Myelin Gene Expression Research Articles

Biallelic EPB41L3 variants underlie a developmental disorder with seizures and myelination defects

Abstract Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3: NM_012307.5), also known as DAL-1, encodes the ubiquitously expressed, neuronally enriched 4.1B protein, part of the 4.1 superfamily of membrane-cytoskeleton adaptors. 4.1B plays key roles in cell spreading, migration, and cytoskeletal scaffolding that support oligodendrocyte axon adhesions essential for proper myelination. We herein describe six individuals from five unrelated families with global developmental delay, intellectual disability, seizures, hypotonia, neuroregression, and delayed myelination. Exome sequencing identified biallelic variants in EPB41L3 in all affected individuals: two nonsense (c.466C>T, p.(R156*); c.2776C>T, p.(R926*)) and three frameshift (c.666delT, p.(F222Lfs*46); c.2289dupC, p.(V764Rfs*19); c.948_949delTG, p.(A317Kfs*33)). Quantitative-real time PCR and Western blot analysis in human fibroblasts harbouring EPB41L3:c.666delT, p.(F222Lfs*46) indicate ablation of EPB41L3 mRNA and 4.1B protein expression. Inhibition of the nonsense mediated decay (NMD) pathway led to an upregulation of EPB41L3:c.666delT transcripts, supporting NMD as a pathogenic mechanism. Epb41l3-deficient mouse oligodendroglia cells showed significant reduction in mRNA expression of key myelin genes, reduced branching, and increased apoptosis. Our report provides the first clinical description of an autosomal recessive disorder associated with variants in EPB41L3, which we refer to as EPB41L3-associated developmental disorder (EADD). Moreover, our functional studies substantiate the pathogenicity of EPB41L3 hypothesized loss-of-function variants.

Transcription Factors Sox8 and Sox10 Contribute with Different Importance to the Maintenance of Mature Oligodendrocytes.

Myelin-forming oligodendrocytes in the vertebrate nervous system co-express the transcription factor Sox10 and its paralog Sox8. While Sox10 plays crucial roles throughout all stages of oligodendrocyte development, including terminal differentiation, the loss of Sox8 results in only mild and transient perturbations. Here, we aimed to elucidate the roles and interrelationships of these transcription factors in fully differentiated oligodendrocytes and myelin maintenance in adults. For that purpose, we conducted targeted deletions of Sox10, Sox8, or both in the brains of two-month-old mice. Three weeks post-deletion, none of the resulting mouse mutants exhibited significant alterations in oligodendrocyte numbers, myelin sheath counts, myelin ultrastructure, or myelin protein levels in the corpus callosum, despite efficient gene inactivation. However, differences were observed in the myelin gene expression in mice with Sox10 or combined Sox8/Sox10 deletion. RNA-sequencing analysis on dissected corpus callosum confirmed substantial alterations in the oligodendrocyte expression profile in mice with combined deletion and more subtle changes in mice with Sox10 deletion alone. Notably, Sox8 deletion did not affect any aspects of the expression profile related to the differentiated state of oligodendrocytes or myelin integrity. These findings extend our understanding of the roles of Sox8 and Sox10 in oligodendrocytes into adulthood and have important implications for the functional relationship between the paralogs and the underlying molecular mechanisms.

Pathologic TDP-43 downregulates myelin gene expression in the monkey brain.

Growing evidence indicates that non-neuronal oligodendrocyte plays an important role in Amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. In patient's brain, the impaired myelin structure is a pathological feature with the observation of TDP-43 in cytoplasm of oligodendrocyte. However, the mechanism underlying the gain of function by TDP-43 in oligodendrocytes, which are vital for the axonal integrity, remains unclear. Recently, we found that the primate-specific cleavage of truncated TDP-43 fragments occurred in cytoplasm of monkey neural cells. This finding opened up the avenue to investigate the myelin integrity affected by pathogenic TDP-43 in oligodendrocytes. In current study, we demonstrated that the truncated TDP-35 in oligodendrocytes specifically, could lead to the dysfunctional demyelination in corpus callosum of monkey. As a consequence of the interaction of myelin regulatory factor with the accumulated TDP-35 in cytoplasm, the downstream myelin-associated genes expression was downregulated at the transcriptional level. Our study aims to investigate the potential effect on myelin structure injury, affected by the truncated TDP-43 in oligodendrocyte, which provided the additional clues on the gain of function during the progressive pathogenesis and symptoms in TDP-43 related diseases.

