Regenerative medicine in multiple sclerosis: identifying pharmacological targets of adult neural stem cell differentiation

Progressive axonal loss from chronic demyelination in multiple sclerosis (MS) is the key contributor to clinical decline. Failure to regenerate myelin by adult oligodendrocyte precursor cells (OPCs), a widely distributed neural stem cell population in the adult brain, is one of the major causes of axonal degeneration. In order to develop successful therapies to protect the integrity of axons in MS, it is important to identify and understand the key molecular pathways involved in myelin regeneration (remyelination) by adult OPCs. This review highlights recent findings on the critical signaling pathways associated with OPC differentiation following CNS demyelination. We discuss the role of LINGO-1, Notch, Wnt, and retinoid X receptor (RXR) signaling, and how they might be useful pharmacological targets to overcoming remyelination failure in MS.     

PDGF is Required for Remyelination-Promoting IgM Stimulation of Oligodendrocyte Progenitor Cell Proliferation

Background

Promotion of remyelination is a major goal in treating demyelinating diseases such as multiple sclerosis (MS). The recombinant human monoclonal IgM, rHIgM22, targets myelin and oligodendrocytes (OLs) and promotes remyelination in animal models of MS. It is unclear whether rHIgM22-mediated stimulation of lesion repair is due to promotion of oligodendrocyte progenitor cell (OPC) proliferation and survival, OPC differentiation into myelinating OLs or protection of mature OLs. It is also unknown whether astrocytes or microglia play a functional role in IgM-mediated lesion repair.

Methods

We assessed the effect of rHIgM22 on cell proliferation in mixed CNS glial and OPC cultures by tritiated-thymidine uptake and by double-label immunocytochemistry using the proliferation marker, Ki-67. Antibody-mediated signaling events, OPC differentiation and OPC survival were investigated and quantified by Western blots.

Results

rHIgM22 stimulates OPC proliferation in mixed glial cultures but not in purified OPCs. There is no proliferative response in astrocytes or microglia. rHIgM22 activates PDGFαR in OPCs in mixed glial cultures. Blocking PDGFR-kinase inhibits rHIgM22-mediated OPC proliferation in mixed glia. We confirm in isolated OPCs that rHIgM22-mediated anti-apoptotic signaling and inhibition of OPC differentiation requires PDGF and FGF-2. We observed no IgM-mediated effect in mature OLs in the absence of PDGF and FGF-2.

Conclusion

Stimulation of OPC proliferation by rHIgM22 depends on co-stimulatory astrocytic and/or microglial factors. We demonstrate that rHIgM22-mediated activation of PDGFαR is required for stimulation of OPC proliferation. We propose that rHIgM22 lowers the PDGF threshold required for OPC proliferation and protection, which can result in remyelination of CNS lesions.     

Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation

Oligodendroglial progenitor/precursor cells (OPCs) represent the main cellular source for the generation of new myelinating oligodendrocytes in the adult central nervous system (CNS). In demyelinating diseases such as multiple sclerosis (MS) myelin repair activities based on recruitment, activation and differentiation of resident OPCs can be observed. However, the overall degree of successful remyelination is limited and the existence of an MS-derived anti-oligodendrogenic milieu prevents OPCs from contributing to myelin repair. It is therefore of considerable interest to understand oligodendroglial homeostasis and maturation processes in order to enable the development of remyelination therapies. Mesenchymal stem cells (MSC) have been shown to exert positive immunomodulatory effects, reduce demyelination, increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. We here addressed whether MSC secreted factors can boost the OPC’s oligodendrogenic capacity in a myelin non-permissive environment. To this end, we analyzed cellular morphologies, expression and regulation of key factors involved in oligodendroglial fate and maturation of primary rat cells upon incubation with MSC-conditioned medium. This demonstrated that MSC-derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. Accelerated maturation resulted in elevated levels of myelin expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as Id2 and Id4. We thus conclude that apart from their suggested application as potential anti-inflammatory and immunomodulatory MS treatment, these cells might also be exploited to support endogenous myelin repair activities.     

Phenotypic changes, signaling pathway, and functional correlates of GPR17-expressing neural precursor cells during oligodendrocyte differentiation

