Endothelial Netrin‐4 regulates oligodendrocyte precursor cell proliferation and differentiation via ET ‐1 signaling in preterm white matter injury

Myelin repair, which is known as remyelination, is critical to the treatment of neurodegenerative diseases, and myelination depends on not only the differentiation of oligodendrocyte precursor cells toward oligodendrocytes but also the renewal of oligodendrocyte precursor cells under pathological conditions. However, simultaneously promoting the differentiation and proliferation of oligodendrocyte precursor cells in lesions remains an unmet challenge and might affect demyelinating diseases. Kidney-tonifying herbs of traditional Chinese medicine (TCM) are effective in improving the symptoms of degenerative patients. However, herbs or compounds with dual functions are unverified. The purpose of this study was to find a kidney-tonifying TCM that synchronously improved the differentiation and proliferation of oligodendrocyte precursor cells under pathological conditions. Compounds with dual functions were screened from highly frequently used kidney-tonifying TCM, and the effects of the obtained compound on remyelination were investigated in an in vitro oligodendrocyte precursor cell differentiation model under pathological conditions and in demyelinating mice in vivo. The compound icaritin, which is an active component of Yin-Yang-Huo (the leaves of Epimedium brevicornu Maxim), demonstrated multiple effects on the remyelination process, including enhancing oligodendrocyte precursor cell proliferation, facilitating the differentiation of neural progenitor cells toward oligodendrocyte precursor cells and further toward oligodendrocytes, and maturation of oligodendrocytes under corticosterone- or glutamate-induced pathological conditions. Importantly, icaritin effectively rescued behavioral functions and increased the formation of myelin in a cuprizone-induced demyelination mouse model. The multiple effects of icaritin make it a promising lead compound for remyelination therapy.

Preterm cerebral white matter injury (WMI), a major form of prenatal brain injury, may potentially be treated by oligodendrocyte (OL) precursor cell (OPC) transplantation. However, the defective differentiation of OPCs during WMI seriously hampers the clinical application of OPC transplantation. Thus, improving the ability of transplanted OPCs to differentiate is critical to OPC transplantation therapy for WMI. We established a hypoxia-ischemia-induced preterm WMI model in mice and screened the molecules affected by WMI using single-cell RNA sequencing. We revealed that endothelin (ET)-1 and endothelin receptor B (ETB) are a pair of signaling molecules responsible for the interaction between neurons and OPCs and that preterm WMI led to an increase in the number of ETB-positive OPCs and premyelinating OLs. Furthermore, the maturation of OLs was reduced by knocking out ETB but promoted by stimulating ET-1/ETB signaling. Our research reveals a new signaling module for neuron-OPC interaction and provides new insight for therapy targeting preterm WMI.

Interferon-beta treatment normalises the inhibitory effect of serum from multiple sclerosis patients on oligodendrocyte progenitor proliferation

BackgroundIn premature newborn infants, preterm white matter injury (PWMI) causes motor and cognitive disabilities. Accumulating evidence suggests that PWMI may result from defected differentiation of oligodendrocyte precursor cells (OPCs) and impaired maturation of oligodendrocytes. However, the underlying mechanisms remain unclear.MethodsUsing RNAscope, we analyzed the expression level of RNA-binding protein LIN28A in individual OPCs. Knockout of one or both alleles of Lin28a in OPCs was achieved by administrating tamoxifen to NG2CreER::Ai14::Lin28aflox/+ or NG2CreER::Ai14::Lin28aflox/flox mice. Lentivirus expressing FLEX-Lin28a was used in NG2CreER mice to overexpress LIN28A in OPCs. A series of behavioral tests were performed to assess the cognitive functions of mice. Two-tailed unpaired t-tests was carried out for statistical analysis between groups.ResultsWe found that the expression of Lin28a was decreased in OPCs in a PWMI mouse model. Knockout of one or both alleles of Lin28a in OPCs postnatally resulted in reduced OPC differentiation, decreased myelinogenesis and impaired cognitive functions. Supplementing LIN28A in OPCs postnatally was able to promote OPC differentiation and enhance myelinogenesis, thus rescuing the cognitive functions in PWMI mice.ConclusionOur study reveals that LIN28A is critical in regulating postnatal myelinogenesis. Overexpression of LIN28A in OPCs rescues cognitive deficits in PWMI mice by promoting myelinogenesis, thus providing a potential strategy for the treatment of PWMI.