Sox10 Activity and the Timing of Schwann Cell Differentiation Are Controlled by a Tle4-Dependent Negative Feedback Loop.

The HMG-domain containing transcription factor Sox10 plays a crucial role in regulating Schwann cell survival and differentiation and is expressed throughout the entire Schwann cell lineage. While its importance in peripheral myelination is well established, little is known about its role in the early stages of Schwann cell development. In a search for direct target genes of Sox10 in Schwann cell precursors, the transcriptional co-repressor Tle4 was identified. At least two regions upstream of the Tle4 gene appear involved in mediating the Sox10-dependent activation. Once induced, Tle4 works in tandem with the bHLH transcriptional repressor Hes1 and exerts a dual inhibitory effect on Sox10 by preventing the Sox10 protein from transcriptionally activating maturation genes and by suppressing Sox10 expression through known enhancers of the gene. This mechanism establishes a regulatory barrier that prevents premature activation of factors involved in differentiation and myelin formation by Sox10 in immature Schwann cells. The identification of Tle4 as a critical downstream target of Sox10 sheds light on the gene regulatory network in the early phases of Schwann cell development. It unravels an elaborate regulatory circuitry that fine-tunes the timing and extent of Schwann cell differentiation and myelin gene expression.

Exosomal miR-23a-3p derived from human umbilical cord mesenchymal stem cells promotes remyelination in central nervous system demyelinating diseases by targeting Tbr1/Wnt pathway

Oligodendrocyte precursor cells are present in the adult central nervous system, and their impaired ability to differentiate into myelinating oligodendrocytes can lead to demyelination in patients with multiple sclerosis, accompanied by neurological deficits and cognitive impairment. Exosomes, small vesicles released by cells, are known to facilitate intercellular communication by carrying bioactive molecules. In this study, we utilized exosomes derived from human umbilical cord mesenchymal stem cells (HUMSCs-Exos). We performed sequencing and bioinformatics analysis of exosome-treated cells to demonstrate that HUMSCs-Exos can stimulate myelin gene expression in oigodendrocyte precursor cells. Functional investigations revealed that HUMSCs-Exos activate the Pi3k/Akt pathway and regulate the Tbr1/Wnt signaling molecules through the transfer of miR-23a-3p, promoting oligodendrocytes differentiation and enhancing the expression of myelin-related proteins. In an experimental autoimmune encephalomyelitis model, treatment with HUMSCs-Exos significantly improved neurological function and facilitated remyelination. This study provides cellular and molecular insights into the use of cell-free exosome therapy for central nervous system demyelination associated with multiple sclerosis, demonstrating its great potential for treating demyelinating and neurodegenerative diseases.

Astrocyte ensheathment of calyx-forming axons of the auditory brainstem precedes accelerated expression of myelin genes and myelination by oligodendrocytes.

Early postnatal brain development involves complex interactions among maturing neurons and glial cells that drive tissue organization. We previously analyzed gene expression in tissue from the mouse medial nucleus of the trapezoid body (MNTB) during the first postnatal week to study changes that surround rapid growth of the large calyx of Held (CH) nerve terminal. Here, we present genes that show significant changes in gene expression level during the second postnatal week, a developmental timeframe that brackets the onset of airborne sound stimulation and the early stages of myelination. Gene Ontology analysis revealed that many of these genes are related to the myelination process. Further investigation of these genes using a previously published cell type-specific bulk RNA-Seq data set in cortex and our own single-cell RNA-Seq data set in the MNTB revealed enrichment of these genes in the oligodendrocyte lineage (OL) cells. Combining the postnatal day (P)6-P14 microarray gene expression data with the previously published P0-P6 data provided fine temporal resolution to investigate the initiation and subsequent waves of gene expression related to OL cell maturation and the process of myelination. Many genes showed increasing expression levels between P2 and P6 in patterns that reflect OL cell maturation. Correspondingly, the first myelin proteins were detected by P4. Using a complementary, developmental series of electron microscopy 3D image volumes, we analyzed the temporal progression of axon wrapping and myelination in the MNTB. By employing a combination of established ultrastructural criteria to classify reconstructed early postnatal glial cells in the 3D volumes, we demonstrated for the first time that astrocytes within the mouse MNTB extensively wrap the axons of the growing CH terminal prior to OL cell wrapping and compaction of myelin. Our data revealed significant expression of several myelin genes and enrichment of multiple genes associated with lipid metabolism in astrocytes, which may subserve axon wrapping in addition to myelin formation. The transition from axon wrapping by astrocytes to OL cells occurs rapidly between P4 and P9 and identifies a potential new role of astrocytes in priming calyceal axons for subsequent myelination.

