Attempts to Overcome Remyelination Failure: Toward Opening New Therapeutic Avenues for Multiple Sclerosis

Abstract

Multiple sclerosis (MS) is a chronic immune-mediated disorder of the central nervous system that results in destruction of the myelin sheath wrapped around the axons and eventual axon degeneration. The disease is pathologically heterogeneous; however, perhaps its most frustrating aspect is the lack of efficient regenerative response for remyelination. Current treatment strategies are based on anti-inflammatory or immunomodulatory medications that have the potential to reduce the numbers of newly evolving lesions. However, therapies are still required that can repair already damaged myelin for which current treatments are not effective. A prerequisite for the development of such new treatments is understanding the reasons for insufficient endogenous repair. This review briefly summarizes the currently suggested causes of remyelination failure in MS and possible solutions.

Introduction

In healthy central nervous system (CNS), myelin allows transmission of impulses via saltatory conduction, in a quick and energy-efficient manner. Myelin also provides physical protection and metabolic support for axons. Most of severe neurological diseases such as multiple sclerosis (MS) are associated with the pathological damage or loss of myelin sheaths, a process called demyelination. The mammalian CNS has a remarkable ability for repair and regenerate damaged myelin. This process, which is called remyelination, is a default spontaneous or intrinsic process by which new myelin sheaths are generated around demyelinated axons in the adult CNS, reinstating the saltatory conduction and resolving functional deficits (Shen et al. 2008).

MS is the most common demyelinating disorder of the CNS. The pathological hallmarks of MS are inflammation, breakdown of the blood–brain barrier (BBB), demyelination, remyelination, multifocal lesions, and axonal degeneration. In some MS patients, remyelination occurs successfully early in the disease; however, for the majority of patients, this recovery process gradually fails while demyelination continues. This may lead to subsequent degeneration of axons and eventually loss of neurons. Neuronal loss might underlie the clinical disability observed in patients following chronic demyelination, highlighting the need for treatments that promote both remyelination and neuronal survival. Current treatments for MS are mainly immunomodulatory or anti-inflammatory drugs, which act primarily by modulating or suppressing the immune system and inflammation. However, although they can decelerate the demyelinating course and reduce clinical deterioration, approaches that promote endogenous remyelination for already established demyelinated lesions are still lacking.

Remyelination is carried out by an endogenous population of adult oligodendrocyte progenitor cells (OPCs). OPCs are widespread throughout the CNS, encompassing approximately 3–4% of cells in the gray matter and 7–8% in white matter (Horner et al. 2000; Dawson et al. 2003). Moreover, adult neural precursor cells from the subventricular zone contribute significantly to oligodendrocyte regeneration and remyelination (Xing et al. 2014; Mozafari et al. 2010,2011). Unlike neurons, which have limited capacity to regenerate after CNS injury, adult OPCs have significant ability to proliferate and generate mature oligodendrocytes, capable of remyelinating the injured axons (Moyon et al. 2015). Despite this regenerative ability of these cells, still the question remains: why do many axons remain demyelinated in the CNS of MS patients? Understanding why endogenous remyelination often fails in MS is crucial to the development of effective remyelination therapies.

For remyelination to occur, OPCs must proliferate and migrate to the lesion then differentiate into mature oligodendrocytes. Mature oligodendrocytes extend processes toward axons, make contact to them and enwrap them with concentric layers of myelin membrane, and finally compact these layers into functional myelin (Miron et al. 2011). In theory, remyelination can be blocked at any point in the remyelination process including proliferation of OPCs, their recruitment towards the lesion, their differentiation into oligodendrocytes, and attachment of mature oligodendrocytes to the axons. Recently, several laboratories have begun to explore the molecular pathology that limits remyelination in hopes of development approaches for therapeutically enhancing remyelination in MS patients. In the following, we discuss some postulates for failure of remyelination in MS (Fig. 1).

Fig. 1.

Transforming growth factor-β 1 (TGF-β1) secreted by resident glial cells stimulates astrocytes in MS lesions to re-express the Notch ligand Jagged 1. Contact-mediated activation Notch signaling by ligand Jagged 1 inhibits OPC differentiation. Lingo-1, expressed by neurons and oligodendrocytes, is up-regulated in MS lesions, leading to OPC maturation inhibition and ultimate remyelination failure. The compounds of extracellular matrix including CSPGs, Hyaluronan, Tenascin C and R, produced through leakage from the damaged vasculature of BBB and secreted from reactive glial cells are often inhibitory to oligodendrocyte process extension and remyelination of axons. Deregulation of OPC guidance factors including semaphorins, netrins, and various chemokines are also contributed to remyelination failure. Production of PSA-NCAM by damaged axons is another inhibitor of remyelination through impairment of oligodendrocyte attachment to axons. Myelin debris remained following myelin sheet unravels and disperses from axons which are another inhibitory block for remyelination

