The Impact of Mouse Preterm Birth Induction by RU-486 on Microglial Activation and Subsequent Hypomyelination.

Preterm birth (PTB) represents 15 million births every year worldwide and is frequently associated with maternal/fetal infections and inflammation, inducing neuroinflammation. This neuroinflammation is mediated by microglial cells, which are brain-resident macrophages that release cytotoxic molecules that block oligodendrocyte differentiation, leading to hypomyelination. Some preterm survivors can face lifetime motor and/or cognitive disabilities linked to periventricular white matter injuries (PWMIs). There is currently no recommendation concerning the mode of delivery in the case of PTB and its impact on brain development. Many animal models of induced-PTB based on LPS injections exist, but with a low survival rate. There is a lack of information regarding clinically used pharmacological substances to induce PTB and their consequences on brain development. Mifepristone (RU-486) is a drug used clinically to induce preterm labor. This study aims to elaborate and characterize a new model of induced-PTB and PWMIs by the gestational injection of RU-486 and the perinatal injection of pups with IL-1beta. A RU-486 single subcutaneous (s.c.) injection at embryonic day (E)18.5 induced PTB at E19.5 in pregnant OF1 mice. All pups were born alive and were adopted directly after birth. IL-1beta was injected intraperitoneally from postnatal day (P)1 to P5. Animals exposed to both RU-486 and IL-1beta demonstrated microglial reactivity and subsequent PWMIs. In conclusion, the s.c. administration of RU-486 induced labor within 24 h with a high survival rate for pups. In the context of perinatal inflammation, RU-486 labor induction significantly decreases microglial reactivity in vivo but did not prevent subsequent PWMIs.

Ontogenetic oligodendrocyte maturation through gestational iron deprivation: The road not taken.

Developmental iron deficiency (dID) models facilitate the study of specific oligodendrocyte (OL) requirements for their progression to a mature state and subsequent contribution to myelination. In the current work, we used the dID model in transgenic mice expressing green fluorescence protein under the CNPase promoter allowing the identification of cells belonging to the oligodendroglial lineage, and the visualization of the entire myelin structure and single OL morphology. The present work evaluates dID effects on OL complexity in different brain areas. Control animals showed an increase in OL complexity both during development and along the anterior-posterior axis. In contrast, dID animals exhibited an initial increase in CNPase+ cells with prevalence of immature-OL (i-OL), an effect later compensated during development by selective death of those i-OL. As a consequence, developmental behavior was impaired in terms of body balance, muscle response, and sensorimotor functions. To explore why i-OL fail to mature in dID, expression levels of transcriptional factors involved in the maturation of the OL lineage were studied. In nuclear fractions, dID animals showed an increase in Hes5, which prevents the maturation of i-OL, and a decrease in Sox10, a positive regulator of OL maturation. The cytoplasmic fractions showed a decrease in Olig1, which is critical for precursor cell differentiation into premyelinating OL. Overall, the expression levels of Hes5, Sox10, and Olig1 in dID conditions correlated with an unfavorable OL maturation profile. In sum, the current results provide further evidence of dID impact on myelination, keeping OL away from the maturational path.

The Differentiation of Rat Oligodendroglial Cells Is Highly Influenced by the Oxygen Tension: In Vitro Model Mimicking Physiologically Normoxic Conditions.