The developing and mature central nervous system contains neural precursor cells expressing the proteoglycan NG2. Some of these cells continuously differentiate to myelin-forming oligodendrocytes; knowledge of the destiny of NG2(+) precursors would benefit from the characterization of new key functional players. In this respect, the G protein-coupled membrane receptor GPR17 has recently emerged as a new timer of oligodendrogliogenesis. Here, we used purified oligodendrocyte precursor cells (OPCs) to fully define the immunophenotype of the GPR17-expressing cells during OPC differentiation, unveil its native signaling pathway, and assess the functional consequences of GPR17 activation by its putative endogenous ligands, uracil nucleotides and cysteinyl leukotrienes (cysLTs). GPR17 presence was restricted to very early differentiation stages and completely segregated from that of mature myelin. Specifically, GPR17 decorated two subsets of slowly proliferating NG2(+) OPCs: (i) morphologically immature cells expressing other early proteins like Olig2 and PDGF receptor-α, and (ii) ramified preoligodendrocytes already expressing more mature factors, like O4 and O1. Thus, GPR17 is a new marker of these transition stages. In OPCs, GPR17 activation by either uracil nucleotides or cysLTs resulted in potent inhibition of intracellular cAMP formation. This effect was counteracted by GPR17 antagonists and receptor silencing with siRNAs. Finally, uracil nucleotides promoted and GPR17 inhibition, by either antagonists or siRNAs, impaired the normal program of OPC differentiation. These data have implications for the in vivo behavior of NG2(+) OPCs and point to uracil nucleotides and cysLTs as main extrinsic local regulators of these cells under physiological conditions and during myelin repair.     

LINGO-1, a transmembrane signaling protein, inhibits oligodendrocyte differentiation and myelination through intercellular self-interactions

Overcoming remyelination failure is a major goal of new therapies for demyelinating diseases like multiple sclerosis. LINGO-1, a key negative regulator of myelination, is a transmembrane signaling protein expressed in both neurons and oligodendrocytes. In neurons, LINGO-1 is an integral component of the Nogo receptor complex, which inhibits axonal growth via RhoA. Because the only ligand-binding subunit of this complex, the Nogo receptor, is absent in oligodendrocytes, the extracellular signals that inhibit myelination through a LINGO-1-mediated mechanism are unknown. Here we show that LINGO-1 inhibits oligodendrocyte terminal differentiation through intercellular interactions and is capable of a self-association in trans. Consistent with previous reports, overexpression of full-length LINGO-1 inhibited differentiation of oligodendrocyte precursor cells (OPCs). Unexpectedly, treatment with a soluble recombinant LINGO-1 ectodomain also had an inhibitory effect on OPCs and decreased myelinated axonal segments in cocultures with neurons from dorsal root ganglia. We demonstrated LINGO-1-mediated inhibition of OPCs through intercellular signaling by using a surface-bound LINGO-1 construct expressed ectopically in astrocytes. Further investigation showed that the soluble LINGO-1 ectodomain can interact with itself in trans by binding to CHO cells expressing full-length LINGO-1. Finally, we observed that soluble LINGO-1 could activate RhoA in OPCs. We propose that LINGO-1 acts as both a ligand and a receptor and that the mechanism by which it negatively regulates OPC differentiation and myelination is mediated by a homophilic intercellular interaction. Disruption of this protein-protein interaction could lead to a decrease of LINGO-1 inhibition and an increase in myelination.     

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.     

The inhibitory effects of chondroitin sulfate proteoglycans on oligodendrocytes

The formation of the glial scar following a spinal cord injury presents a significant barrier to the regenerative process. It is primarily composed of chondroitin sulfate proteoglycans (CSPGs) that can inhibit axonal sprouting and regeneration. Although the inhibitory effects on neurons are well documented, little is known about their effects on oligodendrocyte progenitor cells (OPCs). In this study, we examined the effects of CSPGs on OPC process outgrowth and differentiation in vitro. The results show that specific CSPGs, in particularly those highly up-regulated following spinal cord injury, inhibit OPC process outgrowth and differentiation, and that treatment with chondroitinase ABC can completely reverse this inhibition. Additionally, treatment with the Rho kinase inhibitor Y-27632 also reverses the observed inhibition, implicating the activation of Rho kinase in the CSPG inhibition of OPC growth. Taken together, these findings demonstrate that the CSPGs found within the glial scar are not only inhibitory to neurons, but also to OPCs. Moreover, this study shows that chondroitinase ABC treatment, having shown promise in promoting axonal regeneration, may also enhance remyelination.     

Hyaluronan accumulates in demyelinated lesions and inhibits oligodendrocyte progenitor maturation

Demyelination is the hallmark of numerous neurodegenerative conditions, including multiple sclerosis. Oligodendrocyte progenitors (OPCs), which normally mature into myelin-forming oligodendrocytes, are typically present around demyelinated lesions but do not remyelinate affected axons. Here, we find that the glycosaminoglycan hyaluronan accumulates in demyelinated lesions from individuals with multiple sclerosis and in mice with experimental autoimmune encephalomyelitis. A high molecular weight (HMW) form of hyaluronan synthesized by astrocytes accumulates in chronic demyelinated lesions. This form of hyaluronan inhibits remyelination after lysolecithin-induced white matter demyelination. OPCs accrue and do not mature into myelin-forming cells in demyelinating lesions where HMW hyaluronan is present. Furthermore, the addition of HMW hyaluronan to OPC cultures reversibly inhibits progenitor-cell maturation, whereas degrading hyaluronan in astrocyte-OPC cocultures promotes oligodendrocyte maturation. HMW hyaluronan may therefore contribute substantially to remyelination failure by preventing the maturation of OPCs that are recruited to demyelinating lesions.     