Oligodendrocyte precursor cells (OPCs) proliferation and differentiation are essential for remyelination after white matter injury. Astrocytes could promote oligodendrogenesis after white matter damage whereas the underlying mechanisms are unknown. In this study, the role of astrocytic connexin43 (Cx43) hemichannels involved in OPC proliferation and differentiation in chronic hypoxia was evaluated. In an astrocyte-OPC co-culture chronic hypoxia model, OPCs became proliferative but failed to mature into oligodendrocytes. Application of astrocytic Cx43 blockers attenuated astrocyte activation, suppressed Cx43 hemichannel uptake activity and glutamate release induced by hypoxia, as well as improved OPC differentiation. Moreover, AMPA but not NMDA glutamate receptor antagonist rescued OPC differentiation in hypoxia. In conclusion, these findings suggested that astrocytic Cx43 hemichannel inhibition could potentially improve OPC maturation by attenuating AMPAR-mediated glutamate signaling. Astrocytic Cx43 hemichannels could serve as a potential therapeutic target for remyelination after chronic hypoxia.

To evaluate preterm white matter injury (PWMI) in neonatal rats using multimodal magnetic resonance imaging (MRI) combined with histological assessments and to explore its underlying mechanisms. Healthy 3-day-old Sprague-Dawley neonatal rats were randomly divided into a sham operation group and a PWMI group (n=12 in each group). A PWMI model was established in neonatal rats through hypoxia-ischemia. Laser speckle imaging was used to observe changes in cerebral oxygen saturation and blood flow at different time points post-modeling. Multimodal MRI was employed to assess the condition of white matter injury, while hematoxylin-eosin staining was utilized to observe morphological changes in the striatal area on the injured side. Immunofluorescence staining was performed to detect the proliferation and differentiation of oligodendrocyte precursor cells. At 0, 6, 12, 24, and 72 hours post-modeling, the relative blood flow and relative oxygen saturation on the injured side in the PWMI group were significantly lower than those in the sham operation group (P<0.05). At 24 hours post-modeling, T2-weighted imaging showed high signals in the white matter of the injured side in the PWMI group, with relative apparent diffusion coefficient values and Lorenz differential values being lower than those in the sham operation group (P<0.001); additionally, the arrangement of nerve cells in the PWMI group was disordered, and the number of EdU+PDGFR-α+ cells was higher than that in the sham operation group (P<0.001). At 28 days post-modeling, the relative fractional anisotropy values, the number of EdU+Olig2+ cells, and the fluorescence intensity of myelin basic protein and neurofilament protein 200 in the white matter region of the PWMI group were all lower than those in the sham operation group (P<0.001). Multimodal MRI can evaluate early and long-term changes in PWMI in neonatal rat models in vivo, providing both imaging and pathological evidence for the diagnosis and treatment of PWMI in neonates. Hypoxia-ischemia inhibits the proliferation and differentiation of oligodendrocyte precursor cells in neonatal rats, leading to PWMI.

