Isolation and Culture of Mouse Primary Microglia and Oligodendrocyte Precursor Cells.

Microglia and oligodendrocyte precursor cells (OPCs) are critical glia subsets in the central nervous system (CNS) and are actively engaged in a body of diseases, such as stroke, Alzheimer's disease, multiple sclerosis, etc. Microglia and OPC serve as compelling tools for the study of CNS diseases as well as the repair and damage of myelin sheath in vitro. In this protocol, we summarized a method which is capable of using the same batch of new-born mice to isolate and culture microglia and OPCs. It integrates the characteristics of practicality, convenience, and efficiency, providing a convenient, easy, and reliable technique for research.

Interaction between Oligodendrocytes and Interneurons in Brain Development and Related Neuropsychiatric Disorders.

A variety of neurological and psychiatric disorders have recently been shown to be highly associated with the abnormal development and function of oligodendrocytes (OLs) and interneurons. OLs are the myelin-forming cells in the central nervous system (CNS), while interneurons are important neural types gating the function of excitatory neurons. These two types of cells are of great significance for the establishment and function of neural circuits, and they share similar developmental origins and transcriptional architectures, and interact with each other in multiple ways during development. In this review, we compare the similarities and differences in these two cell types, providing an important reference and further revealing the pathogenesis of related brain disorders.

Conversion of Astrocyte Cell Lines to Oligodendrocyte Progenitor Cells Using Small Molecules and Transplantation to Animal Model of Multiple Sclerosis.

Astrocytes, the most prevalent cells in the central nervous system (CNS), can be transformed into neurons and oligodendrocyte progenitor cells (OPCs) using specific transcription factors and some chemicals. In this study, we present a cocktail of small molecules that target different signaling pathways to promote astrocyte conversion to OPCs. Astrocytes were transferred to an OPC medium and exposed for five days to a small molecule cocktail containing CHIR99021, Forskolin, Repsox, LDN, VPA and Thiazovivin before being preserved in the OPC medium for an additional 10 days. Once reaching the OPC morphology, induced cells underwent immunocytofluorescence evaluation for OPC markers while checked for lacking the astrocyte markers. To test the in vivo differentiation capabilities, induced OPCs were transplanted into demyelinated mice brains treated with cuprizone over 12 weeks. Two distinct lines of astrocytes demonstrated the potential of conversion to OPCs using this small molecule cocktail as verified by morphological changes and the expression of PDGFR and O4 markers as well as the terminal differentiation to oligodendrocytes expressing MBP. Following transplantation into demyelinated mice brains, induced OPCs effectively differentiated into mature oligodendrocytes. The generation of OPCs from astrocytes via a small molecule cocktail may provide a new avenue for producing required progenitors necessary for myelin repair in diseases characterized by the loss of myelin such as multiple sclerosis.

Iron deficiency in astrocytes alters cellular status and impacts on oligodendrocyte differentiation.

Iron deficiency (ID) has been shown to affect central nervous system (CNS) development and induce hypomyelination. Previous work from our laboratory in a gestational ID model showed that both oligodendrocyte (OLG) and astrocyte (AST) maturation was impaired. To explore the contribution of AST iron to the myelination process, we generated an in vitro ID model by silencing divalent metal transporter 1 (DMT1) in AST (siDMT1 AST) or treating AST with Fe3+ chelator deferoxamine (DFX; DFX AST). siDMT1 AST showed no changes in proliferation but remained immature. Co-cultures of oligodendrocyte precursors cells (OPC) with siDMT1 AST and OPC cultures incubated with siDMT1 AST-conditioned media (ACM) rendered a reduction in OPC maturation. These findings correlated with a decrease in the expression of AST-secreted factors IGF-1, NRG-1, and LIF, known to promote OPC differentiation. siDMT1 AST also displayed increased mitochondrial number and reduced mitochondrial size as compared to control cells. DFX AST also remained immature and DFX AST-conditioned media also hampered OPC maturation in culture, in keeping with a decrease in the expression of AST-secreted growth factors IGF-1, NRG-1, LIF, and CNTF. DFX AST mitochondrial morphology and number showed results similar to those observed in siDMT1 AST. In sum, our results show that ID, induced through two different methods, impacts AST maturation and mitochondrial functioning, which in turn hampers OPC differentiation.