Remyelination in the CNS: from biology to therapy

Remyelination involves reinvesting demyelinated axons with new myelin sheaths. In stark contrast to the situation that follows loss of neurons or axonal damage, remyelination in the CNS can be a highly effective regenerative process. It is mediated by a population of precursor cells called oligodendrocyte precursor cells (OPCs), which are widely distributed throughout the adult CNS. However, despite its efficiency in experimental models and in some clinical diseases, remyelination is often inadequate in demyelinating diseases such as multiple sclerosis (MS), the most common demyelinating disease and a cause of neurological disability in young adults. The failure of remyelination has profound consequences for the health of axons, the progressive and irreversible loss of which accounts for the progressive nature of these diseases. The mechanisms of remyelination therefore provide critical clues for regeneration biologists that help them to determine why remyelination fails in MS and in other demyelinating diseases and how it might be enhanced therapeutically.     

CXCR7 Is Involved in Human Oligodendroglial Precursor Cell Maturation

Differentiation of oligodendroglial precursor cells (OPCs), a crucial prerequisite for central nervous system (CNS) remyelination in diseases such as Multiple Sclerosis (MS), is modulated by a multitude of extrinsic and intrinsic factors. In a previous study we revealed that the chemokine CXCL12 stimulates rodent OPC differentiation via activation of its receptor CXCR7. We could now demonstrate that CXCR7 is also expressed on NogoA- and Nkx2.2-positive oligodendroglial cells in human MS brains and that stimulation of cultured primary fetal human OPCs with CXCL12 promotes their differentiation as measured by surface marker expression and morphologic complexity. Pharmacological inhibition of CXCR7 effectively blocks these CXCL12-dependent effects. Our findings therefore suggest that a specific activation of CXCR7 could provide a means to promote oligodendroglial differentiation facilitating endogenous remyelination activities.     

Cdk2 loss accelerates precursor differentiation and remyelination in the adult central nervous system

The specific functions of intrinsic regulators of oligodendrocyte progenitor cell (OPC) division are poorly understood. Type 2 cyclin-dependent kinase (Cdk2) controls cell cycle progression of OPCs, but whether it acts during myelination and repair of demyelinating lesions remains unexplored. Here, we took advantage of a viable Cdk2(-/-) mutant mouse to investigate the function of this cell cycle regulator in OPC proliferation and differentiation in normal and pathological conditions. During central nervous system (CNS) development, Cdk2 loss does not affect OPC cell cycle, oligodendrocyte cell numbers, or myelination. However, in response to CNS demyelination, it clearly alters adult OPC renewal, cell cycle exit, and differentiation. Importantly, Cdk2 loss accelerates CNS remyelination of demyelinated axons. Thus, Cdk2 is dispensable for myelination but is important for adult OPC renewal, and could be one of the underlying mechanisms that drive adult progenitors to differentiate and thus regenerate myelin.     

Interactions between oligodendrocyte precursors control the onset of CNS myelination

The formation of CNS myelin is dependent on the differentiation of oligodendrocyte precursor cells (OPCs) and oligodendrocyte maturation. How the initiation of myelination is regulated is unclear, but it is likely to depend on the development of competence by oligodendrocytes and receptivity by target axons. Here we identify an additional level of control of oligodendrocyte maturation mediated by interactions between the different cellular components of the oligodendrocyte lineage. During development oligodendrocyte precursors mature through a series of stages defined by labeling with monoclonal antibodies A2B5 and O4. Newly differentiated oligodendrocytes begin to express galactocerebroside recognized by O1 antibodies and subsequently mature to myelin basic protein (MBP)-positive cells prior to formation of compact myelin. Using an in vitro brain slice culture system that supports robust myelination, the consequences of ablating cells at different stages of the oligodendrocyte lineage on myelination have been assayed. Elimination of all OPC lineage cells through A2B5+, O4+, and O1+ complement-mediated cell lysis resulted in a delay in development of MBP cells and myelination. Selective elimination of early OPCs (A2B5+) also unexpectedly resulted in delayed MBP expression compared to controls suggesting that early OPCs contribute to the timing of myelination onset. By contrast, elimination of differentiated (O1+) immature oligodendrocytes permanently inhibited the appearance of MBP+ cells suggesting that oligodendrocytes are critical to facilitate the maturation of OPCs. These data illuminate that the presence of intra-lineage feed-forward and feedback cues are important for timely myelination by oligodendrocytes.     