Cerebral palsy (CP), mainly resulting from preterm white matter injury (PWMI), remains a leading neurodevelopmental disorder. While oligodendrocyte precursor cell (OPC) differentiation failure is central to PWMI pathology, the metabolic mechanisms remain unclear. Here, untargeted lipidomic and metabolomic profiling of serum samples from retrospective and prospective cohorts of preterm infants identified a CP-associated metabolic signature, highlighting phosphatidic acid (PA) as a top candidate that was consistently elevated and showed strong discriminative potential. Increased PA levels were validated in both serum and brains of PWMI mice and in OPCs subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), where PA impaired OPC differentiation and myelination. Mechanistically, PA interacted with and stabilized the E3 ubiquitin ligase TRIM59, increasing its protein abundance and half-life without affecting mRNA levels. Elevated TRIM59 promoted proteasomal degradation of the oligodendrocyte lineage transcription factor Olig2, a key regulator of OPC maturation. Inhibition of PA synthesis restored Olig2 expression, improved myelination, and rescued differentiation deficits in PWMI mice. Collectively, this study identifies PA as a potential metabolic risk factor associated with preterm CP and uncovers a PA-TRIM59-Olig2 signaling axis linking lipid metabolism to OPC differentiation failure and PWMI.

The massive loss of oligodendrocytes caused by various pathological factors is a basic feature of many demyelinating diseases of the central nervous system (CNS). Based on a variety of studies, it is now well established that impairment of oligodendrocyte precursor cells (OPCs) to differentiate and remyelinate axons is a vital event in the failed treatment of demyelinating diseases. Recent evidence suggests that Foxg1 is essential for the proliferation of certain precursors and inhibits premature neurogenesis during brain development. To date, very little attention has been paid to the role of Foxg1 in the proliferation and differentiation of OPCs in demyelinating diseases of the CNS. Here, for the first time, we examined the effects of Foxg1 on demyelination and remyelination in the brain using a cuprizone (CPZ)-induced mouse model. In this work, 7-week-old Foxg1 conditional knockout and wild-type (WT) mice were fed a diet containing 0.2% CPZ w/w for 5 weeks, after which CPZ was withdrawn to enable remyelination. Our results demonstrated that, compared with WT mice, Foxg1-knockout mice exhibited not only alleviated demyelination but also accelerated remyelination of the demyelinated corpus callosum. Furthermore, we found that Foxg1 knockout decreased the proliferation of OPCs and accelerated their differentiation into mature oligodendrocytes both in vivo and in vitro. Wnt signaling plays a critical role in development and in a variety of diseases. GSK-3β, a key regulatory kinase in the Wnt pathway, regulates the ability of β-catenin to enter nuclei, where it activates the expression of Wnt target genes. We then used SB216763, a selective inhibitor of GSK-3β activity, to further demonstrate the regulatory mechanism by which Foxg1 affects OPCs in vitro. The results showed that SB216763 clearly inhibited the expression of GSK-3β, which abolished the effect of the proliferation and differentiation of OPCs caused by the knockdown of Foxg1. These results suggest that Foxg1 is involved in the proliferation and differentiation of OPCs through the Wnt signaling pathway. The present experimental results are some of the first to suggest that Foxg1 is a new therapeutic target for the treatment of demyelinating diseases of the CNS.

Excessive release of glutamate, oxidative stress, inflammation after ischemic brain injury can lead to demyelination. Astrocytes participate in the maturation and differentiation of oligodendrocyte precursor cells (OPCs), and play multiple roles in the process of demyelination and remyelination. Here, we studied the role of Astrocyte-derived exosomes (AS-Exo) under ischemic conditions in proliferation, differentiation and migration of OPCs in vitro. Exosomes were collected from astrocytes supernatant by differential centrifugation from control astrocytes (CTexo), mild hypoxia astrocytes (O2R24exo) which were applied oxygen-glucose deprivation for 2h and reperfusion for 24h (OGD2hR24h) and severe hypoxia astrocytes (O4R24exo) which were applied oxygen-glucose deprivation for 4h and reperfusion for 24h (OGD4hR24h). Exosomes (20µg/ml) were co-cultured with OPCs for 24h and their proliferation, differentiation and migration were detected. The results showed that AS-Exo under severe hypoxia (O4R24exo) inhibit the proliferation of OPCs. Meanwhile, all exosomes from three groups can promote OPCs differentiation and migration. Compared to control, the expressions of MAG and MBP, markers of mature oligodendrocytes, were significantly increased in AS-Exo treatment groups. AS-Exo treatment significantly increased chemotaxis for OPCs. AS-Exo improve OPCs' differentiation and migration, whereas AS-Exo with severe hypoxic precondition suppress OPCs' proliferation. AS-Exo may be a potential therapeutic target for myelin regeneration and repair in white matter injury or other demyelination related diseases.