BRG1 programs PRC2-complex repression and controls oligodendrocyte differentiation and remyelination.

Chromatin-remodeling protein BRG1/SMARCA4 is pivotal for establishing oligodendrocyte (OL) lineage identity. However, its functions for oligodendrocyte-precursor cell (OPC) differentiation within the postnatal brain and during remyelination remain elusive. Here, we demonstrate that Brg1 loss profoundly impairs OPC differentiation in the brain with a comparatively lesser effect in the spinal cord. Moreover, BRG1 is critical for OPC remyelination after injury. Integrative transcriptomic/genomic profiling reveals that BRG1 exhibits a dual role by promoting OPC differentiation networks while repressing OL-inhibitory cues and proneuronal programs. Furthermore, we find that BRG1 interacts with EED/PRC2 polycomb-repressive-complexes to enhance H3K27me3-mediated repression at gene loci associated with OL-differentiation inhibition and neurogenesis. Notably, BRG1 depletion decreases H3K27me3 deposition, leading to the upregulation of BMP/WNT signaling and proneurogenic genes, which suppresses OL programs. Thus, our findings reveal a hitherto unexplored spatiotemporal-specific role of BRG1 for OPC differentiation in the developing CNS and underscore a new insight into BRG1/PRC2-mediated epigenetic regulation that promotes and safeguards OL lineage commitment and differentiation.

Discovery and Optimization of Selective Brain-Penetrant EBP Inhibitors that Enhance Oligodendrocyte Formation.

The inhibition of emopamil binding protein (EBP), a sterol isomerase within the cholesterol biosynthesis pathway, promotes oligodendrocyte formation, which has been proposed as a potential therapeutic approach for treating multiple sclerosis. Herein, we describe the discovery and optimization of brain-penetrant, orally bioavailable inhibitors of EBP. A structure-based drug design approach from literature compound 1 led to the discovery of a hydantoin-based scaffold, which provided balanced physicochemical properties and potency and an improved in vitro safety profile. The long half-lives of early hydantoin-based EBP inhibitors in rodents prompted an unconventional optimization strategy, focused on increasing metabolic turnover while maintaining potency and a brain-penetrant profile. The resulting EBP inhibitor 11 demonstrated strong in vivo target engagement in the brain, as illustrated by the accumulation of EBP substrate zymostenol after repeated dosing. Furthermore, compound 11 enhanced the formation of oligodendrocytes in human cortical organoids, providing additional support for our therapeutic hypothesis.

The proton-sensing receptors TDAG8 and GPR4 are differentially expressed in human and mouse oligodendrocytes: Exploring their role in neuroinflammation and multiple sclerosis.

Acidosis is one of the hallmarks of demyelinating central nervous system (CNS) lesions in multiple sclerosis (MS). The response to acidic pH is primarily mediated by a family of G protein-coupled proton-sensing receptors: OGR1, GPR4 and TDAG8. These receptors are inactive at alkaline pH, reaching maximal activation at acidic pH. Genome-wide association studies have identified a locus within the TDAG8 gene associated with several autoimmune diseases, including MS. Accordingly, we here found that expression of TDAG8, as opposed to GPR4 or OGR1, is upregulated in MS plaques. This led us to investigate the expression of TDAG8 in oligodendrocytes using mouse and human in vitro and in vivo models. We observed significant upregulation of TDAG8 in human MO3.13 oligodendrocytes during maturation and in response to acidic conditions. However, its deficiency did not impact normal myelination in the mouse CNS, and its expression remained unaltered under demyelinating conditions in mouse organotypic cerebellar slices. Notably, our data revealed no expression of TDAG8 in primary mouse oligodendrocyte progenitor cells (OPCs), in contrast to its expression in primary human OPCs. Our investigations have revealed substantial species differences in the expression of proton-sensing receptors in oligodendrocytes, highlighting the limitations of the employed experimental models in fully elucidating the role of TDAG8 in myelination and oligodendrocyte biology. Consequently, the study does not furnish robust evidence for the role of TDAG8 in such processes. Nonetheless, our findings tentatively point towards a potential association between TDAG8 and myelination processes in humans, hinting at a potential link between TDAG8 and the pathophysiology of MS and warrants further research.