Oligodendrocyte progenitor cells (OPCs) constitute one of the main populations of dividing cells in the central nervous system (CNS). Physiologically, OPCs give rise to mature, myelinating oligodendrocytes and confer trophic support to their neighboring cells within the nervous tissue. OPCs are known to be extremely sensitive to the influence of exogenous clues which might affect their crucial biological processes, like survival, proliferation, differentiation, and the ability to generate a myelin membrane. Alterations in their differentiation influencing their final potential for myelinogenesis are usually the leading cause of CNS dys- and demyelination, contributing to the development of leukodystrophic disorders. The evaluation of the mechanisms that cause oligodendrocytes to malfunction requires detailed studies based on designed in vitro models. Since OPCs readily respond to changes in local homeostasis, it is crucial to establish restricted culture conditions to eliminate the potential stimuli that might influence oligodendrocyte biology. Additionally, the in vitro settings should mimic the physiological conditions to enable the obtained results to be translated to future preclinical studies. Therefore, the aim of our study was to investigate OPC differentiation in physiological normoxia (5% O2) and a restricted in vitro microenvironment. To evaluate the impact of the combined microenvironmental clues derived from other components of the nervous tissue, which are also influenced by the local oxygen concentration, the process of generating OPCs was additionally analyzed in organotypic hippocampal slices. The obtained results show that OPC differentiation, although significantly slowed down, proceeded correctly through its typical stages in the physiologically relevant conditions created in vitro. The established settings were also conducive to efficient cell proliferation, exerting also a neuroprotective effect by promoting the proliferation of neurons. In conclusion, the performed studies show how oxygen tension influences OPC proliferation, differentiation, and their ability to express myelin components, and should be taken into consideration while planning preclinical studies, e.g., to examine neurotoxic compounds or to test neuroprotective strategies.

Activation of oligodendroglial Stat3 is required for efficient remyelination.

Multiple sclerosis is the most prevalent demyelinating disease of the central nervous system (CNS) and is histologically characterized by perivascular demyelination as well as neurodegeneration. While the degree of axonal damage is correlated with clinical disability, it is believed that remyelination can protect axons from degeneration and slow disease progression. Therefore, understanding the intricacies associated with myelination and remyelination may lead to therapeutics that can enhance the remyelination process and slow axon degeneration and loss of function. Ciliary neurotrophic factor (CNTF) family cytokines such as leukemia inhibitory factor (LIF) and interleukin 11 (IL-11) are known to promote oligodendrocyte maturation and remyelination in experimental models of demyelination. Because CNTF family member binding to the gp130 receptor results in activation of the JAK2/Stat3 pathway we investigated the necessity of oligodendroglial Stat3 in transducing the signal required for myelination and remyelination. We found that Stat3 activation in the CNS coincides with myelination during development. Stimulation of oligodendrocyte precursor cells (OPCs) with CNTF or LIF promoted OPC survival and final differentiation, which was completely abolished by pharmacologic blockade of Stat3 activation with JAK2 inhibitor. Similarly, genetic ablation of Stat3 in oligodendrocyte lineage cells prevented CNTF-induced OPC differentiation in culture. In vivo, while oligodendroglial Stat3 signaling appears to be dispensable for developmental CNS myelination, it is required for oligodendrocyte regeneration and efficient remyelination after toxin-induced focal demyelination in the adult brain. Our data suggest a critical function for oligodendroglial Stat3 signaling in myelin repair.

Glial cholesterol redistribution in hypoxic injury in vitro influences oligodendrocyte maturation and myelination.

Hypoxic insult to the fetal brain causes loss of vulnerable premyelinating oligodendrocytes and arrested oligodendrocyte differentiation. Astrocytes influence oligodendrocyte differentiation and the astrocytic response to hypoxia could affect oligodendrocyte maturation under hypoxia. To identify pathways by which astrocytes influence oligodendroglial maturation in hypoxic injury, human fetal neural stem cell-derived astrocytes were exposed to 0.2 % oxygen for 48 hours. Transcriptomic analysis revealed the upregulation of the cholesterol-biosynthesis pathway in hypoxia-exposed astrocytes. Hypoxia-exposed primary astrocytes and astrocytic cell line (SVG) showed increased expression of hydroxy-methyl-glutaryl-CoA reductase (HMGCR), squalene epoxidase (SQLE), apolipoprotein E (apoE) and ATP-binding cassette transporter 1 (ABCA1) on qPCR and Western blot. Hypoxic SVG also showed increased cholesterol content in cells and culture supernatants and increased cell surface expression of ABCA1. Interestingly hypoxia-exposed premyelinating oligodendrocytes (Mo3.13) showed reduced cholesterol along with decreased expression of HMGCR and SQLE on qPCR and Western blot. Exogenous cholesterol increased the differentiation of Mo3.13 as measured by increased expression of myelin basic protein (MBP) on flow cytometry. Hypoxia exposure resulted in increased cholesterol transport from astrocytes to oligodendrocytes in cocultures with BODIPY-cholesterol labelled SVG and membrane-labelled Mo3.13. As exogenous cholesterol enhanced oligodendrocyte differentiation, our findings indicate that increased cholesterol synthesis by astrocytes and transport to oligodendrocytes could supplement oligodendroglial maturation in conditions of hypoxic brain injury in neonates.