Objectives: The pathologic changes of demyelination after spinal cord injury (SCI) significantly impair functional recovery of lesioned spinal cord. At present, transplantation of myelinating cells is regarded as a promising strategy for treating demyelination following SCI. Hence, the In vitro culture and growth, differentiation and proliferation of oligodendrocyte precursor cells (OPCs) were intensively investigated in this study.Methods: In vitro cells from cerebral cortices of neonatal rats were primarily cultured and OPCs were then separated by shaking process and differential adhesion. Following cultured in the conditional medium, growth pattern and differentiation of OPCs were continuously studied by both light microscopy and scanning electron microscopy. Furthermore, maturation of OPCs was detected immunochemically and proliferative ability of OPCs In vitro was also evaluated by methyl thiazolyl tetrazolium (MTT) assay.Results: The distinct stratification of glial cells usually developed around 9-10 days in the primary culture. The OPCs were found primarily living on the surface of confluent astrocytes and these cells typically displayed the simple appearance of immature cells. Furthermore, the OPCs progressively developed in the conditional medium, and these differentiated cells underwent dramatic changes of morphology and also expressed different specific markers. Moreover, the OPCs also proved by MTT assay to proliferate significantly while cultured In vitro.Discussion: Demyelination prevents recovery of neural function following SCI. Demyelination has already become a potential therapeutic target for this insidious and challenging problem. The In vitro culture and biological characteristics of OPCs are fundamental and necessary for further investigation of cell transplantation in vivo. Growth pattern, differentiation and proliferation are very vital for therapeutical effects of OPCs following transplantation after SCI.

Preterm white matter injury (PWMI) is a leading cause of cerebral palsy and chronic neurological disabilities in premature infants. It is characterized by defects in oligodendrocyte precursor cell (OPC) differentiation and dysmyelination. Currently, there are no effective therapeutic strategies available in clinical practice. Lipid homeostasis plays a crucial role in myelin development, yet the function of Lipin1 - a key phosphatidic acid phosphatase involved in phospholipid synthesis - remains unclear. In this study, we identified a significant downregulation of Lipin1 in OPCs from PWMI mice, which impaired OPC differentiation and myelin formation. Conversely, Lipin1 overexpression in these mice promoted OPC maturation and enhanced myelin development. We found evidence that N-acetyltransferase 10 (NAT10) acts as a regulator of Lipin1 expression through RNA pull-down and mass spectrometry. NAT10-mediated N4-acetylcytidine (ac4C) modification enhanced Lipin1 mRNA stability and translation, and NAT10 knockdown in OPCs impaired myelination, highlighting its crucial role in Lipin1-mediated myelination. Our study revealed that the downregulation of Lipin1 impaired OPC differentiation and myelination in PWMI, with NAT10-mediated ac4C modification playing a critical role in regulating Lipin1 expression. These findings highlight Lipin1 and NAT10 as promising therapeutic targets for treating myelination defects in PWMI, warranting further investigation into their potential in preterm birth-related neurological disorders.