Ageing impairs the regenerative capacity of regulatory T cells in mouse central nervous system remyelination.

Myelin regeneration (remyelination) is essential to prevent neurodegeneration in demyelinating diseases such as Multiple Sclerosis, however, its efficiency declines with age. Regulatory T cells (Treg) recently emerged as critical players in tissue regeneration, including remyelination. However, the effect of ageing on Treg-mediated regenerative processes is poorly understood. Here, we show that expansion of aged Treg does not rescue age-associated remyelination impairment due to an intrinsically diminished capacity of aged Treg to promote oligodendrocyte differentiation and myelination in male and female mice. This decline in regenerative Treg functions can be rescued by a young environment. We identified Melanoma Cell Adhesion Molecule 1 (MCAM1) and Integrin alpha 2 (ITGA2) as candidates of Treg-mediated oligodendrocyte differentiation that decrease with age. Our findings demonstrate that ageing limits the neuroregenerative capacity of Treg, likely limiting their remyelinating therapeutic potential in aged patients, and describe two mechanisms implicated in Treg-driven remyelination that may be targetable to overcome this limitation.

PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

Oligodendrocyte progenitor cells (OPCs) differentiate into myelin-producing cells and modulate neuronal activity. Defects in OPC development are associated with neurological diseases. N6-methyladenosine (m6A) contributes to neural development; however, the mechanism by which m6A regulates OPC development remains unclear. Here, we demonstrate that PRRC2B is an m6A reader that regulates OPC development and myelination. Nestin-Cre-mediated Prrc2b deletion affects neural stem cell self-renewal and glial differentiation. Moreover, the oligodendroglia lineage-specific deletion of Prrc2b reduces the numbers of OPCs and oligodendrocytes, causing hypomyelination and impaired motor coordination. Integrative methylated RNA immunoprecipitation sequencing, RNA sequencing, and RNA immunoprecipitation sequencing analyses identify Sox2 as the target of PRRC2B. Notably, PRRC2B, displaying separate and cooperative functions with PRRC2A, stabilizes mRNA by binding to m6A motifs in the coding sequence and 3' UTR of Sox2. In summary, we identify the posttranscriptional regulation of PRRC2B in OPC development, extending the understanding of PRRC2 family proteins and providing a therapeutic target for myelin-related disorders.

The use of a SOX10 reporter toward ameliorating oligodendrocyte lineage differentiation from human induced pluripotent stem cells.

Oligodendrocytes (OLs) are key players in the central nervous system, critical for the formation and maintenance of the myelin sheaths insulating axons, ensuring efficient neuronal communication. In the last decade, the use of human induced pluripotent stem cells (iPSCs) has become essential for recapitulating and understanding the differentiation and role of OLs in vitro. Current methods include overexpression of transcription factors for rapid OL generation, neglecting the complexity of OL lineage development. Alternatively, growth factor-based protocols offer physiological relevance but struggle with efficiency and cell heterogeneity. To address these issues, we created a novel SOX10-P2A-mOrange iPSC reporter line to track and purify oligodendrocyte precursor cells. Using this reporter cell line, we analyzed an existing differentiation protocol and shed light on the origin of glial cell heterogeneity. Additionally, we have modified the differentiation protocol, toward enhancing reproducibility, efficiency, and terminal maturity. Our approach not only advances OL biology but also holds promise to accelerate research and translational work with iPSC-derived OLs.