Adolescent binge ethanol impacts H3K9me3-occupancy at synaptic genes and the regulation of oligodendrocyte development.

Introduction: Binge drinking in adolescence can disrupt myelination and cause brain structural changes that persist into adulthood. Alcohol consumption at a younger age increases the susceptibility of these changes. Animal models to understand ethanol's actions on myelin and white matter show that adolescent binge ethanol can alter the developmental trajectory of oligodendrocytes, myelin structure, and myelin fiber density. Oligodendrocyte differentiation is epigenetically regulated by H3K9 trimethylation (H3K9me3). Prior studies have shown that adolescent binge ethanol dysregulates H3K9 methylation and decreases H3K9-related gene expression in the PFC. Methods: Here, we assessed ethanol-induced changes to H3K9me3 occupancy at genomic loci in the developing adolescent PFC. We further assessed ethanol-induced changes at the transcription level with qPCR time course approaches in oligodendrocyte-enriched cells to assess changes in oligodendrocyte progenitor and oligodendrocytes specifically. Results: Adolescent binge ethanol altered H3K9me3 regulation of synaptic-related genes and genes specific for glutamate and potassium channels in a sex-specific manner. In PFC tissue, we found an early change in gene expression in transcription factors associated with oligodendrocyte differentiation that may lead to the later significant decrease in myelin-related gene expression. This effect appeared stronger in males. Conclusion: Further exploration in oligodendrocyte cell enrichment time course and dose response studies could suggest lasting dysregulation of oligodendrocyte maturation at the transcriptional level. Overall, these studies suggest that binge ethanol may impede oligodendrocyte differentiation required for ongoing myelin development in the PFC by altering H3K9me3 occupancy at synaptic-related genes. We identify potential genes that may be contributing to adolescent binge ethanol-related myelin loss.

Understanding the Links among Maternal Diet, Myelination, and Depression: Preclinical and Clinical Overview.

Depression is one of the most common mental disorders in the general population, and multiple mechanisms are involved in the etiology of this disease, including myelination. According to the Developmental Origins of Health and Disease (DOHaD) hypothesis, maternal diet affects the lifetime of the individual during adulthood and may contribute to the development of neuropsychiatric disorders. Additionally, the intensive processes of myelination contribute to the development of the central nervous system in the perinatal period, while any alterations during this crucial process providing the physiological functioning of neurons may lead to neuropsychiatric disorders in the next generation. The present review summarizes the current knowledge on the role of the myelin-related changes in depression, as well as the crosstalk among maternal malnutrition, myelination, and depression in preclinical and clinical settings.