Glioma is currently an incurable disease. Identifying the cell of origin for glioma could provide critical insights for developing effective therapies. We previously used a mouse genetic system termed Mosaic Analysis using Double Markers (MADM) to introduce p53 and NF1 mutations into neural stem cells to determine which cell type is responsible for gliomagenesis. The unequivocal GFP-labeling of sparse mutant cells generated by MADM enabled us to study the entire course of tumor development, from pre-transforming stages to full malignancy. Based on thorough histological, transcriptomic, and genetic analyses we identified oligodendrocyte precursor cells (OPCs) as the cell of origin in this glioma model. Here, we sought to understand the distinct role of p53 and NF1 in the transformation of OPCs since understanding these roles can give us a more thorough insight into how future therapies may be designed. The MADM system generates sparse GFP+ null and RFP+ sibling WT OPCs in an otherwise colorless, heterozygous mouse, allowing us to assess the impact of TSG loss on the capacity of cells to proliferate, survive, and differentiate. The loss of NF1 alone significantly increased mutant OPC proliferation with a proportional decrease in differentiation. However, massively over-expanded NF1-null OPCs did not transform into malignant gliomas, implicating the critical role of p53 in tumor suppression. Surprisingly, the loss of p53 alone had no effect on mutant OPC number, proliferation, or differentiation, suggesting that p53 function as a braking system that won't manifest its activity in the absence of driver mutation, such as NF1 loss. Next, we set out to test whether restoring either TSG function in tumor OPC cell lines would have any therapeutic efficacy. The restoration of wild type p53 led to a significant increase in both cell-cycle arrest and cell death. Since p53 function is commonly altered from mutations in patients, rather than complete loss of the protein, we tested whether restoring mutant p53 function with PRIMA-1 would have the same biological response as WT p53. We found that the restoration of mutant p53 function resulted in similar levels of cell cycle arrest and cell death as WT p53. This finding suggests that rescuing p53 function using small molecules may be of high value for future therapies. It also suggests that the loss of p53 activity is not a permanent fixture of tumor cells but that p53 can still exert it's tumor suppressor activity after transformation. Next we tested the therapeutic effects of the restoration of the GAP domain of NF1, which should reduce Ras activity in tumor OPCs. We found that the expression of WT but not the “GAP-dead” NF1-GAP led to a decrease in proliferation and an increase in differentiation of glioma cells. To test pharmacological compounds, we explore the possibility of targeting downstream effectors of Ras signaling, specifically mTOR inhibition because phospho-Akt level is elevated in tumor OPCs compared to WT OPCs. When we inhibited mTOR activity in MADM tumor mice at an age that is critical for mutant OPC expansion prior to transformation, we found that it blocked the ability of mutant OPCs to over-expand, suggesting that mTOR inhibition could potentially prevent gliomagenesis. In summary our data demonstrates that while NF1 loss increases OPC proliferation and decreases differentiation, p53 loss is critical for transformation and that restoring the function of either of these pathways has profound effects on tumor OPC biology that could be translated into future therapeutic strategies. Citation Format: Phillippe P. Gonzalez, Hui Zong. Deciphering individual roles of p53 and NF1 in the cell of origin for malignant glioma and the implications of targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B16.

MicroRNA-219 (miR-219) regulates the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) during central nervous system (CNS) development. OPCs only differentiate into oligodendrocytes (OLs) in the healthy CNS, but can generate astrocytes (As) after injury. We hypothesized that miR-219 may modulate OPC proliferation and differentiation in a cervical C5 contusion spinal cord injury (SCI) model. After injury, we observed a decrease in the miR-219 level and quantity of OLs and an increase in the number of OPCs and As. Silencing of miR-219 by its antagomir in vivo produced similar results, but of greater magnitude. Overexpression of miR-219 by its agomir in vivo increased the number of OLs and suppressed generation of OPCs and As. Luxol fast blue staining confirmed that SCI caused demyelination and that the extent of demyelination was attenuated by miR-219 overexpression, but aggravated by miR-219 reduction. Monocarboxylate transporter 1 (MCT-1) may be implicated in the regulation of OPC proliferation and differentiation mediated by miR-219 following contusion SCI. Collectively, our data suggest that miR-219 may mediate SCI-induced OPC proliferation and differentiation, and MCT-1 may participate in this process as a target of miR-219.