Inhibition of SUMOylation promotes remyelination and reduces IL-17 mediated autoimmune inflammation: Novel approach toward treatment of inflammatory CNS demyelinating disease

Small ubiquitin like modifiers (SUMO) are reversible posttranslational modifiers of intracellular proteins. In the CNS, expression of myelin genes is regulated by state of SUMOylation of their respective transcription factors. In the immune system, deSUMOylation activates innate immune responses and promotes anti-viral immunity. However, the role played by SUMO in an adaptive immune response and in the development of T cell mediated autoimmune disease has not been previously described.TAK981 is a synthetic small molecule which by forming adducts with SUMO proteins prevents SUMOylation. We examined the expression of myelin genes and their transcription factors following culture with TAK981 in Oligodendrocyte Precursor Cells (OPC). We found that myelin basic protein (MBP), a key myelin protein, is upregulated in OPC in the presence of TAK981. We also found increased expression of transcription factors Sox10 and Myrf, which engage in the expression of MBP. In the Cuprizone model of demyelination/remyelination, animals which were treated with TAK981 showed increased remyelination in areas of demyelination and an increase in the number of maturing oligodendrocytes compared to vehicle treated controls. In in vitro cultures of lymphocytes, TAK981 reduced the expression of TH17 in T cells in mice immunized with MOGp35–55. Following in vivo treatment with TAK981, there was a significant reduction in the clinical and pathological severity in mice immunized to develop experimental allergic encephalitis (EAE).The dual effects of deSUMOylation on remyelination and in regulating an autoimmune adaptive response offers a novel approach to the management of human inflammatory demyelinating diseases such as multiple sclerosis.

Inhibiting microglia exacerbates the early effects of cuprizone in males in a rat model of multiple sclerosis, with no effect in females.

Hyper-activity of the brain's innate immune cells, microglia, is a hallmark of multiple sclerosis (MS). However, it is not clear whether this involvement of microglia is beneficial or detrimental or whether manipulating microglial activity may be therapeutic. We investigated if inhibiting microglial activity with minocycline prevents the early changes in oligodendrocyte and myelin-related markers associated with a demyelinating challenge in adult female and male rats. Cuprizone reduced the expression of myelin and oligodendrocyte genes in both females and males, reflective of cuprizone intoxication and the early phases demyelination, and reduced the number of oligodendrocytes in the corpus callosum. However, we see notable differences in the role for microglia in this response between females and males. In males, myelin and oligodendrocyte genes, as well as oligodendrocytes were also reduced by minocycline treatment; an effect that was not seen in females. In males, but not females, early changes in oligodendrocyte and myelin-related genes were associated with microglial proliferation in corpus callosum, and this increase was reversed by minocycline. These data indicate sex-specific effects of inhibiting microglia on the early changes leading to demyelination in an MS model and suggest microglia may play a key role in myelin stability in males but not in females. This highlights a strong need for sex-specific understanding of disease development in MS and suggest that treatments targeting microglia may be more effective in males than in females due to differing mechanisms of disease progression.

Suppression of Experimental Autoimmune Encephalomyelitis in Mice by β-Hydroxy β-Methylbutyrate, a Body-Building Supplement in Humans.

