Oligodendrocytes Research Articles

Chronic demyelination and myelin repair after spinal cord injury in mice: A potential link for glutamatergic axon activity.

Our prior work examining endogenous repair after spinal cord injury (SCI) in mice revealed that large numbers of new oligodendrocytes (OLs) are generated in the injured spinal cord, with peak oligodendrogenesis between 4 and 7 weeks post-injury (wpi). We also detected new myelin formation over 2 months post-injury (mpi). Our current work significantly extends these results, including quantification of new myelin through 6 mpi and concomitant examination of indices of demyelination. We also examined electrophysiological changes during peak oligogenesis and a potential mechanism driving OL progenitor cell (OPC) contact with axons. Results reveal peak in remyelination occurs during the 3rd mpi, and that myelin generation continues for at least 6 mpi. Further, motor evoked potentials significantly increased during peak remyelination, suggesting enhanced axon potential conduction. Interestingly, two indices of demyelination, nodal protein spreading and Nav1.2 upregulation, were also present chronically after SCI. Nav1.2 was expressed through 10 wpi and nodal protein disorganization was detectable throughout 6 mpi suggesting chronic demyelination, which was confirmed with EM. Thus, demyelination may continue chronically, which could trigger the long-term remyelination response. To examine a potential mechanism that may initiate post-injury myelination, we show that OPC processes contact glutamatergic axons in the injured spinal cord in an activity-dependent manner. Notably, these OPC/axon contacts were increased 2-fold when axons were activated chemogenetically, revealing a potential therapeutic target to enhance post-SCI myelin repair. Collectively, results show the surprisingly dynamic nature of the injured spinal cord over time and that the tissue may be amenable to treatments targeting chronic demyelination.

Role of Gltp in Maturation of Oligodendrocytes Under the Regulation of Nkx2.2.

Myelin, a lipid-enriched multi-layer membrane structure, allows for rapid long-distance saltatory conduction of neuronal impulses. Although glycolipids are the predominant types of lipids in the myelin bilayer, the role of glycolipid transfer protein (GLTP), which selectively mediates the transfer of various glycolipids between phospholipid bilayer, in myelin development and maintenance remains unknown at present. In this study, we identified Gltp as the key lipid metabolism gene in myelin-forming oligodendrocytes (OLs) through integrated omics analysis across independent transcriptomic and single-cell sequencing studies. Gene expression analysis revealed that Gltp is selectively expressed in the differentiated OLs. Functional study demonstrated that its expression is essential for the differentiation of OLs, and promotes the outgrowth of OL membrane. Moreover, we found that the expression of Gltp is regulated by OL-lineage transcriptional factors, such as NKX2.2, OLIG2, SOX10, and MYRF. These findings provide important insights into the unrecognized functions of Gltp in OL differentiation and maturation.

Oligodendroglia Confer Neuroprotection to NSC-34 Motor Neuronal Cells Against the Toxic Insults of Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder with multifactorial pathomechanisms affecting not only motor neurons but also glia. Both astrocytes and microglia get activated and contribute significantly to neurodegeneration. The role of oligodendroglia in such a situation remains obscure, especially in the sporadic form of ALS (SALS), which contributes to 90% of cases. Here, we have investigated the role of oligodendroglia in SALS pathophysiology using a human oligodendroglial cell line, MO3.13, by exposing the cells to cerebrospinal fluid from SALS patients (ALS-CSF; 10% v/v for 48 h). ALS-CSF significantly reduced the viability of MO3.13 cells and down-regulated the expression of oligodendroglia-specific proteins, namely, CNPase and Olig2. Furthermore, to investigate the effect of the observed oligodendroglial changes on motor neurons, NSC-34 motor neuronal cells were co-cultured/supplemented with conditioned/spent medium of MO3.13 cells upon exposure to ALS-CSF. Live cell imaging experiments revealed protection to NSC-34 cells against ALS-CSF toxicity upon co-culture with MO3.13 cells. This was evidenced by the absence of neuronal cytoplasmic vacuolation and beading of neurites, which instead resulted in better neuronal differentiation. Enhanced lactate levels and increased expression of its transporter, MCT-1, with sustained expression of trophic factors, namely, GDNF and BDNF, by MO3.13 cells hint towards metabolic and trophic support provided by the surviving oligodendroglia. Similar metabolic changes were seen in the lumbar spinal cord oligodendroglia of rat neonates intrathecally injected with ALS-CSF. The findings indicate that oligodendroglia are indeed rescuer to the degenerating motor neurons when the astrocytes and microglia turn topsy-turvy.