Contrasting somatic mutation patterns in aging human neurons and oligodendrocytes.

Characterizing somatic mutations in the brain is important for disentangling the complex mechanisms of aging, yet little is known about mutational patterns in different brain cell types. Here, we performed whole-genome sequencing (WGS) of 86 single oligodendrocytes, 20 mixed glia, and 56 single neurons from neurotypical individuals spanning 0.4-104 years of age and identified >92,000 somatic single-nucleotide variants (sSNVs) and small insertions/deletions (indels). Although both cell types accumulate somatic mutations linearly with age, oligodendrocytes accumulated sSNVs 81% faster than neurons and indels 28% slower than neurons. Correlation of mutations with single-nucleus RNA profiles and chromatin accessibility from the same brains revealed that oligodendrocyte mutations are enriched in inactive genomic regions and are distributed across the genome similarly to mutations in brain cancers. In contrast, neuronal mutations are enriched in open, transcriptionally active chromatin. These stark differences suggest an assortment of active mutagenic processes in oligodendrocytes and neurons.

Extracellular vesicles released by microglia and macrophages carry endocannabinoids which foster oligodendrocyte differentiation.

Introduction: Microglia and macrophages can influence the evolution of myelin lesions through the production of extracellular vesicles (EVs). While microglial EVs promote in vitro differentiation of oligodendrocyte precursor cells (OPCs), whether EVs derived from macrophages aid or limit OPC maturation is unknown. Methods: Immunofluorescence analysis for the myelin protein MBP was employed to evaluate the impact of EVs from primary rat macrophages on cultured OPC differentiation. Raman spectroscopy and liquid chromatography-mass spectrometry was used to define the promyelinating lipid components of myelin EVs obtained in vitro and isolated from human plasma. Results and discussion: Here we show that macrophage-derived EVs do not promote OPC differentiation, and those released from macrophages polarized towards an inflammatory state inhibit OPC maturation. However, their lipid cargo promotes OPC maturation in a similar manner to microglial EVs. We identify the promyelinating endocannabinoids anandamide and 2-arachidonoylglycerol in EVs released by both macrophages and microglia in vitro and circulating in human plasma. Analysis of OPC differentiation in the presence of the endocannabinoid receptor antagonists SR141716A and AM630 reveals a key role of vesicular endocannabinoids in OPC maturation. From this study, EV-associated endocannabinoids emerge as important mediators in microglia/macrophage-oligodendrocyte crosstalk, which may be exploited to enhance myelin repair.

Recognizing Alzheimer's disease from perspective of oligodendrocytes: Phenomena or pathogenesis?

BACKGROUND: Accumulation of amyloid beta, tau hyperphosphorylation, and microglia activation are the three highly acknowledged pathological factors of Alzheimer's disease (AD). However, oligodendrocytes (OLs) were also widely investigated in the pathogenesis and treatment for AD. AIMS: We aimed to update the regulatory targets of the differentiation and maturation of OLs, and emphasized the key role of OLs in the occurrence and treatment of AD. METHODS: This review first concluded the targets of OL differentiation and maturation with AD pathogenesis, and then advanced the key role of OLs in the pathogenesis of AD based on both clinic and basic experiments. Later, we extensively discussed the possible application of the current progress in the diagnosis and treatment of this complex disease. RESULTS: Molecules involving in OLs' differentiation or maturation, including various transcriptional factors, cholesterol homeostasis regulators, and microRNAs could also participate in the pathogenesis of AD. Clinical data point towards the impairment of OLs in AD patients. Basic research further supports the central role of OLs in the regulation of AD pathologies. Additionally, classic drugs, including donepezil, edaravone, fluoxetine, and clemastine demonstrate their potential in remedying OL impairment in AD models, and new therapeutics from the perspective of OLs is constantly being developed. CONCLUSIONS: We believe that OL dysfunction is one important pathogenesis of AD. Factors regulating OLs might be biomarkers for early diagnosis and agents stimulating OLs warrant the development of anti-AD drugs.