Neurodegenerative diseases are closely associated with myelin loss, and the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) are crucial to remyelination. However, the regulatory mechanisms involved remain incompletely understood. This study aims to investigate how astrocytes (ASTs) regulate the secretion of chitinase-3-like protein 1 (CHI3L1) via connexin 47 (Cx47)-mediated exosome signaling, and its subsequent effect on OPC proliferation. Primary cells were isolated from postnatal day 1 Sprague-Dawley (P1SD) rats to establish 3 culture conditions: OPCs alone (Group O), OPCs in direct contact with ASTs (Group C), and OPCs cultured with AST-conditioned medium (Group A). Cellular morphology and proliferation were assessed using optical microscopy, 5-ethynyl-2'- deoxyuridine (EdU) incorporation, and flow cytometry. RNA sequencing (RNA-Seq) and bioinformatics analysis (BA) were conducted to identify differentially expressed genes (DEGs) among groups. Protein expression and cell cycle distribution were analyzed by Western blotting (WB) and flow cytometry. Exosomes were isolated and purified via differential centrifugation, characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), and CHI3L1 expression in exosomes was verified via WB. Cx47 was silenced using small interfering RNA (siRNA) to evaluate its effect on OPC proliferation and exosome secretion. Artificial exosomes were constructed by encapsulating CHI3L1 in single unilamellar vesicles (SUVs), whose structure and size were validated by NTA and TEM. Following Cx47 knockdown, artificial exosomes were added back, and OPC proliferation was assessed via flow cytometry and EdU assay. Direct co-cultured with ASTs (Group C) resulted in significantly enhanced OPC proliferation compared to the Group O and Group A (P<0.05). RNA-Seq and WB analyses revealed that ASTs promote OPC proliferation and exosome secretion enriched in CHI3L1 through Cx47. Cx47 knockdown by siRNA led to significant decreases in OPC proliferation and exosome release (P<0.05). The inhibitory effect of Cx47 silencing on OPC proliferation was partially reversed by supplementation with either isolated exosomes or exogenous CHI3L1. This study reveals a novel mechanism by which ASTs regulate OPC proliferation: Through direct contact, ASTs enhance the secretion of CHI3L1-rich exosomes via Cx47, thereby converting intercellular contact signals into secretory signals that promote OPC proliferation. As a key exosomal molecule, CHI3L1 may play an important role in neural function and remyelination and warrants further investigation.

Because successful remyelination does not occur following traumatic spinal cord injury, patients suffer from long tract dysfunction. However, demyelination is followed by remyelination in early multiple sclerosis. Oligodendrocyte precursor cells constitute a large cell population in the adult mammalian central nervous system. We demonstrated the proliferation, migration, and differentiation of oligodendrocyte precursor cells in chemically induced demyelination, a model for multiple sclerosis, and reported that Nkx2.2 expression may regulate oligodendrocyte precursor cell differentiation, making it a key factor in the differentiation. To investigate what factors disturb remyelination in spinal cord injury, we examined the oligodendrocyte precursor cell proliferation and differentiation, and the expression of Nkx2.2 using contusive injury in rats as a model for traumatic spinal cord injury. This study showed that oligodendrocyte precursor cells proliferated after contusive injury but did not subsequently differentiate. The number of Nkx2.2-positive oligodendrocyte precursor cells did not significantly change in the tissue surrounding the lesion. Within the demyelinating lesion, the peak of Nkx2.2-positive oligodendrocyte precursor cell was delayed, and its level was lower than in the chemical models. No clearly recognizable oligodendrocytes were found in the demyelinating lesion throughout the observation period. To assess whether environmental changes differ between these two models, mRNA expressions of various cytokines were evaluated and compared. IL-1beta and IL-6 mRNA significantly increased in the contusion-induced injury model, 6 h after the injury. These results suggest that environmental factors such as cytokines may affect Nkx2.2 expression or oligodendrocyte precursor cell differentiation in the contusion-induced spinal cord injury model.