Alpha-asaronol promoted oligodendrocyte precursor cell differentiation and improved myelination as an activator PPARγ

Preterm white matter injury (PWMI), characterized by oligodendrocyte precursor cell (OPC) differentiation disorder and dysmyelination, is a prevalent demyelinating disease of the central nervous system in premature infants, necessitating the development of mitigating strategies. Convincing evidence suggests that peroxisome proliferator-activated receptor γ (PPARγ) activation is a stimulative factor against the hindered process of oligodendrocyte (OL) differentiation. However, much remains unknown about its promotive mechanism. Our previous study indicated that alpha-asaronol (α-asaronol) could alleviate myelination disorder in a neonatal PWMI rat model, but the mechanism remained unclear. In this study, we demonstrated that α-asaronol attenuated cognitive deficits, repaired myelin damage, and stimulated OL differentiation in the corpus callosum of PWMI rats. Co-immunoprecipitation analysis confirmed that α-asaronol induced the binding of PPARγ with its coactivator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), which in turn activated oligodendroglial PPARγ. This activation subsequently upregulated the expression of phosphatase and tensin homolog (PTEN) and pro-differentiation-associated genes of Cnp1 and Klk6 and downregulated the expression of Clk1. However, the benefits of α-asaronol were blocked by GW9662, an antagonist of PPARγ. Moreover, α-asaronol also promoted OPC differentiation under oxygen-glucose deprivation conditions. In conclusion, α-asaronol can promote OL differentiation and myelination and alleviate cognitive deficits in neonatal PWMI rats by activating PPARγ and modulating OL differentiation-associated gene expression. This study suggests that α-asaronol may be a potential therapeutic drug for myelination failure in PWMI.

Blockade of L-Type Ca2+ Channel Activity Alleviates Oligodendrocyte Pathology following Brain Injury in Male Rats.

A growing body of studies suggests that Ca2+ signaling controls a variety of biological processes in brain elements. Activation of L-type voltage-operated Ca2+ channels (VOCCs) plays a role in the development of oligodendrocyte (OL) lineage loss, and indicates that the blocking of these channels may be an effective way to inhibit OL lineage cell loss. For this study, 10.5-day-old male Sprague-Dawley rats were used to generate cerebellar tissue slices. The slice tissues were cultured and randomly allocated to one of four groups (six each) and treated as follows: Group I, (sham control); Group II, 0.1% dimethyl sulfoxide (DMSO) only (vehicle control); Group III, injury (INJ); Group IV, (INJ and treatment with NIF). The injury was simulated by exposing the slice tissues to 20 min of oxygen-glucose deprivation (OGD). At 3 days post-treatment, the survival, apoptosis, and proliferation of the OL lineages were measured and compared. Results: In the INJ group, there was a decrease in mature myelin basic protein+ OLs (MBP+ OLs) and their precursors, NG2+ OPCs (Nerve-glia antigen 2+ oligodendrocyte precursor cell), compared with controls. A significant elevation was observed in the NG2+ OPCs and apoptotic MBP+ OLs as confirmed by a TUNEL assay. However, the cell proliferation rate was decreased in NG2+ OPCs. NIF increased OL survival as measured by apoptosis rate in both OL lineages and preserved the rate of proliferation in the NG2+ OPCs. Conclusions: Activation of L-type VOCCs may contribute to OL pathology in association with reduced mitosis of OPCs following brain injury as a strategy to treat demyelinating diseases.