Although several immunomodulatory drugs are available for multiple sclerosis (MS), most present significant side effects with long-term use. Therefore, delineation of nontoxic drugs for MS is an important area of research. β-Hydroxy β-methylbutyrate (HMB) is accessible in local GNC stores as a muscle-building supplement in humans. This study underlines the importance of HMB in suppressing clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of MS. Dose-dependent study shows that oral HMB at a dose of 1 mg/kg body weight/d or higher significantly suppresses clinical symptoms of EAE in mice. Accordingly, orally administered HMB attenuated perivascular cuffing, preserved the integrity of the blood-brain barrier and blood-spinal cord barrier, inhibited inflammation, maintained the expression of myelin genes, and blocked demyelination in the spinal cord of EAE mice. From the immunomodulatory side, HMB protected regulatory T cells and suppressed Th1 and Th17 biasness. Using peroxisome proliferator-activated receptor (PPAR)α-/- and PPARβ-/- mice, we observed that HMB required PPARβ, but not PPARα, to exhibit immunomodulation and suppress EAE. Interestingly, HMB reduced the production of NO via PPARβ to protect regulatory T cells. These results describe a novel anti-autoimmune property of HMB that may be beneficial in the treatment of MS and other autoimmune disorders.

Mineralocorticoid receptor knockout in Schwann cells alters myelin sheath thickness.

Myelination allows fast and synchronized nerve influxes and is provided by Schwann cells (SCs) in the peripheral nervous system. Glucocorticoid hormones are major regulators of stress, metabolism and immunity affecting all tissues. They act by binding to two receptors, the low-affinity glucocorticoid receptor (GR) and the high-affinity mineralocorticoid receptor (MR). Little is known about the effect of glucocorticoid hormones on the PNS, and this study focuses on deciphering the role of MR in peripheral myelination. In this work, the presence of a functional MR in SCs is demonstrated and the expression of MR protein in mouse sciatic nerve SC is evidenced. Besides, knockout of MR in SC (SCMRKO using Cre-lox system with DesertHedgeHog (Dhh) Cre promoter) was undertaken in mice. SCMRKO was not associated with alterations of performance in motor behavioral tests on 2- to 6-month-old male mice compared to their controls. No obvious modifications of myelin gene expression or MR signaling gene expression were observed in the SCMRKO sciatic nerves. Nevertheless, Gr transcript and GR protein amounts were significantly increased in SCMRKO nerves compared to controls, suggesting a possible compensatory effect. Besides, an increase in myelin sheath thickness was noted for axons with perimeters larger than 15 µm in SCMRKO illustrated by a significant 4.5% reduction in g-ratio (axon perimeter/myelin sheath perimeter). Thus, we defined MR as a new player in peripheral system myelination and in SC homeostasis.

The TGR5 Agonist INT-777 Promotes Peripheral Nerve Regeneration by Activating cAMP-dependent Protein Kinase A in Schwann Cells.

Schwann cell (SC) myelination is a pivotal event in the normal physiological functioning of the peripheral nervous system (PNS), where myelination is finely controlled by a series of factors within SCs to ensure timely onset and correct myelin thickness for saltatory conduction. Among these, cyclic AMP (cAMP) is a promising factor for driving myelin gene expression in SCs. It has been shown that TGR5 activation is often associated with increased production of cAMP. Therefore, we speculated that the G-protein-coupled receptor (TGR5) might be involved in the PNS myelination. To test this hypothesis, sciatic nerve crush-injured mice were treated with INT-777, a specific agonist of TGR5, which significantly improved remyelination and functional recovery. Furthermore, rats that underwent sciatic nerve transection were treated with INT-777, which also promoted nerve regeneration and functional recovery. In primary SCs, the stimulatory effect of INT-777 on myelin gene expression was largely counteracted by H89, a potent inhibitor of cAMP-dependent protein kinase A (PKA). Additionally, INT-777 stimulated cell migration was blunted in the presence of H89. Overall, these data indicate that INT-777 is capable of promoting peripheral nerve regeneration and functional recovery after injury, and these benefits are likely due to the activation of the TGR5/cAMP/PKA axis. As such, INT-777, together with other TGR5 agonists, may hold great therapeutic potential for treating peripheral nerve injury.

Evaluation of in vivo lithium chloride effects as a GSK3-β inhibitor on human adipose derived stem cells differentiation into oligodendrocytes and re-myelination in an animal model of multiple sclerosis

The application of neuroprotective agents in combination with stem cells is considered a potential effective treatment for multiple sclerosis (MS). Therefore, the effects of lithium chloride as a neuroprotective agent and a GSK3-β inhibitor were evaluated in combination with human adipose derived stem cells on re-myelination, oligodendrocyte differentiation, and functional recovery. After inducing a mouse model of MS and proving it by the hanging wire test, the mice were randomly assigned to five experimental groups: Cup, Sham, Li, hADSC, and Li + hADSC. Additionally, a control group with normal feeding was considered. Finally, toluidine blue staining was carried out to estimate the level of myelination. Furthermore, immunofluorescent staining was used to evaluate the mean of OLIG2 and MOG positive cells. The mRNA levels of β-Catenin, myelin and oligodendrocyte specific genes were determined via the Real-Time PCR. The results of the hanging wire test and toluidine blue staining showed a significant increase in myelin density and improvements in motor function in groups, which received lithium and stem cells, particularly in the Li + hADSC group compared with the untreated groups (P < 0.01). Moreover, immunostaining results indicated that the mean percentages of MOG and OLIG2 positive cells were significantly higher in the Li + hADSC group than in the other groups (P < 0.01). Finally, gene expression studies indicated that the use of lithium could increase the expression of β-Catenin, myelin and oligodendrocyte specific genes. The use of Lithium Chloride can increase stem cells differentiation into oligodendrocytes and improve re-myelination in MS.

KLF9 and KLF13 transcription factors boost myelin gene expression in oligodendrocytes as partners of SOX10 and MYRF.

Differentiated oligodendrocytes produce myelin and thereby ensure rapid nerve impulse conduction and efficient information processing in the vertebrate central nervous system. The Krüppel-like transcription factor KLF9 enhances oligodendrocyte differentiation in culture, but appears dispensable in vivo. Its mode of action and role within the oligodendroglial gene regulatory network are unclear. Here we show that KLF9 shares its expression in differentiating oligodendrocytes with the closely related KLF13 protein. Both KLF9 and KLF13 bind to regulatory regions of genes that are important for oligodendrocyte differentiation and equally recognized by the central differentiation promoting transcription factors SOX10 and MYRF. KLF9 and KLF13 physically interact and synergistically activate oligodendrocyte-specific regulatory regions with SOX10 and MYRF. Similar to KLF9, KLF13 promotes differentiation and myelination in primary oligodendroglial cultures. Oligodendrocyte differentiation is also altered in KLF13-deficient mice as demonstrated by a transiently reduced myelin gene expression during the first postnatal week. Considering mouse phenotypes, the similarities in expression pattern and genomic binding and the behaviour in functional assays, KLF9 and KLF13 are important and largely redundant components of the gene regulatory network in charge of oligodendrocyte differentiation and myelination.

Korean Red Ginseng and Rb1 facilitate remyelination after cuprizone diet-induced demyelination

BackgroundDemyelination has been observed in neurological disorders, motivating researchers to search for components for enhancing remyelination. Previously we found that Rb1, a major ginsenoside in Korean Red Ginseng (KRG), enhances myelin formation. However, it has not been studied whether Rb1 or KRG function in remyelination after demyelination in vivo. MethodsMice were fed 0.2% cuprizone-containing chow for 5 weeks and returned to normal chow with daily oral injection of vehicle, KRG, or Rb1 for 3 weeks. Brain sections were stained with luxol fast blue (LFB) staining or immunohistochemistry. Primary oligodendrocyte or astrocyte cultures were subject to normal or stress condition with KRG or Rb1 treatment to measure gene expressions of myelin, endoplasmic reticulum (ER) stress, antioxidants and leukemia inhibitory factor (LIF). ResultsCompared to the vehicle, KRG or Rb1 increased myelin levels at week 6.5 but not 8, when measured by the LFB+ or GST-pi+ area within the corpus callosum. The levels of oligodendrocyte precursor cells, astrocytes, and microglia were high at week 5, and reduced afterwards but not changed by KRG or Rb1. In primary oligodendrocyte cultures, KRG or Rb1 increased expression of myelin genes, ER stress markers, and antioxidants. Interestingly, under cuprizone treatment, elevated ER stress markers were counteracted by KRG or Rb1. Under rotenone treatment, reduced myelin gene expressions were recovered by Rb1. In primary astrocyte cultures, KRG or Rb1 decreased LIF expression. ConclusionKRG and Rb1 may improve myelin regeneration during the remyelination phase in vivo, potentially by directly promoting myelin gene expression.