TBBPA targets converging key events of human oligodendrocyte development resulting in two novel AOPs

Myelinating oligodendrocytes (OLs) establish saltatory nerve conduction during white matter development. Thus, interference with oligodendrogenesis leads to an adverse outcome on brain performance in the child due to aberrant myelination. An intertwined network of hormonal, transcriptional and biosynthetic processes regulates OL development, thereby simultaneously creating various routes of interference for environmental toxicants. The flame retardant tetrabromobisphenol A (TBBPA) is debated as an endocrine disruptor, especially of the thyroid hormone (TH) system. We identified how TBBPA interferes with the establishment of a population of maturing OLs by two independent modes-of-action (MoA), dependent and independent of TH signaling. Combining the previously published oligodendrocyte maturation assay (NPC6) with large-scale transcriptomics, we describe TBBPA as a TH disruptor, impairing human OL maturation in vitro by dysregulation of oligodendrogenesis-associated genes (i.e., MBP, KLF9 and EGR1). Furthermore, TBBPA disrupts a gene expression network regulating cholesterol homeostasis, reducing OL numbers independently of TH signaling. These two MoA converge in a novel putative adverse outcome pathway (AOP) network on the key event (KE) hypomyelination. Comparative analyses of human and rat neural progenitor cells (NPCs) revealed that human oligodendrogenesis is more sensitive to endocrine disruption by TBBPA. Therefore, ethical, cost-efficient and species-overarching in vitro assays are needed for developmental neurotoxicity hazard assessment. By incorporation of large-scale transcriptomic analyses, we brought the NPC6 assay to a higher readiness level for future applications in a regulatory context. The combination of phenotypic and transcriptomic analyses helps to study MoA to eventually build AOPs for a better understanding of neurodevelopmental toxicity.

Investigation of neuro-inflammatory parameters in a cuprizone induced mouse model of multiple sclerosis.

Cuprizone, copper chelator, treatment of mouse is a toxic model of multiple sclerosis (MS) in which oligodendrocyte death, demyelination and remyelination can be observed. Understanding T and B cell subset as well as their cytokines involved in MS pathogenesis still requires further scrutiny to better understand immune component of MS. The study presented here, aimed to evaluate relevant cytokines, lymphocytes, and gene expressions profiles during demyelination and remyelination in the cuprizone mouse model of MS. Eighty male C57BL/6J mice fed with 0.2% cuprizone for eight weeks. Cuprizone has been removed from the diet in the following eight weeks. Cuprizone treated and control mice sacrificed biweekly, and corpus callosum of the brain was investigated by staining. Lymphocyte cells of mice analyzed by flow cytometry with CD3e, CD11b, CD19, CD80, CD86, CD4, CD25 and FOXP3 antibodies. IFN-gamma, IL-1alpha, IL-2, IL-5, IL-6, IL-10, IL-17, TNF-alpha cytokines were analyzed in plasma samples. Neuregulin 1 (Nrg1), ciliary neurotrophic factor (Cntf) and C-X-C chemokine receptor type 4 (Cxcr4) gene expressions in corpus callosum sections of the mice brain were quantified. Histochemistry analysis showed that demyelination began at the fourth week of cuprizone administration and total demyelination occurred at the twelfth week in chronic model. Remyelination occurred at the fourth week of following withdrawal of cuprizone from diet. The level of mature and activated T cells, regulatory T cells, T helper cells and mature B cells increased during demyelination and decreased when cuprizone removed from diet. Further, both type 1 and type 2 cytokines together with the proinflammatory cytokines increased. The level of oligodendrocyte maturation and survival genes showed differential gene expression in parallel to that of demyelination and remyelination. In conclusion, for the first-time, involvement of both cellular immune response and antibody response as well as oligodendrocyte maturation and survival factors having role in demyelination and remyelination of cuprizone mouse model of MS have been shown.

GDE2-Dependent Activation of Canonical Wnt Signaling in Neurons Regulates Oligodendrocyte Maturation.