The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS

Remyelination of primary demyelinated lesions is a common feature of experimental models of multiple sclerosis (MS) and is also suggested to be the normal response to demyelination during the early stages of MS itself. Many lines of evidence have shown that remyelination is preceded by the division of endogenous oligodendrocyte precursor cells (OPCs) in the lesion and its borders. It is suggested that this rapid response of OPCs to repopulate the lesion site and their subsequent differentiation into new oligodendrocytes is the key to the rapid remyelination. Antibodies to the NG2 chondroitin sulphate proteoglycan have proved exceedingly useful in following and quantitating the response of endogenous OPCs to demyelination. Here we review the literature on the response of NG2-expressing OPCs to demyelination and provide some new evidence on their response to the chronic inflammatory demyelinating environment seen in recombinant myelin oligodendrocyte glycoprotein (MOG) induced experimental allergic encephalomyelitis (EAE) in the DA rat. NG2-expressing OPCs responded to the inflammatory demyelination in this model by becoming reactive and increasing in number in a very focal manner. Evidence of NG2⁺OPCs in lesioned areas beginning to express the oligodendrocyte marker CNP was also seen. The response of OPCs appeared to occur following successive relapses but did not always lead to remyelination, with areas of chronic demyelination observed in the spinal cord. The presence of OPCs in the adult human CNS is clearly of vital importance for repair in multiple sclerosis (MS). As in rat tissue, the antibody labels an evenly distributed cell population present in both white and grey matter, distinct from HLA-DR⁺microglia. NG2⁺cells are sparsely distributed in the centre of chronic MS lesions. These cells apparently survive demyelination and exhibit a multi-processed or bipolar morphology in the very hypocellular environment of the lesion.     

Protocol to Isolate a Large Amount of Functional Oligodendrocyte Precursor Cells from the Cerebral Cortex of Adult Mice and Humans

During development, oligodendrocytes are generated from oligodendrocyte precursor cells (OPCs), a cell type that is a significant proportion of the total cells (3-8%) in the adult central nervous system (CNS) of both rodents and humans. Adult OPCs are responsible for the spontaneous remyelination that occurs in demyelinating diseases like Multiple Sclerosis (MS) and they constitute an interesting source of cells for regenerative therapy in such conditions. However, there is little data regarding the neurobiology of adult OPCs isolated from mice since an efficient method to isolate them has yet to be established. We have designed a protocol to obtain viable adult OPCs from the cerebral cortex of different mouse strains and we have compared its efficiency with other well-known methods. In addition, we show that this protocol is also useful to isolate functional OPCs from human brain biopsies. Using this method we can isolate primary cortical OPCs in sufficient quantities so as to be able to study their survival, maturation and function, and to facilitate an evaluation of their utility in myelin repair.     

Prostacyclin promotes oligodendrocyte precursor recruitment and remyelination after spinal cord demyelination

Adult oligodendrocyte precursor cells (OPCs) are located adjacent to demyelinated lesion and contribute to myelin repair. The crucial step in remyelination is the migration of OPCs to the demyelinated area; however, the mechanism of OPC migration remains to be fully elucidated. Here we show that prostacyclin (prostaglandin I₂, PGI₂) promotes OPC migration, thereby promoting remyelination and functional recovery in mice after demyelination induced by injecting lysophosphatidylcholine (LPC) into the spinal cord. Prostacyclin analogs enhanced OPC migration via a protein kinase A (PKA)-dependent mechanism, and prostacyclin synthase expression was increased in the spinal cord after LPC injection. Notably, pharmacological inhibition of prostacyclin receptor (IP receptor) impaired remyelination and motor recovery, whereas the administration of a prostacyclin analog promoted remyelination and motor recovery after LPC injection. Our results suggest that prostacyclin could be a key molecule for facilitating the migration of OPCs that are essential for repairing demyelinated areas, and it may be useful in treating disorders characterized by demyelination.     

Therapeutic Effect of Transplanted Human Wharton’s Jelly Stem Cell-Derived Oligodendrocyte Progenitor Cells (hWJ-MSC-derived OPCs) in an Animal Model of Multiple Sclerosis

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). A potential new therapeutic approach for MS is cell transplantation which may promote remyelination. We transplanted human Wharton’s jelly stem cell-derived oligodendrocyte progenitor cells (hWJ-MSC-derived OPCs) into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We studied the effect of the transplanted OPCs on the functional and pathological manifestations of the disease. Transplanted hWJ-MSC-derived OPCs significantly reduced the clinical signs of EAE. Histological examinations showed that remyelination was significantly increased after transplantation. These results suggest that hWJ-MSC-derived OPCs promote the regeneration of myelin sheaths in the brain.     

Silencing or knocking out the Na(+)/Ca(2+) exchanger-3 (NCX3) impairs oligodendrocyte differentiation

Changes in intracellular [Ca(2+)](i) levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation.