SOX17 transcription factor negatively regulates oligodendrocyte precursor cell differentiation.

Oligodendrocyte development is a critical process timely and spatially regulated to ensure proper myelination of the central nervous system. HMG-box transcription factors are key regulators of oligodendrocyte lineage progression. Among these factors, Sox17 was previously identified as a positive regulator of oligodendrocyte development. However, the role of Sox17 in oligodendroglial cell lineage progression and differentiation is still poorly understood. To define the functional role of Sox17, we generated new transgenic mouse models with inducible overexpression of Sox17, specifically in oligodendroglial cells. Here, we report that gain of Sox17 function has no effect on oligodendrocyte progenitor cells (OPCs) specification. During early postnatal development, Sox17 overexpression increases the pool of OPCs at the expense of differentiated oligodendrocytes. However, the oligodendroglial cell population, OPC proliferation and apoptosis remained unchanged in Sox17 transgenic mice. RNA sequencing, quantitative RT-PCR and immunohistochemical analysis showed that Sox17 represses the expression of the major myelin genes, resulting in a severe CNS hypomyelination. Overall, our data highlight an unexpected role for Sox17 as a negative regulator of OPC differentiation and myelination, suggesting stage specific functions for this factor during oligodendroglial cell lineage progression.

Vitamin D receptor-retinoid X receptor heterodimer signaling regulates oligodendrocyte progenitor cell differentiation.

The mechanisms regulating differentiation of oligodendrocyte (OLG) progenitor cells (OPCs) into mature OLGs are key to understanding myelination and remyelination. Signaling via the retinoid X receptor γ (RXR-γ) has been shown to be a positive regulator of OPC differentiation. However, the nuclear receptor (NR) binding partner of RXR-γ has not been established. In this study we show that RXR-γ binds to several NRs in OPCs and OLGs, one of which is vitamin D receptor (VDR). Using pharmacological and knockdown approaches we show that RXR-VDR signaling induces OPC differentiation and that VDR agonist vitamin D enhances OPC differentiation. We also show expression of VDR in OLG lineage cells in multiple sclerosis. Our data reveal a role for vitamin D in the regenerative component of demyelinating disease and identify a new target for remyelination medicines.

Alteration of synaptic connectivity of oligodendrocyte precursor cells following demyelination.

Oligodendrocyte precursor cells (OPCs) are a major source of remyelinating oligodendrocytes in demyelinating diseases such as Multiple Sclerosis (MS). While OPCs are innervated by unmyelinated axons in the normal brain, the fate of such synaptic contacts after demyelination is still unclear. By combining electrophysiology and immunostainings in different transgenic mice expressing fluorescent reporters, we studied the synaptic innervation of OPCs in the model of lysolecithin (LPC)-induced demyelination of corpus callosum. Synaptic innervation of reactivated OPCs in the lesion was revealed by the presence of AMPA receptor-mediated synaptic currents, VGluT1+ axon-OPC contacts in 3D confocal reconstructions and synaptic junctions observed by electron microscopy. Moreover, 3D confocal reconstructions of VGluT1 and NG2 immunolabeling showed the existence of glutamatergic axon-OPC contacts in post-mortem MS lesions. Interestingly, patch-clamp recordings in LPC-induced lesions demonstrated a drastic decrease in spontaneous synaptic activity of OPCs early after demyelination that was not caused by an impaired conduction of compound action potentials. A reduction in synaptic connectivity was confirmed by the lack of VGluT1+ axon-OPC contacts in virtually all rapidly proliferating OPCs stained with EdU (50-ethynyl-20-deoxyuridine). At the end of the massive proliferation phase in lesions, the proportion of innervated OPCs rapidly recovers, although the frequency of spontaneous synaptic currents did not reach control levels. In conclusion, our results demonstrate that newly-generated OPCs do not receive synaptic inputs during their active proliferation after demyelination, but gain synapses during the remyelination process. Hence, glutamatergic synaptic inputs may contribute to inhibit OPC proliferation and might have a physiopathological relevance in demyelinating disorders.

Demyelination causes adult CNS progenitors to revert to an immature state and express immune cues that support their migration.