Neurons and oligodendrocytes communicate to regulate oligodendrocyte development and ensure appropriate axonal myelination. Here, we show that Glycerophosphodiester phosphodiesterase 2 (GDE2) signaling underlies a neuronal pathway that promotes oligodendrocyte maturation through the release of soluble neuronally derived factors. Mice lacking global or neuronal GDE2 expression have reduced mature oligodendrocytes and myelin proteins but retain normal numbers of oligodendrocyte precursor cells (OPCs). Wild-type (WT) OPCs cultured in conditioned medium (CM) from Gde2-null (Gde2KO) neurons exhibit delayed maturation, recapitulating in vivo phenotypes. Gde2KO neurons show robust reduction in canonical Wnt signaling, and genetic activation of Wnt signaling in Gde2KO neurons rescues in vivo and in vitro oligodendrocyte maturation. Phosphacan, a known stimulant of oligodendrocyte maturation, is reduced in CM from Gde2KO neurons but is restored when Wnt signaling is activated. These studies identify GDE2 control of Wnt signaling as a neuronal pathway that signals to oligodendroglia to promote oligodendrocyte maturation.

Effects of early postnatal sevoflurane exposure on oligodendrocyte maturation and myelination in cerebral white matter of the rat.

General anaesthesia may impose significant neurocognitive risks on the developing brain. As brain injury in preterm neonates has a particular predilection for cerebral white matter, we aimed to evaluate the effects of sevoflurane on oligodendrocyte maturation and myelination in a preterm-equivalent rat model. Two-day-old postnatal (P2) Sprague-Dawley rats were exposed to 3.3 % (approximately 1 minimum alveolar concentration [MAC]) or 4.9 % (approximately 1.5 MAC) sevoflurane for 2 h. Physiologic parameters were measured at the end of sevoflurane anaesthesia. Oligodendrocyte proliferation, maturation, and myelination were evaluated by immunofluorescence with specific markers at different time points. Open field test and Morris water maze tests were performed to access behavior changes from P29 to P36. Arterial blood gases values and blood glucose levels were within the normal physiologic range. As compared to control, 4.9 %, but not 3.3 % sevoflurane disturbed oligodendrocyte maturation at P14, resulting in hypomyelination and axonal damage in cerebral white matter at P28. Rats exposed to 4.9 %, but not 3.3 % sevoflurane showed decreased explorative activity and increased anxiety-like behaviour, as well as learning and memory impairments. Furthermore, 4.9 %, but not 3.3 % sevoflurane inhibited oligodendrocyte proliferation in the developing white matter of the rat brain at 12 h post-anaesthesia, with further evidence of widespread reactive astrogliosis. High concentration of sevoflurane (4.9 %) exposure in early postnatal rats may disrupt oligodendrocyte maturation and myelination. Our study has aimed a spotlight on the need for safe and rational use of analgesics in neonates, especially preterm infants.

Ryanodine receptor- and sodium-calcium exchanger-mediated spontaneous calcium activity in immature oligodendrocytes in cultures.

Myelination in the central nervous system depends on interactions between axons and oligodendrocyte precursor cells (OPCs). Action potentials in an axon can be followed by release of biologically active substances, like glutamate, which can instruct OPCs to start myelination. Myelin Basic Protein (MBP) is an "executive molecule of myelin" required for the formation of compact myelin. As cells of the oligodendrocyte lineage (OLCs) are capable of producing MBP in pure oligodendrocyte cultures, i.e. without neurons, we investigated Ca2+ signaling in developing OLCs in cultures. We show that spontaneous Ca2+ transients (CTs) occur at very low frequency in both bipolar OPCs and mature oligodendrocytes. In contrast immature OLCs (imOLCs), cells with several thick processes, demonstrate a relatively high frequency of CTs. Moreover, CT frequency in imOLC processes is much higher as compared with the somatic CT frequency. Somatic CTs are almost completely blocked by thapsigargin, an antagonist of sarco-(endo-) plasmic reticulum Ca2+ ATPase, and ryanodine, a blocker of ryanodine receptors, indicating an involvement of Ca2+ release from the endoplasmic reticulum. Ryanodine strongly reduces CT frequency in imOLC processes. Ouabain, an antagonist of Na+, K+-ATPase (NKA), applied at low concentration increases CT frequency, while KB-R7943, a blocker of reverse mode of Na+, Ca2+ exchanger (NCX), decreases CT frequency. We suggest that local RyR-NCX-(NKA?) interaction might underlie the generation of CTs in imOLC in the absence of neurons, and this activity influences oligodendrocyte maturation.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Reduce Neuroinflammation, Promote Neural Cell Proliferation and Improve Oligodendrocyte Maturation in Neonatal Hypoxic-Ischemic Brain Injury.