SRF transcriptionally regulates the oligodendrocyte cytoskeleton during CNS myelination.

Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)-a transcription factor known to regulate expression of actin and actin regulators in other cell types-as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the mechanistic role of SRF in oligodendrocyte lineage cells. Here, we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in oligodendrocyte precursor cells and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Surprisingly, oligodendrocyte-restricted loss of SRF results in upregulation of gene signatures associated with aging and neurodegenerative diseases. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies an essential pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease.

Deletion of Slc9a1 in Cx3cr1+ cells stimulated microglial subcluster CREB1 signaling and microglia-oligodendrocyte crosstalk.

Microglial Na/H exchanger-1 (NHE1) protein, encoded by Slc9a1, plays a role in white matter demyelination of ischemic stroke brains. To explore underlying mechanisms, we conducted single cell RNA-seq transcriptome analysis in conditional Slc9a1 knockout (cKO) and wild-type (WT) mouse white matter tissues at 3 days post-stroke. Compared to WT, Nhe1 cKO brains expanded a microglial subgroup with elevated transcription of white matter myelination genes including Spp1, Lgals3, Gpnmb, and Fabp5. This subgroup also exhibited more acidic pHi and significantly upregulated CREB signaling detected by ingenuity pathway analysis and flow cytometry. Moreover, the Nhe1 cKO white matter tissues showed enrichment of a corresponding oligodendrocyte subgroup, with pro-phagocytosis and lactate shuffling gene expression, where activated CREB signaling is a likely upstream regulator. These findings demonstrate that attenuation of NHE1-mediated H+ extrusion acidifies microglia/macrophage and may underlie the stimulation of CREB1 signaling, giving rise to restorative microglia-oligodendrocyte interactions for remyelination.

Oligodendrocyte-derived exosomes-containing SIRT2 ameliorates depressive-like behaviors and restores hippocampal neurogenesis and synaptic plasticity via the AKT/GSK-3ß pathway in depressed mice.

AIMS: To investigate the antidepressant role of oligodendrocyte-derived exosomes (ODEXs)-containing sirtuin 2 (SIRT2) and the underlying mechanism both in vivo and in vitro. METHODS: Oligodendrocyte-derived exosomes isolated from mouse serum were administered to mice with chronic unpredictable mild stress (CUMS)-induced depression via the tail vein. The antidepressant effects of ODEXs were assessed through behavioral tests and quantification of alterations in hippocampal neuroplasticity. The role of SIRT2 was confirmed using the selective inhibitor AK-7. Neural stem/progenitor cells (NSPCs) were used to further validate the impact of overexpressed SIRT2 and ODEXs on neurogenesis and synapse formation in vitro. RESULTS: Oligodendrocyte-derived exosome treatment alleviated depressive-like behaviors and restored neurogenesis and synaptic plasticity in CUMS mice. SIRT2 was enriched in ODEXs, and blocking SIRT2 with AK-7 reversed the antidepressant effects of ODEXs. SIRT2 overexpression was sufficient to enhance neurogenesis and synaptic protein expression. Mechanistically, ODEXs mediated transcellular delivery of SIRT2, targeting AKT deacetylation and AKT/GSK-3ß signaling to regulate neuroplasticity. CONCLUSION: This study establishes how ODEXs improve depressive-like behaviors and hippocampal neuroplasticity and might provide a promising therapeutic approach for depression.

Oligodendrocyte Maturation Alters the Cell Death Mechanisms That Cause Demyelination.