The declining efficiency of myelin regeneration in individuals with multiple sclerosis has stimulated a search for ways by which it might be therapeutically enhanced. Here we have used gene expression profiling on purified murine oligodendrocyte progenitor cells (OPCs), the remyelinating cells of the adult CNS, to obtain a comprehensive picture of how they become activated after demyelination and how this enables them to contribute to remyelination. We find that adult OPCs have a transcriptome more similar to that of oligodendrocytes than to neonatal OPCs, but revert to a neonatal-like transcriptome when activated. Part of the activation response involves increased expression of two genes of the innate immune system, IL1ß and CCL2, which enhance the mobilization of OPCs. Our results add a new dimension to the role of the innate immune system in CNS regeneration, revealing how OPCs themselves contribute to the postinjury inflammatory milieu by producing cytokines that directly enhance their repopulation of areas of demyelination and hence their ability to contribute to remyelination.

Gain of Olig2 function in oligodendrocyte progenitors promotes remyelination.

The basic helix-loop-helix transcription factor Olig2 is a key determinant for the specification of neural precursor cells into oligodendrocyte progenitor cells. However, the functional role of Olig2 in oligodendrocyte migration and differentiation remains elusive both during developmental myelination and under demyelinating conditions of the adult central nervous system. To decipher Olig2 functions, we generated transgenic mice (TetOlig2:Sox10(rtTA/+)) overexpressing Olig2 in Sox10(+) oligodendroglial cells in a doxycycline inducible manner. We show that Olig2 overexpression increases the generation of differentiated oligodendrocytes, leading to precocious myelination of the central nervous system. Unexpectedly, we found that gain of Olig2 function in oligodendrocyte progenitor cells enhances their migration rate. To determine whether Olig2 overexpression in adult oligodendrocyte progenitor cells promotes oligodendrocyte regeneration for myelin repair, we induced lysophosphatidylcholine demyelination in the corpus callosum of TetOlig2:Sox10(rtTA/+) and control mice. We found that Olig2 overexpression enhanced oligodendrocyte progenitor cell differentiation and remyelination. To assess the relevance of these findings in demyelinating diseases, we also examined OLIG2 expression in multiple sclerosis lesions. We demonstrate that OLIG2 displays a differential expression pattern in multiple sclerosis lesions that correlates with lesion activity. Strikingly, OLIG2 was predominantly detected in NOGO-A(+) (now known as RTN4-A) maturing oligodendrocytes, which prevailed in active lesion borders, rather than chronic silent and shadow plaques. Taken together, our data provide proof of principle indicating that OLIG2 overexpression in oligodendrocyte progenitor cells might be a possible therapeutic mechanism for enhancing myelin repair.

Early netrin-1 expression impairs central nervous system remyelination.

OBJECTIVE: Chronically demyelinated multiple sclerosis (MS) lesions are frequently characterized by scarce undifferentiated oligodendrocyte progenitor cells (OPCs), suggesting the exhaustion of a local OPC pool followed by failure of recruitment and differentiation. Stimulating prompt OPC recruitment following demyelination could improve myelin repair by providing sufficient numbers of remyelinating cells during the repair-permissive period. Understanding mechanisms that determine this process may have important therapeutic implications. We therefore investigated the role of the guidance molecule netrin-1 in OPC recruitment and central nervous system (CNS) remyelination. METHODS: Netrin-1 expression was analyzed immunohistochemically in different types of MS lesions and in the murine lysolecithin model of demyelination. The influence of netrin-1 on CNS remyelination was examined using gain and loss of function experiments. RESULTS: We show that in MS lesions, astrocytes upregulate netrin-1 expression early during demyelination and netrin-1 receptors are expressed by OPCs. In contrast, in the efficiently repairing lysolecithin model of demyelination (astrocyte-free), netrin-1 expression is absent during early phases and detected concomitant with completion of OPC recruitment. In vitro migration assays demonstrated that netrin-1 is a chemorepellent for migrating adult OPCs. In mouse lesions, antibody-mediated disruption of netrin-1 function at the peak phase of recruitment increased OPC numbers. Conversely, lentiviral-mediated induction of netrin-1 expression prior to OPC recruitment reduced the number of cells recruited and impaired remyelination. INTERPRETATION: Our findings support the conclusion that netrin-1 expression within demyelinating MS plaques blocks OPC recruitment, which with repeated demyelinating episodes contributes to permanent remyelination failure.

Astrocyte-derived endothelin-1 inhibits remyelination through notch activation.

Oligodendrocyte progenitor cells (OPCs) can repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. However, the OPC potential to differentiate can be prevented by inhibitory signals present in the pathological lesion environment. Identification of these signals is essential to promote OPC differentiation and lesion repair. We identified an endogenous inhibitor of remyelination, Endothelin-1 (ET-1), which is highly expressed in reactive astrocytes of demyelinated lesions. Using both gain- and loss-of-function approaches, we demonstrate that ET-1 drastically reduces the rate of remyelination. We also discovered that ET-1 acts mechanistically by promoting Notch activation in OPCs during remyelination through induction of Jagged1 expression in reactive astrocytes. Pharmacological inhibition of ET signaling prevented Notch activation in demyelinated lesions and accelerated remyelination. These findings reveal that ET-1 is a negative regulator of OPC differentiation and remyelination and is potentially a therapeutic target to promote lesion repair in demyelinated tissue.

Tocopherol derivative TFA-12 promotes myelin repair in experimental models of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is associated with demyelination and axonal loss, resulting in severe neurological handicap. Current MS therapies mostly target neuroinflammation but have only a little impact on CNS myelin repair. Progress toward treatments that enhance remyelination would therefore represent major advances in MS treatment. Here, we examined the ability of TFA-12, a new synthetic compound belonging to tocopherol long-chain fatty alcohols, to promote oligodendrocyte regeneration and remyelination in experimental models of MS. We showed that TFA-12 significantly ameliorates neurological deficit and severity of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) in mice. Histological evaluation of mouse EAE spinal cords showed that TFA-12 treatment reduces inflammation, astrogliosis, and myelin loss. Additionally, we demonstrated that TFA-12 accelerates remyelination of focal demyelinated lesions induced by lysolecithin injections. We also found that this compound induces the differentiation of oligodendrocyte precursor cells into mature oligodendrocytes through the inhibition of the Notch/Jagged1 signaling pathway. Altogether, our data provide important proof of principle indicating that TFA-12 could be a potential therapeutic compound for myelin repair in MS.

Ascl1/Mash1 promotes brain oligodendrogenesis during myelination and remyelination.

Oligodendrocytes are the myelin-forming cells of the CNS. They differentiate from oligodendrocyte precursor cells (OPCs) that are produced from progenitors throughout life but more actively during the neonatal period and in response to demyelinating insults. An accurate regulation of oligodendrogenesis is required to generate oligodendrocytes during these developmental or repair processes. We hypothesized that this regulation implicates transcription factors, which are expressed by OPCs and/or their progenitors. Ascl1/Mash1 is a proneural transcription factor previously implicated in embryonic oligodendrogenesis and operating in genetic interaction with Olig2, an essential transcriptional regulator in oligodendrocyte development. Herein, we have investigated the contribution of Ascl1 to oligodendrocyte development and remyelination in the postnatal cortex. During the neonatal period, Ascl1 expression was detected in progenitors of the cortical subventricular zone and in cortical OPCs. Different genetic approaches to delete Ascl1 in cortical progenitors or OPCs reduced neonatal oligodendrogenesis, showing that Ascl1 positively regulated both OPC specification from subventricular zone progenitors as well as the balance between OPC differentiation and proliferation. Examination of remyelination processes, both in the mouse model for focal demyelination of the corpus callosum and in multiple sclerosis lesions in humans, indicated that Ascl1 activity was upregulated along with increased oligodendrogenesis observed in remyelinating lesions. Additional genetic evidence indicated that remyelinating oligodendrocytes derived from Ascl1(+) progenitors/OPCs and that Ascl1 was required for proper remyelination. Together, our results show that Ascl1 function modulates multiple steps of OPC development in the postnatal brain and in response to demyelinating insults.

Inflammation-induced subventricular zone dysfunction leads to olfactory deficits in a targeted mouse model of multiple sclerosis.

Neural stem cells (NSCs) persist in defined brain niches, including the subventricular zone (SVZ), throughout adulthood and generate new neurons destined to support specific neurological functions. Whether brain diseases such as multiple sclerosis (MS) are associated with changes in adult NSCs and whether this might contribute to the development and/or persistence of neurological deficits remains poorly investigated. We examined SVZ function in mice in which we targeted an MS-like pathology to the forebrain. In these mice, which we refer to herein as targeted EAE (tEAE) mice, there was a reduction in the number of neuroblasts compared with control mice. Altered expression of the transcription factors Olig2 and Dlx2 in the tEAE SVZ niche was associated with amplification of pro-oligodendrogenic transit-amplifying cells and decreased neuroblast generation, which resulted in persistent reduction in olfactory bulb neurogenesis. Altered SVZ neurogenesis led to impaired long-term olfactory memory, mimicking the olfactory dysfunction observed in MS patients. Importantly, we also found that neurogenesis was reduced in the SVZ of MS patients compared with controls. Thus, our findings suggest that neuroinflammation induces functional alteration of adult NSCs that may contribute to olfactory dysfunction in MS patients.

Retinoid X receptor gamma signaling accelerates CNS remyelination.

The molecular basis of CNS myelin regeneration (remyelination) is poorly understood. We generated a comprehensive transcriptional profile of the separate stages of spontaneous remyelination that follow focal demyelination in the rat CNS and found that transcripts that encode the retinoid acid receptor RXR-γ were differentially expressed during remyelination. Cells of the oligodendrocyte lineage expressed RXR-γ in rat tissues that were undergoing remyelination and in active and remyelinated multiple sclerosis lesions. Knockdown of RXR-γ by RNA interference or RXR-specific antagonists severely inhibited oligodendrocyte differentiation in culture. In mice that lacked RXR-γ, adult oligodendrocyte precursor cells efficiently repopulated lesions after demyelination, but showed delayed differentiation into mature oligodendrocytes. Administration of the RXR agonist 9-cis-retinoic acid to demyelinated cerebellar slice cultures and to aged rats after demyelination caused an increase in remyelinated axons. Our results indicate that RXR-γ is a positive regulator of endogenous oligodendrocyte precursor cell differentiation and remyelination and might be a pharmacological target for regenerative therapy in the CNS.

Gain-of-function of Olig transcription factors enhances oligodendrogenesis and myelination.

The basic helix-loop-helix transcription factors Olig1 and Olig2 are required for oligodendrocyte specification and differentiation during central nervous system (CNS) development but the effects of overexpression of these factors in murine development are not well understood. To test whether Olig1 and Olig2 may reprogram CNS stem/progenitors toward an oligodendroglial fate for myelination, we generated transgenic mice with doxycycline (Dox)-inducible expression of Olig1 or Olig2 in nestin-expressing stem/progenitor cells of the CNS. Overexpression of Olig1 or Olig2 from E8.5 to E12.5 was sufficient to promote the generation of platelet-derived growth factor receptor alpha + oligodendrocyte precursors (OPCs) in the spinal cord. We also demonstrated that overexpression of Olig2, but not Olig1, enhanced the stem/progenitor cell proliferation and generation of motoneuron precursors and inhibited the development of V3 interneurons. In the postnatal brain, Dox-inducible expression of Olig2 but not Olig1 in nestin+ stem/progenitors of the subventricular zone increased the generation of OPCs that migrated and differentiated into mature oligodendrocytes in the corpus callosum, cortex and olfactory bulb, leading to increased and precocious myelination. Altogether, our data indicate that Olig2 is a potential therapeutic target to enhance myelination and remyelination in the CNS.

Directing human neural stem/precursor cells into oligodendrocytes by overexpression of Olig2 transcription factor.

Multipotential neural stem/precursor cells of the central nervous system were extensively studied for their properties of generating myelinating oligodendrocytes both in vitro and in vivo upon engraftment in animal models of myelin disorders, such as leucodystrophy and multiple sclerosis. These studies provided proof-of-principle that efficient myelination can be achieved by cell transplantation. However, one major drawback of cell-based therapy of myelin diseases is the difficulty in generating oligodendrocytes efficiently from human fetal neural stem/precursor cells (hNPC). Here we explored whether overexpression of the basic helix-loop-helix (bHLH) transcription factor Olig2 in fetal hNPC could enhance the generation of oligodendrocytes both in vitro and in vivo. We report that transduction of hNPC with Olig2-encoding lentiviral vectors enhances their commitment toward an oligodendroglial fate. Moreover, Olig2-transduced hNPC, grafted into the dysmyelinated shiverer mouse brain, survived up to 9 weeks, migrated extensively, and differentiated into MBP(+) myelinating oligodendrocytes. In contrast, control hNPC remained at a less mature stage and generated very few myelinating oligodendrocytes. Our study indicates that bHLH transcription factors, such as Olig2, are interesting targets for directing hNPC into myelinating oligodendrocytes.

The role of SVZ-derived neural precursors in demyelinating diseases: from animal models to multiple sclerosis.

We will review the role of endogenous neural stem cells in myelin repair both in animal models of demyelination and multiple sclerosis. The mammalian sub-ventricular zone (SVZ) is the largest germinative zone of the adult brain, which contains a well characterized stem cell niche. While most studies highlight the neurogenic potential of SVZ progenitors, recent data indicate that SVZ cells become reactived in response to different pathological cues, like trauma, ischemia, neurodegeneration, inflammation and demyelination. Experimental models of demyelination in rodent demonstrate enhanced proliferation and recruitment of SVZ progenitors into myelin lesions, in response to demyelination. Moreover, cell lineage tracing experiments showed that SVZ progenitor cells can give rise to oligodendrocytes in demyelinated lesions, that could potentially contribute to remyelination. To examine the relevance of these studies in myelin diseases, we recently examined the human SVZ in post-mortem MS brains. The human SVZ is characterized by a ribbon of SVZ astrocytes lining the ependymal border of the lateral ventricles and which behave as multipotential progenitors in vitro. We showed that cellular density and proliferation were enhanced in MS SVZ compared to non-neurological controls. This high cellular density was correlated with the increased number of progenitor cells in MS SVZ, as well as in sub-ventricular lesions. Interestingly, some of these progenitors expressed transcription factors involved in oligodendrogenesis, such as Sox9, Olig2 and Sox10. These data indicate that gliogenesis occurs also in MS SVZ and suggest the recruitment of SVZ-derived oligodendrocyte precursors to peri-ventricular demyelinated lesions. Further investigation of adult neural stem cells and their progenitors in the brain of rodents and non-human primates should help to gain insights in their process of activation in response to demyelination and their role in myelin repair.

Activation of the subventricular zone in multiple sclerosis: evidence for early glial progenitors.

In multiple sclerosis (MS), oligodendrocyte and myelin destruction lead to demyelination with subsequent axonal loss. Experimental demyelination in rodents has highlighted the activation of the subventricular zone (SVZ) and the involvement of progenitor cells expressing the polysialylated form of neural cell adhesion molecule (PSA-NCAM) in the repair process. In this article, we studied the distribution of early PSA-NCAM(+) progenitors in the SVZ and MS lesions in human postmortem brains. Compared with controls, MS SVZ showed a 2- to 3-fold increase in cell density and proliferation, which correlated with enhanced numbers of PSA-NCAM(+) and glial fibrillary acidic protein-positive (GFAP(+)) cells. PSA-NCAM(+) progenitors mainly were Sox9(+), and a few expressed Sox10 and Olig2, markers of oligodendroglial specification. PSA-NCAM(+) progenitors expressing Sox10 and Olig2 also were detected in demyelinated MS lesions. In active and chronic active lesions, the number of PSA-NCAM(+) progenitors was 8-fold higher compared with chronic silent lesions, shadow plaques, and normal-appearing white matter. In active and chronic active lesions, PSA-NCAM(+) progenitors were more frequent in periventricular lesions (30-50%) than in lesions remote from the ventricular wall. These data indicate that, as in rodents, activation of gliogenesis in the SVZ occurs in MS and suggest the mobilization of SVZ-derived early glial progenitors to periventricular lesions, where they could give rise to oligodendrocyte precursors. These early glial progenitors could be a potential target for therapeutic strategies designed to promote myelin repair in MS.

Identification of Sox17 as a transcription factor that regulates oligodendrocyte development.

Microarray analysis of oligodendrocyte lineage cells purified by fluorescence-activated cell sorting (FACS) from 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-enhanced green fluorescent protein (EGFP) transgenic mice revealed Sox17 (SRY-box containing gene 17) gene expression to be coordinately regulated with that of four myelin genes during postnatal development. In CNP-EGFP-positive (CNP-EGFP+) cells, Sox17 mRNA and protein levels transiently increased between postnatal days 2 and 15, with white matter O4+ preoligodendrocytes expressing greater Sox17 levels than Nkx2.2+ (NK2 transcription factor related, locus 2) NG2+, or GalC+ (galactocerebroside) cells. In spinal cord, Sox17 protein expression was undetectable in the primary motor neuron domain between embryonic days 12.5 and 15.5 but was evident in Nkx2.2+ and CC1+ cells. In cultured oligodendrocyte progenitor cells (OPCs), Sox17 levels were maximal in O4+ cells and peaked during the phenotypic conversion from bipolar to multipolar. Parallel increases in Sox17 and p27 occurred before MBP protein expression, and Sox17 upregulation was prevented by conditions inhibiting differentiation. Sox17 downregulation with small interfering RNAs increased OPC proliferation and decreased lineage progression after mitogen withdrawal, whereas Sox17 overexpression in the presence of mitogen had opposite effects. Sox17 overexpression enhanced myelin gene expression in OPCs and directly stimulated MBP gene promoter activity. These findings support important roles for Sox17 in controlling both oligodendrocyte progenitor cell cycle exit and differentiation.