Inward rectifying Kir4.1 channels regulate oligodendrocyte precursor cell differentiation and CNS myelination in vivo

The functions of Kir4.1 in oligodendrocyte development have been in controversial. We recently reported that inhibiting Kir4.1 impeded oligodendrocyte precursor cell (OPC) differentiation and oligodendrocyte (OL) maturation, due to Kir4.1 altering intracellular pH of OPCs through Na+/H+ exchangers. However, our conclusion was limited by in vitro observation, thereby it becomes necessary to seek in vivo evidence to determine the roles of Kir4.1 on OPC development and CNS myelination. Here, we used Olig1-Cre to knockout Kir4.1 in OPCs from the early developmental stage. We found that the cell-specific deletion of Kir4.1 significantly impeded OPC differentiation and reduced the number of mature OLs in the cerebral cortex and the corpus callosum. Hence, our in vivo evidence supports that Kir4.1 can regulate OPC differentiation and is essential to CNS myelination.

Decreased RNA polymerase III Expression Leads to Several Defects in Oligodendrocyte-Lineage Development (P12-9.009)

<h3>Objective:</h3> To characterize the role of RNA polymerase III (Pol III) in oligodendrocyte development. <h3>Background:</h3> Hypomyelinating leukodystrophies are a heterogeneous group of rare, genetic white matter disorders characterized by insufficient myelin deposition in development. They typically present in early development and lead to progressive neurodegeneration and premature death in the months to years following onset. RNA polymerase III-related hypomyelinating leukodystrophy (POLR3-HLD) is one of the most common hypomyelinating leukodystrophies and arises from biallelic pathogenic variants in genes encoding subunits of Pol III, a ubiquitous enzymatic complex. Myelin, produced by oligodendrocytes (OLs) in the central nervous system, insulates axons, enabling efficient propagation of action potentials. As the constitutive nature of Pol III would suggest a multi-system organ disease, it is unclear why OLs are particularly vulnerable to reduced Pol III activity to explain the devastating hypomyelination seen in POLR3-HLD. <h3>Design/Methods:</h3> Oligodendrocyte precursor cells (OPCs) undergo a complex developmental process to become OLs. Therefore, we hypothesized that reduced Pol III activity would lead to defective OL-lineage development, either in the 1) migration and proliferation of OPCs, 2) maturation of OPCs into OLs and/or 3) myelination. To investigate this, primary murine OPCs were isolated and transfected with siRNA targeting <i>Polr3a, Polr3b</i> or <i>Polr1c</i> and subjected to various <i>in vitro</i> assays. <h3>Results:</h3> Our data shows that reducing Pol III activity leads to a maturation defect, as evidenced by reduced morphological complexity and decreased expression of late-stage OL markers. Apoptotic marker staining indicates these cells remain alive. Analysis of the OPC stage demonstrated abnormal proliferation, while migration was unaffected. Ongoing myelination assays suggest this altered oligodendrogenesis impacts myelination. <h3>Conclusions:</h3> Our results suggest a role for Pol III in normal OL development, providing valuable insight into the cellular mechanisms underlying myelination failure in POLR3-HLD. <b>Disclosure:</b> Ms. Macintosh has nothing to disclose. Mackenzie A. Michell-Robinson has nothing to disclose. Dr. Chen has nothing to disclose. Geneviève Bernard has nothing to disclose.