Background: Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of childhood mortality and disability. Stem cell-based regenerative therapies seem promising to prevent long-term neurological deficits. Our previous work in neonatal HI revealed an unexpected interaction between mesenchymal stem/stromal cells (MSCs) and the brains' microenvironment leading to an altered therapeutic efficiency. MSCs are supposed to mediate most of their therapeutic effects in a paracrine mode via extracellular vesicles (EVs), which might be an alternative to cell therapy. In the present study, we investigated the impact of MSC-EVs on neonatal HI-induced brain injury. Methods: Nine-day-old C57BL/6 mice were exposed to HI through ligation of the right common carotid artery followed by 1 h hypoxia (10% oxygen). MSC-EVs were injected intraperitoneally 1, 3, and 5 days after HI. One week after HI, brain injury was evaluated by regional neuropathological scoring, atrophy measurements and immunohistochemistry to assess effects on neuronal, oligodendrocyte and vessel densities, proliferation, oligodendrocyte maturation, myelination, astro-, and microglia activation. Immunohistochemistry analyses were complemented by mRNA expression analyses for a broad set of M1/M2- and A1/A2-associated molecules and neural growth factors. Results: While total neuropathological scores and tissue atrophy were not changed, MSC-EVs significantly protected from HI-induced striatal tissue loss and decreased micro- and astroglia activation. MSC-EVs lead to a significant downregulation of the pro-inflammatory cytokine TNFa, accompanied by a significant upregulation of the M2 marker YM-1 and the anti-inflammatory cytokine TGFb. MSC-EVs significantly decreased astrocytic expression of the A1 marker C3, concomitant with an increased expression of neural growth factors (i.e., BDNF, VEGF, and EGF). These alterations were associated with an increased neuronal and vessel density, coinciding with a significant increase of proliferating cells in the neurogenic sub-ventricular zone juxtaposed to the striatum. MSC-EV-mediated neuroprotection went along with a significant improvement of oligodendrocyte maturation and myelination. Conclusion: The present study demonstrates that MSC-EVs mediate anti-inflammatory effects, promote regenerative responses and improve key developmental processes in the injured neonatal brain. The present results suggest different cellular target mechanisms of MSC-EVs, preventing secondary HI-induced brain injury. MSC-EV treatment may be a promising alternative to risk-associated cell therapies in neonatal brain injury.

Oligodendrocyte Response to Pathophysiological Conditions Triggered by Episode of Perinatal Hypoxia-Ischemia: Role of IGF-1 Secretion by Glial Cells.

Differentiation of oligodendrocyte progenitors towards myelinating cells is influenced by a plethora of exogenous instructive signals. Insulin-like growth factor 1 (IGF-1) is one of the major factors regulating cell survival, proliferation, and maturation. Recently, there is an ever growing recognition concerning the role of autocrine/paracrine IGF-1 signaling in brain development and metabolism. Since oligodendrocyte functioning is altered after the neonatal hypoxic-ischemic (HI) insult, a question arises if the injury exerts any influence on the IGF-1 secreted by neural cells and how possibly the change in IGF-1 concentration affects oligodendrocyte growth. To quantify the secretory activity of neonatal glial cells, the step-wise approach by sequentially using the in vivo, ex vivo, and in vitro models of perinatal asphyxia was applied. A comparison of the results of in vivo and ex vivo studies allowed evaluating the role of autocrine/paracrine IGF-1 signaling. Accordingly, astroglia were indicated to be the main local source of IGF-1 in the developing brain, and the factor secretion was shown to be significantly upregulated during the first 24 h after the hypoxic-ischemic insult. And conversely, the IGF-1 amounts released by oligodendrocytes and microglia significantly decreased. A morphometric examination of oligodendrocyte differentiation by means of the Sholl analysis showed that the treatment with low IGF-1 doses markedly improved the branching of oligodendroglial cell processes and, in this way, promoted their differentiation. The changes in the IGF-1 amounts in the nervous tissue after HI might contribute to the resulting white matter disorders, observed in newborn children who experienced perinatal asphyxia. Pharmacological modulation of IGF-1 secretion by neural cells could be reasonable solution in studies aimed at searching for therapies alleviating the consequences of perinatal asphyxia.