Variants in the zinc transporter TMEM163 cause a hypomyelinating leukodystrophy.

Hypomyelinating leukodystrophies comprise a subclass of genetic disorders with deficient myelination of the CNS white matter. Here we report four unrelated families with a hypomyelinating leukodystrophy phenotype harbouring variants in TMEM163 (NM_030923.5). The initial clinical presentation resembled Pelizaeus-Merzbacher disease with congenital nystagmus, hypotonia, delayed global development and neuroimaging findings suggestive of significant and diffuse hypomyelination. Genomic testing identified three distinct heterozygous missense variants in TMEM163 with two unrelated individuals sharing the same de novo variant. TMEM163 is highly expressed in the CNS particularly in newly myelinating oligodendrocytes and was recently revealed to function as a zinc efflux transporter. All the variants identified lie in highly conserved residues in the cytoplasmic domain of the protein, and functional in vitro analysis of the mutant protein demonstrated significant impairment in the ability to efflux zinc out of the cell. Expression of the mutant proteins in an oligodendroglial cell line resulted in substantially reduced mRNA expression of key myelin genes, reduced branching and increased cell death. Our findings indicate that variants in TMEM163 cause a hypomyelinating leukodystrophy and uncover a novel role for zinc homeostasis in oligodendrocyte development and myelin formation.

A TCF7L2-responsive suppression of both homeostatic and compensatory remyelination in Huntington disease mice.

Huntington's disease (HD) is characterized by defective oligodendroglial differentiation and white matter disease. Here, we investigate the role of oligodendrocyte progenitor cell (OPC) dysfunction in adult myelin maintenance in HD. We first note a progressive, age-related loss of myelin in both R6/2 and zQ175 HD mice compared with wild-type controls. Adult R6/2 mice then manifest a significant delay in remyelination following cuprizone demyelination. RNA-sequencing and proteomic analysis of callosal white matter and OPCs isolated from both R6/2 and zQ175 mice reveals a systematic downregulation of genes associated with oligodendrocyte differentiation and myelinogenesis. Gene co-expression and network analysis predicts repressed Tcf7l2 signaling as a major driver of this expression pattern. Invivo Tcf7l2 overexpression restores both myelin gene expression and remyelination in demyelinated R6/2 mice. These data causally link impaired TCF7L2-dependent transcription to the poor development and homeostatic retention of myelin in HD and provide a mechanism for its therapeutic restoration.

Deletion of the Sodium-Dependent Glutamate Transporter GLT-1 in Maturing Oligodendrocytes Attenuates Myelination of Callosal Axons During a Postnatal Phase of Central Nervous System Development.

The sodium-dependent glutamate transporter GLT-1 (EAAT2, SLC1A2) has been well-described as an important regulator of extracellular glutamate homeostasis in the central nervous system (CNS), a function that is performed mainly through its presence on astrocytes. There is, however, increasing evidence for the expression of GLT-1 in CNS cells other than astrocytes and in functional roles that are mediated by mechanisms downstream of glutamate uptake. In this context, GLT-1 expression has been reported for both neurons and oligodendrocytes (OLGs), and neuronal presynaptic presence of GLT-1 has been implicated in the regulation of glutamate uptake, gene expression, and mitochondrial function. Much less is currently known about the functional roles of GLT-1 expressed by OLGs. The data presented here provide first evidence that GLT-1 expressed by maturing OLGs contributes to the modulation of developmental myelination in the CNS. More specifically, using inducible and conditional knockout mice in which GLT-1 was deleted in maturing OLGs during a peak period of myelination (between 2 and 4 weeks of age) revealed hypomyelinated characteristics in the corpus callosum of preferentially male mice. These characteristics included reduced percentages of smaller diameter myelinated axons and reduced myelin thickness. Interestingly, this myelination phenotype was not found to be associated with major changes in myelin gene expression. Taken together, the data presented here demonstrate that GLT-1 expressed by maturing OLGs is involved in the modulation of the morphological aspects associated with CNS myelination in at least the corpus callosum and during a developmental window that appears of particular vulnerability in males compared to females.

Reactive Astrocytes Derived From Human Induced Pluripotent Stem Cells Suppress Oligodendrocyte Precursor Cell Differentiation.