It takes two to remyelinate: A bioengineered platform to study astrocyte-oligodendrocyte crosstalk and potential therapeutic targets in remyelination

The loss of the myelin sheath insulating axons is the hallmark of demyelinating diseases. These pathologies often lead to irreversible neurological impairment and patient disability. No effective therapies are currently available to promote remyelination.Several elements contribute to the inadequacy of remyelination, thus understanding the intricacies of the cellular and signaling microenvironment of the remyelination niche might help us to devise better strategies to enhance remyelination.Here, using a new in vitro rapid myelinating artificial axon system based on engineered microfibres, we investigated how reactive astrocytes influence oligodendrocyte (OL) differentiation and myelination ability. This artificial axon culture system enables the effective uncoupling of molecular cues from the biophysical properties of the axons, allowing the detailed study of the astrocyte-OL crosstalk. Oligodendrocyte precursor cells (OPCs) were cultured on poly(trimethylene carbonate-co-ε-caprolactone) copolymer electrospun microfibres that served as surrogate axons. This platform was then combined with a previously established tissue engineered glial scar model of astrocytes embedded in 1 % (w/v) alginate matrices, in which astrocyte reactive phenotype was acquired using meningeal fibroblast conditioned medium.OPCs were shown to adhere to uncoated engineered microfibres and differentiate into myelinating OL. Reactive astrocytes were found to significantly impair OL differentiation ability, after six and eight days in a co-culture system. Differentiation impairment was seen to be correlated with astrocytic miRNA release through exosomes. We found significantly reduction on the expression of pro-myelinating miRNAs (miR-219 and miR-338) and an increase in anti-myelinating miRNA (miR-125a-3p) content between reactive and quiescent astrocytes. Additionally, we show that OPC differentiation inhibition could be reverted by rescuing the activated astrocytic phenotype with ibuprofen, a chemical inhibitor of the small rhoGTPase RhoA.Overall, these findings show that modulating astrocytic function might be an interesting therapeutic avenue for demyelinating diseases. The use of these engineered microfibres as an artificial axon culture system will enable the screening for potential therapeutic agents that promote OL differentiation and myelination while providing valuable insight on the myelination/remyelination processes.

NF-κB is a critical mediator of post-mitotic senescence in oligodendrocytes and subsequent white matter loss

BackgroundInflammaging represents an accepted concept where the immune system shifts to a low-grade chronic pro-inflammatory state without overt infection upon aging. In the CNS, inflammaging is mainly driven by glia cells and associated with neurodegenerative processes. White matter degeneration (WMD), a well-known process in the aging brain, manifests in myelin loss finally resulting in motor, sensory and cognitive impairments. Oligodendrocytes (OL) are responsible for homeostasis and maintenance of the myelin sheaths, which is a complex and highly energy demanding process sensitizing these cells to metabolic, oxidative and other forms of stress. Yet, the immediate impact of chronic inflammatory stress like inflammaging on OL homeostasis, myelin maintenance and WMD remains open.MethodsTo functionally analyze the role of IKK/NF-κB signaling in the regulation of myelin homeostasis and maintenance in the adult CNS, we established a conditional mouse model allowing NF-κB activation in mature myelinating oligodendrocytes. IKK2-CAPLP−CreERT2 mice were characterized by biochemical, immunohistochemical, ultrastructural and behavioral analyses. Transcriptome data from isolated, primary OLs and microglia cells were explored by in silico pathway analysis and validated by complementary molecular approaches.ResultsChronic NF-κB activation in mature OLs leads to aggravated neuroinflammatory conditions phenocopying brain inflammaging. As a consequence, IKK2-CAPLP−CreERT2 mice showed specific neurological deficits and impaired motoric learning. Upon aging, persistent NF-κB signaling promotes WMD in these mice as ultrastructural analysis revealed myelination deficits in the corpus callosum accompanied by impaired myelin protein expression. RNA-Seq analysis of primary oligodendrocytes and microglia cells uncovers gene expression signatures associated with activated stress responses and increased post mitotic cellular senescence (PoMiCS) which was confirmed by elevated senescence-associated β-galactosidase activity and SASP gene expression profile. We identified an elevated integrated stress response (ISR) characterized by phosphorylation of eIF2α as a relevant molecular mechanism which is able to affect translation of myelin proteins.ConclusionsOur findings demonstrate an essential role of IKK/NF-κB signaling in mature, post-mitotic OLs in regulating stress-induced senescence in these cells. Moreover, our study identifies PoMICS as an important driving force of age-dependent WMD as well as of traumatic brain injury induced myelin defects.Graphical

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.