Myelinating oligodendrocytes die in human disease and early in aging. Despite this, the mechanisms that underly oligodendrocyte death are not resolved and it is also not clear whether these mechanisms change as oligodendrocyte lineage cells are undergoing differentiation and maturation. Here, we used a combination of intravital imaging, single-cell ablation, and cuprizone-mediated demyelination, in both female and male mice, to discover that oligodendrocyte maturation dictates the dynamics and mechanisms of cell death. After single-cell phototoxic damage, oligodendrocyte precursor cells underwent programmed cell death within hours, differentiating oligodendrocytes died over several days, while mature oligodendrocytes took weeks to die. Importantly cells at each maturation stage all eventually died but did so with drastically different temporal dynamics and morphological features. Consistent with this, cuprizone treatment initiated a caspase-3-dependent form of rapid cell death in differentiating oligodendrocytes, while mature oligodendrocytes never activated this executioner caspase. Instead, mature oligodendrocytes exhibited delayed cell death which was marked by DNA damage and disruption in poly-ADP-ribose subcellular localization. Thus, oligodendrocyte maturation plays a key role in determining the mechanism of death a cell undergoes in response to the same insult. This means that oligodendrocyte maturation is important to consider when designing strategies for preventing cell death and preserving myelin while also enhancing the survival of new oligodendrocytes in demyelinating conditions.

Engineered PDGFA-ligand-modified exosomes delivery T3 for demyelinating disease targeted therapy.

Demyelination is a proper syndrome in plenty of central nervous system (CNS) diseases, which is the main obstacle to recovery and still lacks an effective treatment. To overcome the limitations of the brain-blood barrier on drug permeability, we modified an exosome secreted by neural stem cells (NSCs), which had transfected with lentivirus armed with platelet-derived growth factors A (PDGFA)-ligand. Through the in vivo and in vitro exosomes targeting test, the migration ability to the lesion areas and OPCs significantly improved after ligand modification. Furthermore, the targeted exosomes loaded with 3,5, 30-L-triiodothyronine (T3) have a critical myelination ability in CNS development, administrated to the cuprizone animal model treatment. The data shows that the novel drug vector loaded with T3 significantly promotes remyelination compared with T3 alone. At the same time, it improved the CNS microenvironment by reducing astrogliosis, inhibiting pro-inflammatory microglia, and alleviating axon damage. This investigation provides a straightforward strategy to produce a targeting exosome and indicates a possible therapeutic manner for demyelinating disease.

Erinacine S, a small active component derived from Hericium erinaceus, protects oligodendrocytes and alleviates mood abnormalities in cuprizone-exposed rodents.

Hericium erinaceus mycelium extract (HEM), containing erinacine A (HeA) and erinacine S (HeS), has shown promise in promoting the differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs), crucial for myelin production in the central nervous system (CNS). The main aim of this study was to characterize the protective effects of HEM and its components on OLs and myelin in demyelinating rodents by exposure to cuprizone (CPZ), a copper chelating agent commonly used to induce demyelination in the corpus callosum of the brain. Rats were fed by CPZ-containing diet and simultaneously orally administered HEM, HeA, or HeS on a daily basis for three weeks. We found that HEM and HeS preserved myelin and OLs in the corpus callosum of CPZ-fed rats, along with reduced microglia and astrocyte activation, and downregulated IL-1ß expression. Furthermore, post-treatment with HeS, in mouse models with acute (6 weeks) or chronic (12 weeks) CPZ-induced demyelination demonstrated oral administration during the final 4 weeks (HeS4/6 or HeS4/12) effectively preserved myelin in the corpus callosum. Additionally, HeS4/6 and HeS4/12 inhibited anxious and depressive-like behaviors in CPZ-fed mice. In summary, simultaneous administration of HEM and HeS in rats during short-term CPZ intoxication preserved OLs and myelin. Furthermore, post-administration of HeS not only inhibited demyelination and gliosis but also alleviated anxiety and depression in both acute and chronic CPZ-fed mice. This study presents compelling evidence supporting the potential of HeS as a promising small active compound for protecting OLs and preserving myelin in demyelinating diseases associated with emotional disorders.