Astrocytes are instrumental in maintaining central nervous system (CNS) homeostasis and responding to injury. A major limitation of studying neurodegenerative diseases like multiple sclerosis (MS) is lack of human pathological specimens obtained during the acute stages, thereby relegating research to post-mortem specimens obtained years after the initiation of pathology. Rodent reactive astrocytes have been shown to be cytotoxic to neurons and oligodendrocytes but may differ from human cells, especially in diseases with genetic susceptibility. Herein, we purified human CD49f+ astrocytes from induced pluripotent stem cells derived from individual patient and control peripheral leukocytes. We compared TNF and IL1α stimulated human reactive astrocytes from seven persons with MS and six non-MS controls and show their transcriptomes are remarkably similar to those described in rodents. The functional effect of astrocyte conditioned media (ACM) was examined in a human oligodendrocyte precursor cell (OPC) line differentiation assay. ACM was not cytotoxic to the OPCs but robustly inhibited the myelin basic protein (MBP) reporter. No differences were seen between MS and control stimulated astrocytes at either the transcript level or in ACM mediated OPC suppression assays. We next used RNAseq to interrogate differentially expressed genes in the OPC lines that had suppressed differentiation from the human ACM. Remarkably, not only was OPC differentiation and myelin gene expression suppressed, but we observed induction of several immune pathways in OPCs exposed to the ACM. These data support the notion that reactive astrocytes can inhibit OPC differentiation thereby limiting their remyelination capacity, and that OPCs take on an immune profile in the context of inflammatory cues.

Cyclin‐dependent kinase 7 contributes to myelin maintenance in the adult central nervous system and promotes myelin gene expression

Mechanisms regulating oligodendrocyte differentiation, developmental myelination and myelin maintenance in adulthood are complex and still not completely described. Their understanding is crucial for the development of new protective or therapeutic strategies in demyelinating pathologies such as multiple sclerosis. In this perspective, we have investigated the role of Cyclin-dependent kinase 7 (Cdk7), a kinase involved in cell-cycle progression and transcription regulation, in the oligodendroglial lineage. We generated a conditional knock-out mouse model in which Cdk7 is invalidated in post-mitotic oligodendrocytes. At the end of developmental myelination, the number and diameter of myelinated axons, as well as the myelin structure, thickness and protein composition, were normal. However, in young adult and in aged mice, there was a higher number of small caliber myelinated axons associated with a decreased mean axonal diameter, myelin sheaths of large caliber axons were thinner, and the level of some major myelin-associated proteins was reduced. These defects were accompanied by the appearance of an abnormal clasping phenotype. We also used an in vitro oligodendroglial model and showed that Cdk7 pharmacological inhibition led to an altered myelination-associated morphological modification combined with a decreased expression of myelin-specific genes. Altogether, we identified novel functions for Cdk7 in CNS myelination.

Exosomal miR-673-5p from fibroblasts promotes Schwann cell-mediated peripheral neuron myelination by targeting the TSC2/mTORC1/SREBP2 axis

Peripheral myelination is a complicated process, wherein Schwann cells (SCs) promote the formation of the myelin sheath around the axons of peripheral neurons. Fibroblasts are the second resident cells in the peripheral nerves; however, the precise function of fibroblasts in SC-mediated myelination has rarely been examined. Here, we show that exosomes derived from fibroblasts boost myelination-related gene expression in SCs. We used exosome sequencing, together with bioinformatic analysis, to demonstrate that exosomal microRNA miR-673-5p is capable of stimulating myelin gene expression in SCs. Subsequent functional studies revealed that miR-673-5p targets the regulator of mechanistic target of the rapamycin (mTOR) complex 1 (mTORC1) tuberous sclerosis complex 2 in SCs, leading to the activation of downstream signaling pathways including mTORC1 and sterol-regulatory element binding protein 2. In vivo experiments further confirmed that miR-673-5p activates the tuberous sclerosis complex 2/mTORC1/sterol-regulatory element binding protein 2 axis, thus promoting the synthesis of cholesterol and related lipids and subsequently accelerating myelin sheath maturation in peripheral nerves. Overall, our findings revealed exosome-mediated cross talk between fibroblasts and SCs that plays a pivotal role in peripheral myelination. We propose that exosomes derived from fibroblasts and miR-673-5p might be useful for promoting peripheral myelination in translational medicine.