Abstract 6614: Spatial transcriptomic driven mechanistic model to investigate and predict pathogenesis of oligodendroglioma

Abstract Rationale: Oligodendrogliomas represent the second most common glioma in adults after glioblastoma. Recent studies conjecture that oligodendrocyte progenitor cells (OPCs) are precursors for glioma. The etiology of OPC derived oligodendrogliomas, the corresponding genetic program and genetic aberrations, and the underlying mechanisms leading to the pathogenesis of oligodendroglioma is yet to be explored. We hypothesize that genetic mechanisms mediating oligodendrogenesis (the process of OPCs maturing to oligodendrocytes (OL)) are temporally and spatially coordinated. In this study, we spearhead a comprehensive computational framework that utilizes a) Machine learning (ML) to interpret spatial transcriptomic (ST) images of a normal whole mouse brain dataset obtained from Vizgen’s MERFISH Mouse Brain Receptor Map and b) mathematical modeling to perturb the cellular mechanisms in the normal brain to understand the abnormalities leading to the pathogenesis of oligodendroglioma. Methods: Using ML for clustering and trajectory inference, we create a comprehensive spatial catalog of cell types. The cell types are validated using canonical genes from the scRNAseq mouse brain data from CZI Biohub’s Tabula Muris Consortium. From the cell types, we extract potential OL pseudotime differentiation (from immature to mature OLs) trajectories. These trajectories are used to develop a phenotype structured partial differentiation equation (PDE) model of oligodendrogenesis. This can be used to simulate both spatial and phenotypic interactions, differentiation status, and the pathogenesis of oligodendroglioma by mathematically perturbing the process of cellular proliferation and differentiation using genes known to cause oligodendroglioma: PDGFRA, OLIG1, MAS1. Results: We mathematically perturb cell differentiation lineages in normal oligodendrogenesis to simulate, and predict the effects of perturbation and how this leads to abnormal (cancer aiding) cell states. Our PDE simulations reinforce that OPC proliferation and OL differentiation occur more in the white matter in the normal mouse brain, and further postulate that OPC and OL differentiation occur more in gray matter in malignant mouse brains. Conclusions: Our study is one of the earliest attempts to integrate qualitative and scalable ML techniques with quantitative and interpretable mathematical modeling at the cellular, omics, and organ level using ST imaging and sequencing data. We develop and calibrate PDE mathematical models for robustly simulating, predicting, and studying the effects of perturbation from sparse, noisy single cell spatial measurements to forecast abnormal behavior in OLs based on spatial and (pseudo)temporal changes. In future work, we will apply our framework to jointly perturb differentiation trajectories of multiple cell types to understand their cumulative effects in tumor progression. Citation Format: Sandhya Prabhakaran, Heyrim Cho, Maximilian Strobl, Russell Rockne, Alexander R. Anderson. Spatial transcriptomic driven mechanistic model to investigate and predict pathogenesis of oligodendroglioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6614.

Progesterone alleviates esketamine-induced hypomyelination via PI3K/Akt signaling pathway in the developing rat brain