Neuroprotection of the preterm brain.

Despite notable advances in the care and survival of preterm infants, a significant proportion of preterm neonates will have life-long cognitive, behavioral, and motor deficits, and robustly effective neuroprotective strategies are still missing. These therapies must target the pathophysiologic mechanisms observed in contemporaneous infants and rely on modern epidemiology, imaging, and experimental models and assessment techniques. Two drugs, magnesium sulfate and caffeine, are already in use in several units, and although their targets are apnea of prematurity and myometrial contractility (respectively), they do offer improved odds of positive outcomes. Nevertheless, these drugs have limited efficacy, and NICU-to-NICU administration varies greatly. As such, there is an obvious need for additional specific neurotherapeutic strategies to further enhance the outcome of this very fragile population of neonates. The chapter reviews these issues, highlights bottlenecks that need to be solved for meaningful progress in the field, and proposes future innovative avenues for intervention, including delayed interventions.

Iron Availability Compromises Not Only Oligodendrocytes But Also Astrocytes and Microglial Cells.

When disrupted, iron homeostasis negatively impacts oligodendrocyte (OLG) differentiation and impairs myelination. To better understand myelin formation and OLG maturation, in vivo and in vitro studies were conducted to evaluate the effect of iron deficiency (ID) not only on OLG maturation but also on astrocytes (AST) and microglial cells (MG). In vivo experiments in an ID model were carried out to describe maturational events during OLG and AST development and the reactive profile of MG during myelination when iron availability is lower than normal. In turn, in vitro assays were conducted to explore proliferating and maturational states of each glial cell type derived from control or ID conditions. Studies targeted NG2, PDGFRα, CNPAse, CC1, and MBP expression in OLG, GFAP and S100 expression in AST, and CD11b, ED1, and cytokine expression in MG, as well as BrDU incorporation in the three cell types. Our results show that ID affected OLG development at early stages, not only reducing their maturation capacity but also increasing their proliferation and affecting their morphological complexity. AST ID proliferated more than control ones and were more immature, much like OLG. Cytokine expression in ID animals reflected an anti-inflammatory state which probably influenced OLG maturation. These results show that ID conditions alter all glial cells and may impact myelin formation, which could be regulated by a mechanism involving a cross talk between AST, MG, and oligodendrocyte progenitors (OPC).

Glial and Neuronal Protein Tyrosine Phosphatase Alpha (PTPα) Regulate Oligodendrocyte Differentiation and Myelination.

