ feeding, microglia and astrocytes were markedly migrated and accumulated in myelin sheath. Simultaneously, the expression of tight junction protein ZO-1 was declined and the infiltration of CD4+IFNγ+ and CD4+IL-17+ T cells was increased in the brain, accompanied by increased production of IFN-γ and IL-17 in the extract of brain. However, the levels of IFN-γ and IL-17 were reduced, while IL-6 and TNF-α were elevated in the supernatant of splenocytes. At the 4th and 6th weeks of feeding, CPZ caused astrocyte activation and upregulated the expression of BDNF, CNTF, and IGF-II, providing a neurotrophic microenvironment in the brain. At this stage, NG2+ and PDGF-Rα+ oligodendroglia progenitor cells were enhanced in the corpus callosum, but the myelin sheath is still severely lost. Therefore, targeting microglia to improve the inflammatory microenvironment should contribute to the remyelination.

Heterogeneous fate choice of genetically modulated adult neural stem cells in gray and white matter of the central nervous system.

Apart from dedicated oligodendroglial progenitor cells, adult neural stem cells (aNSCs) can also give rise to new oligodendrocytes in the adult central nervous system (CNS). This process mainly confers myelinating glial cell replacement in pathological situations and can hence contribute to glial heterogeneity. Our previous studies demonstrated that the p57kip2 gene encodes an intrinsic regulator of glial fate acquisition and we here investigated to what degree its modulation can affect stem cell-dependent oligodendrogenesis in different CNS environments. We therefore transplanted p57kip2 knockdown aNSCs into white and gray matter (WM and GM) regions of the mouse brain, into uninjured spinal cords as well as in the vicinity of spinal cord injuries and evaluated integration and differentiation in vivo. Our experiments revealed that under healthy conditions intrinsic suppression of p57kip2 as well as WM localization promote differentiation toward myelinating oligodendrocytes at the expense of astrocyte generation. Moreover, p57kip2 knockdown conferred a strong benefit on cell survival augmenting net oligodendrocyte generation. In the vicinity of hemisectioned spinal cords, the gene knockdown led to a similar induction of oligodendroglial features; however, newly generated oligodendrocytes appeared to suffer more from the hostile environment. This study contributes to our understanding of mechanisms of adult oligodendrogenesis and glial heterogeneity and further reveals critical factors when considering aNSC mediated cell replacement in injury and disease.

The K2P -channel TASK1 affects Oligodendroglial differentiation but not myelin restoration.

In multiple sclerosis, demyelination occurs as a consequence of chronic autoimmunity in the central nervous system causing progressive neurological impairment in patients. After a demyelinating event, new myelin sheaths are formed by adult oligodendroglial progenitor cells; a process called remyelination. However, remyelination often fails in multiple sclerosis due to insufficient recruitment and differentiation of oligodendroglial precursor cells. A pivotal role for the two-pore-domain potassium (K2P ) channel, TASK1, has already been proven for an animal model of multiple sclerosis. However, the mechanisms underlying the TASK1-mediated effects are still elusive. Here, we tested the role of TASK1 channels in oligodendroglial differentiation and remyelination after cuprizone-induced demyelination in male mice. We found TASK1 channels to be functionally expressed on primary murine and human, pluripotent stem cell-derived oligodendrocytes. Lack of TASK1 channels resulted in an increase of mature oligodendrocytes in vitro as well as a higher number of mature oligodendrocytes and accelerated developmental myelination in vivo. Mechanistically, Task1-deficient cells revealed a higher amount of phosphorylated WNK1, a kinase known to be involved in the downstream signaling of the myelination regulator LINGO-1. Furthermore, we analyzed the effect of genetic TASK1 ablation or pharmacological TASK1 inhibition on disease-related remyelination. Neither channel inhibition nor lack of TASK1 channels promoted remyelination after pathological demyelination. In summary, we conclude that functional TASK1 channels participate in the modulation of differentiating oligodendroglial cells in a previously unknown manner. However, while being involved in developmental myelination our data suggest that TASK1 channels have no major effect on remyelination.

Abstract 1958: Benztropine enhances temozolomide sensitivity by restricting the growth of drug-resistant glioma stem cells

Abstract Glioblastomas (GBMs) are the most aggressive type of primary brain tumor in adults, the second leading cause of cancer-related death in children, and are incurable using currently available treatment modalities. GBMs are characterized by the presence of primitive glioma stem-like cell subpopulations that resist chemotherapy and sustain disease progression. In this study, we found that Benztropine, a muscarinic acetylcholine receptor (mAChR) inhibitor, suppresses glioma cell proliferation and sensitizes these cells to treatment with Temozolomide. Modeling the development of Temozolomide resistance in vitro, we identified a subpopulation of glioma cells, defined by the co-expression of CD44, CD15, and PDGFRa that was resistant to Temozolomide. This chemo-resistant subpopulation was phenotypically similar to oligodendroglial progenitor cells (OPC-like); a stem-like cell population important in the initiation and progression of glioblastoma known to be modulated by mAChR signaling. The addition of the mAChR inhibitor, benztropine, dramatically reduced the emergence of this Temozolomide-resistant sub-population. To begin understanding the mechanism of action of benztropine on GBM cells, we analyzed the effect of this drug on a panel of mitotic drivers that have been implicated in glioma initiation and progression. Sub-lethal doses of Benztropine alone or in combination with temozolomide repressed the PDGF-signaling axis as characterized by a significant reduction of PDGFA secretion and PDGFRA activation. We also developed an in vivo model of Temozolomide resistance to determine whether Benztropine could improve the survival of animals harboring glioma xenografts that had recurred following treatment with Temozolomide. These in vivo studies demonstrated that Benztropine alone improves animal survival as compared with vehicle controls and that Benztropine combined with Temozolomide reduces glioma growth following initial Temozolomide treatment and improves survival in mice xenografted with human GBM cells. Taken together, our results suggest that Benztropine might be targeting glioma stem cells by altering the glioma stem cell microenvironment. These findings support a potential role for the use of adjuvant Benztropine for the clinical management of GBM. Citation Format: Damian A. Almiron Bonnin, Myung Chang Lee, Joseph M. Howard, Alison L. Young, Matthew C. Havrda. Benztropine enhances temozolomide sensitivity by restricting the growth of drug-resistant glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1958.