ABSTRACT The neurodevelopmental toxicity of anesthetics has been confirmed repeatedly, and esketamine is now widely used in pediatric surgeries. Oligodendrocyte precursor cells (OPCs) evolved into mature oligodendrocytes (OLs) and formed myeline sheath during the early brain development. In this study, we investigated whether esketamine exposure interrupted development of OPCs and induced hypomyelination in rats. Further we explored the roles of PI3K/Akt phosphorylation in OPCs development and myelination. Sprague Dawley rats with different ages (postnatal day (P) 1, 3, 7 and 12) were exposed to 40mg/kg esketamine. Progesterone treatment was given (16 mg/kg per day for 3 days) 24 h after esketamine exposure via the intraperitoneal route. Corpus callosum tissues were collected at P8 or P14 for western blot and immunofluorescence analyses. Esketamine exposure at P7 and P12 significantly reduced myelin basic protein (MBP) expression and CC1+ OLs number in corpus callosum. Esketamine exposure at P7 not only aggravated the mature OLs apoptosis, also decreased the OPCs proliferation and differentiation, which was related with dephosphorylation of PI3K/Akt. Progesterone was able to promote OPCs differentiation and ameliorate esketamine-induced hypomyelination by enhancing PI3K/Akt phosphorylation. Stage-dependent abnormality of OPCs/OLs after esketamine leads to the esketamine-induced hypomyelination. Esketamine interrupted OPCs evolution via PI3K/Akt signaling pathway, which can be ameliorated by progesterone.

Oligodendrocyte-mediated myelin plasticity and its role in neural synchronization.

Temporal synchrony of signals arriving from different neurons or brain regions is essential for proper neural processing. Nevertheless, it is not well understood how such synchrony is achieved and maintained in a complex network of time-delayed neural interactions. Myelin plasticity, accomplished by oligodendrocytes (OLs), has been suggested as an efficient mechanism for controlling timing in brain communications through adaptive changes of axonal conduction velocity and consequently conduction time delays, or latencies; however, local rules and feedback mechanisms that OLs use to achieve synchronization are not known. We propose a mathematical model of oligodendrocyte-mediated myelin plasticity (OMP) in which OLs play an active role in providing such feedback. This is achieved without using arrival times at the synapse or modulatory signaling from astrocytes; instead, it relies on the presence of global and transient OL responses to local action potentials in the axons they myelinate. While inspired by OL morphology, we provide the theoretical underpinnings that motivated the model and explore its performance for a wide range of its parameters. Our results indicate that when the characteristic time of OL's transient intracellular responses to neural spikes is between 10 and 40 ms and the firing rates in individual axons are relatively low (10 Hz), the OMP model efficiently synchronizes correlated and time-locked signals while latencies in axons carrying independent signals are unaffected. This suggests a novel form of selective synchronization in the CNS in which oligodendrocytes play an active role by modulating the conduction delays of correlated spike trains as they traverse to their targets.

Endothelin-1-Endothelin receptor B complex contributes to oligodendrocyte differentiation and myelin deficits during preterm white matter injury.

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.

Hesperetin Ameliorates Inhibition of Neuronal and Oligodendroglial Cell Differentiation Phenotypes Induced by Knockdown of Rab2b, an Autism Spectrum Disorder-Associated Gene Product.

Autism spectrum disorder (ASD) is a central nervous system (CNS) neurodevelopmental disorder that includes autism, pervasive developmental disorder, and Asperger's syndrome. ASD is characterized by repetitive behaviors and social communication deficits. ASD is thought to be a multifactorial disorder with a range of genetic and environmental factors/candidates. Among such factors is the rab2b gene, although it remains unclear how Rab2b itself is related to the CNS neuronal and glial developmental disorganization observed in ASD patients. Rab2 subfamily members regulate intracellular vesicle transport between the endoplasmic reticulum and the Golgi body. To the best of our knowledge, we are the first to report that Rab2b positively regulates neuronal and glial cell morphological differentiation. Knockdown of Rab2b inhibited morphological changes in N1E-115 cells, which are often used as the neuronal cell differentiation model. These changes were accomplished with decreased expression levels of marker proteins in neuronal cells. Similar results were obtained for FBD-102b cells, which are used as the model of oligodendroglial cell morphological differentiation. In contrast, knockdown of Rab2a, which is another Rab2 family member not known to be associated with ASD, affected only oligodendroglial and not neuronal morphological changes. In contrast, treatment with hesperetin, a citrus flavonoid with various cellular protective effects, in cells recovered the defective morphological changes induced by Rab2b knockdown. These results suggest that knockdown of Rab2b inhibits differentiation in neuronal and glial cells and may be associated with pathological cellular phenotypes in ASD and that hesperetin can recover their phenotypes at the in vitro level at least.