CNS myelination defects occur in mice deficient in receptor-like protein tyrosine phosphatase alpha (PTPα). Here, we investigated the role of PTPα in oligodendrocyte differentiation and myelination using cells and tissues from wild-type (WT) and PTPα knockout (KO) mice. PTPα promoted the timely differentiation of neural stem cell-derived oligodendrocyte progenitor cells (OPCs). Compared to WT OPCs, KO OPC cultures had more NG2+ progenitors, fewer myelin basic protein (MBP)+ oligodendrocytes, and reduced morphological complexity. In longer co-cultures with WT neurons, more KO than WT OPCs remained NG2+ and while equivalent MBP+ populations of WT and KO cells formed, the reduced area occupied by the MBP+ KO cells suggested that their morphological maturation was impeded. These defects were associated with reduced myelin formation in KO OPC/WT neuron co-cultures. Myelin formation was also impaired when WT OPCs were co-cultured with KO neurons, revealing a novel role for neuronal PTPα in myelination. Canonical Wnt/ß-catenin signaling is an important regulator of OPC differentiation and myelination. Wnt signaling activity was not dysregulated in OPCs lacking PTPα, but suppression of Wnt signaling by the small molecule XAV939 remediated defects in KO oligodendrocyte differentiation and enhanced myelin formation by KO oligodendrocytes. However, the myelin segments that formed were significantly shorter than those produced by WT oligodendrocytes, raising the possibility of a role for glial PTPα in myelin extension distinct from its pro-differentiating actions. Altogether, this study reveals PTPα as a molecular coordinator of oligodendroglial and neuronal signals that controls multiple aspects of oligodendrocyte development and myelination.

After Intracerebral Hemorrhage, Oligodendrocyte Precursors Proliferate and Differentiate Inside White-Matter Tracts in the Rat Striatum.

Damage to myelinated axons contributes to neurological deficits after acute CNS injury, including ischemic and hemorrhagic stroke. Potential treatments to promote re-myelination will require fully differentiated oligodendrocytes, but almost nothing is known about their fate following intracerebral hemorrhage (ICH). Using a rat model of ICH in the striatum, we quantified survival, proliferation, and differentiation of oligodendrocyte precursor cells (OPCs) (at 1, 3, 7, 14, and 28 days) in the peri-hematoma region, surrounding striatum, and contralateral striatum. In the peri-hematoma, the density of Olig2(+) cells increased dramatically over the first 7 days, and this coincided with disorganization and fragmentation of myelinated axon bundles. Very little proliferation (Ki67(+)) of Olig2(+) cells was seen in the anterior subventricular zone from 1 to 28 days. However, by 3 days, many were proliferating in the peri-hematoma region, suggesting that local proliferation expands their population. By 14 days, the density of Olig2(+) cells declined in the peri-hematoma region, and, by 28 days, it reached the low level seen in the contralateral striatum. At these later times, many surviving axons were aligned into white-matter bundles, which appeared less swollen or fragmented. Oligodendrocyte cell maturation was prevalent over the 28-day period. Densities of immature OPCs (NG2(+)Olig2(+)) and mature (CC-1(+)Olig2(+)) oligodendrocytes in the peri-hematoma increased dramatically over the first week. Regardless of the maturation state, they increased preferentially inside the white-matter bundles. These results provide evidence that endogenous oligodendrocyte precursors proliferate and differentiate in the peri-hematoma region and have the potential to re-myelinate axon tracts after hemorrhagic stroke.

Efficient Generation of Viral and Integration-Free Human Induced Pluripotent Stem Cell-Derived Oligodendrocytes.

Here we document three highly reproducible protocols: (1) a culture system for the derivation of human oligodendrocytes (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation-our protocol generates viral- and integration-free OLs that efficiently commit and move forward in the OL lineage, recapitulating all the steps known to occur during in vivo development; (2) a method for the isolation, propagation and maintenance of neural stem cells (NSCs); and (3) a protocol for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling. © 2016 by John Wiley & Sons, Inc.

Efficient Generation of Viral and Integration-Free Human Induced Pluripotent Stem Cell-Derived Oligodendrocytes.

Here we document three highly reproducible protocols: (1) a culture system for the derivation of human oligodendrocytes (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation-our protocol generates viral- and integration-free OLs that efficiently commit and move forward in the OL lineage, recapitulating all the steps known to occur during in vivo development; (2) a method for the isolation, propagation and maintenance of neural stem cells (NSCs); and (3) a protocol for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling. © 2016 by John Wiley & Sons, Inc.