Schwann Cell Remyelination in the Multiple Sclerosis Central Nervous System

Multiple sclerosis (MS) is a central nervous system (CNS) demyelinating disease. Failure to remyelinate successfully is common in MS lesions, often with consequent neuronal/axonal damage. CNS myelin is normally produced by oligodendroglial cells. Remyelination by Schwann cells (SchC) has been reported in spinal cord demyelination, in which SchCs are in close proximity to CNS myelin. We identified an MS cerebral lesion that was remyelinated by SchCs. This prompted us to query the extent of SchC remyelination in the brain and spinal cords of additional autopsied MS specimens. CNS tissues were obtained from the autopsies of 14 MS cases. Remyelinated lesions were identified by Luxol fast blue-periodic-acid Schiff and solochrome cyanine staining. Deparaffinized sections containing remyelinated lesions were stained with anti–glial fibrillary acid protein to identify reactive astrocytes. Glycoprotein P zero (P0) is a protein exclusive to peripheral but not CNS myelin. Areas of SchC remyelination were identified by staining with anti-P0. Myelinated regions in the index case cerebral lesion were confirmed to be of SchC origin using anti-P0 staining. Subsequently, 64 MS lesions from 14 autopsied MS cases were examined, and 23 lesions in 6 cases showed remyelination by SchCs. Lesions from the cerebrum, brainstem, and spinal cord were examined in each case. When present, SchC remyelination was most commonly located adjacent to the venules and associated with a lower surrounding density of glial fibrillary acid protein+ reactive astrocytes than areas of only oligodendroglial cell remyelination. The difference was significant only for spinal cord and brainstem lesions but not for lesions located in the brain. In conclusion, we demonstrated SchC remyelination in the cerebrum, brainstem, and spinal cord of 6 autopsied MS cases. To our knowledge, this is the first report of supratentorial SchC remyelination in MS.

Higher throughput workflow with sensitive, reliable and automatic quantification of myelination in vitro suitable for drug screening

Multiple sclerosis (MS) is the most common demyelinating autoimmune disease of the central nervous system (CNS). Immune-mediated myelin and axonal damage that is accompanied by chronic axonal loss causing destruction of the myelin sheaths are hallmarks of MS. While great strides have been made in understanding the molecular underpinnings of re-/myelination, currently no remyelination therapy is available for MS. As myelination is a complex process that is not fully understood, we sought to develop a systematic, reliable, automated and quantitative higher throughput screening method. We aimed to quantitate myelin sheaths in vitro with high sensitivity at the single cell level suitable for testing small compound libraries. To this end, we miniaturised in vitro retinal ganglion cell-oligodendrocyte precursor cell (RGC–OPC) co-cultures into a multi-well plate format. This allowed us to maintain the reciprocal interaction of live axons and oligodendrocytes (OLs) to ensure compact myelin formation. To quantify our co-cultures, we developed a novel computer vision algorithm to precisely measure myelination. We demonstrated efficacy of our system with known pro-differentiating compounds BQ3020 and XAV939 which exhibited robust, efficient, and dose dependent effects on myelination. Through this combination of experimental and technical advances, we have developed a method allowing systematic and reliable testing of remyelinating compound efficacy.

Interactions of glial cells with neuronal synapses, from astrocytes to microglia and oligodendrocyte lineage cells.

The mammalian brain is a complex organ comprising neurons, glia, and more than 1 × 1014 synapses. Neurons are a heterogeneous group of electrically active cells, which form the framework of the complex circuitry of the brain. However, glial cells, which are primarily divided into astrocytes, microglia, oligodendrocytes (OLs), and oligodendrocyte precursor cells (OPCs), constitute approximately half of all neural cells in the mammalian central nervous system (CNS) and mainly provide nutrition and tropic support to neurons in the brain. In the last two decades, the concept of "tripartite synapses" has drawn great attention, which emphasizes that astrocytes are an integral part of the synapse and regulate neuronal activity in a feedback manner after receiving neuronal signals. Since then, synaptic modulation by glial cells has been extensively studied and substantially revised. In this review, we summarize the latest significant findings on how glial cells, in particular, microglia and OL lineage cells, impact and remodel the structure and function of synapses in the brain. Our review highlights the cellular and molecular aspects of neuron-glia crosstalk and provides additional information on how aberrant synaptic communication between neurons and glia may contribute to neural pathologies.

Nerve growth factor promotes differentiation and protects the oligodendrocyte precursor cells from in vitro hypoxia/ischemia.

Nerve growth factor (NGF) is a pleiotropic molecule acting on different cell types in physiological and pathological conditions. However, the effect of NGF on the survival, differentiation and maturation of oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs), the cells responsible for myelin formation, turnover, and repair in the central nervous system (CNS), is still poorly understood and heavily debated. Here we used mixed neural stem cell (NSC)-derived OPC/astrocyte cultures to clarify the role of NGF throughout the entire process of OL differentiation and investigate its putative role in OPC protection under pathological conditions. We first showed that the gene expression of all the neurotrophin receptors (TrkA, TrkB, TrkC, and p75NTR ) dynamically changes during the differentiation. However, only TrkA and p75NTR expression depends on T3-differentiation induction, as Ngf gene expression induction and protein secretion in the culture medium. Moreover, in the mixed culture, astrocytes are the main producer of NGF protein, and OPCs express both TrkA and p75NTR . NGF treatment increases the percentage of mature OLs, while NGF blocking by neutralizing antibody and TRKA antagonist impairs OPC differentiation. Moreover, both NGF exposure and astrocyte-conditioned medium protect OPCs exposed to oxygenglucose deprivation (OGD) from cell death and NGF induces an increase of AKT/pAKT levels in OPCs nuclei by TRKA activation. This study demonstrated that NGF is implicated in OPC differentiation, maturation, and protection in the presence of metabolic challenges, also suggesting implications for the treatment of demyelinating lesions and diseases.

Erythrocyte Plasma Membrane Lipid Composition Mirrors That of Neurons and Glial Cells in Murine Experimental In Vitro and In Vivo Inflammation.

Lipid membrane turnover and myelin repair play a central role in diseases and lesions of the central nervous system (CNS). The aim of the present study was to analyze lipid composition changes due to inflammatory conditions. We measured the fatty acid (FA) composition in erythrocytes (RBCs) and spinal cord tissue (gas chromatography) derived from mice affected by experimental allergic encephalomyelitis (EAE) in acute and remission phases; cholesterol membrane content (Filipin) and GM1 membrane assembly (CT-B) in EAE mouse RBCs, and in cultured neurons, oligodendroglial cells and macrophages exposed to inflammatory challenges. During the EAE acute phase, the RBC membrane showed a reduction in polyunsaturated FAs (PUFAs) and an increase in saturated FAs (SFAs) and the omega-6/omega-3 ratios, followed by a restoration to control levels in the remission phase in parallel with an increase in monounsaturated fatty acid residues. A decrease in PUFAs was also shown in the spinal cord. CT-B staining decreased and Filipin staining increased in RBCs during acute EAE, as well as in cultured macrophages, neurons and oligodendrocyte precursor cells exposed to inflammatory challenges. This regulation in lipid content suggests an increased cell membrane rigidity during the inflammatory phase of EAE and supports the investigation of peripheral cell membrane lipids as possible biomarkers for CNS lipid membrane concentration and assembly.