Intracellular Pathways that Block Remyelination

Notch1 Pathway

Expressed by oligodendrocytes, Notch1 receptor is a well-known regulator of OPC maturation in the developing CNS (Wang et al. 1998). Notch1 interacts with membrane-bound ligands, Jagged1 and Delta, activating the canonical pathway that increases the downstream effecter, Hes5. In turn, Hes5 inhibits OPC differentiation. When cultured human OPCs were exposed to Jagged1, the OPCs failed to mature (John et al. 2002; Mi et al. 2009). Within and around active MS plaques lacking remyelination, Jagged1 was found to be expressed by reactive astrocytes, via stimulation with transforming growth factor-β1, (TGF- β1) an up-regulated cytokine in MS (John et al. 2002; Mi et al. 2009). Thus, re-expression of Jagged1 has been proposed to hamper remyelination in MS via activation of Notch1 pathway and inhibition of OPCs differentiation (John et al. 2002; Mi et al. 2009). However, the results of available in vivo studies provide conflicting evidence regarding a role for Notch1-Jagged1 in failure of adult CNS remyelination. First, in experimental models of demyelination, alterations of Jagged1 and Notch1 expression were not in agreement with what was found for MS lesions (Stidworthy et al. 2004). The experiment showed that Jagged1 and Notch1 were expressed in OPCs from experimental ethidium bromide-induced demyelination model, despite complete remyelination (Stidworthy et al. 2004), indicating that remyelination may occur in the presence of active Notch signaling. Similarly in EAE model of MS, Notch1-positive oligodendrocytes were exclusively present in lesions with ongoing remyelination and not found in lesions without signs of remyelination (Seifert et al. 2007). Secondly, conditional ablation of Notch1 in PLP-expressing oligodendrocytes of cuprizone-treated transgenic mice (proteolipid protein (PLP)⁻creERNotch^lox/lox mice) did not produce a marked effect on remyelination, based on G ratio and percent remyelination (Stidworthy et al. 2004). However, one possibility explaining this conflicting result is that PLP⁺ oligodendrocytes may be too far along in maturation to respond to Jagged1/Notch1 inhibitory signaling in this experiment. In support of this possibility, conditional deletion of Notch1 in oligodendrocytes using Olig1Cre:Notch1, instead promoted precocious oligodendrocyte maturation (Zhang et al. 2009). Remyelination was extensive in these mice as well. Therefore, Notch1 signaling may block oligodendrocyte maturation in earlier stages of differentiation. In addition, an in vitro myelination experiment targeting Notch1 by siRNA confirmed that Notch1 signals inhibit OPCs differentiation and ultimately remyelination following toxin-induced demyelination of the corpus callosum (Mi et al. 2009). Overall, the reports of available animal studies make the role of Notch1 in remyelination of MS lesions complicated, leaving the role of Notch1 signaling on OPC differentiation during remyelination in vivo inconclusive.

LINGO1 Pathway and Nogo Receptor

LINGO-1 is a CNS-specific single transmembrane glycoprotein expressed in neurons and in oligodendrocytes but absent in astrocytes during development (Mi et al. 2004). It belongs to a large family of Leucine-rich-domain Ig-containing proteins involved in neurogenesis, axon guidance, and myelination during normal development (Mi et al. 2004). LINGO-1 associates with the Nogo-66 receptor complex (NgR1 complex) and exerts control over developmental myelination in vitro and in vivo, through a homophilic–hemophilic interaction and a Ras homolog gene family member A (RhoA)-mediated mechanism (Mi et al. 2005). Various molecular and genetic tools including loss of function studies using siRNA, blocking antibodies, and null-mutant mice (Mi et al. 2005, 2007, 2009) as well as gain of function studies using lentiviral overexpression and transgenic mice (Lee et al. 2007; Mi et al. 2005) have reported LINGO-1 as a negative regulator of OPC differentiation and myelination. In vitro, experimental attenuation of LINGO-1 resulted in down-regulation of RhoA activity, a small GTPase involved in cytoskeletal dynamics, implicated in oligodendrocyte differentiation (Burridge and Wennerberg 2004). This, in turn, propels the in vitro differentiation of OPCs. Conversely, overexpression of LINGO-1 leads to activation of RhoA and inhibition of oligodendrocyte differentiation and myelination (Mi et al. 2005). LINGO-1 expression is reported to be up-regulated during CNS injury across diverse animal models and human CNS diseases (Mi et al. 2007; Inoue et al. 2007; Fernandez-Enright et al. 2014). Because impaired remyelination is a pathological hallmark of MS, antagonizing LINGO-1 was supposed to provide therapeutic target for the treatment of this disease. However, recent studies have reported that this approach failed to show significant therapeutic outcomes in MS patients. Still, poor trial design and not optimally developed drug are two possible causes of this failure.

Moreover, major myelin-associated inhibitory factors, NogoA and myelin-associated glycoprotein (MAG), exhibit their inhibitory effect through binding to NgR1. This receptor is expressed by neurons and other neural cells including neural stem cells, OPCs, astrocytes, Schwann cells, microglia as well as non-neural cells. It is well documented that inhibition of NgR can functionally enhance axonal repair and also lead to repopulation of progenitor cell and increase of myelinogenic potential of oligodendrocytes (Pourabdolhossein et al. 2014; Yang et al. 2010; Steinbach et al. 2011).

Wnt Pathway

Wnt signaling is another well-described developmental pathway implicated in preventing myelination during development and possibly preventing remyelination. This pathway prevents OPCs exit from the cell cycle and arrests their differentiation (Mi et al. 2009). Treatment of cord explants or primary mixed glial cultures with Wnt-conditioned media suppresses oligodendrocyte development. Moreover, pharmacological inhibition of GSK3ß, a downstream of Wnt signaling, resulted in inhibition of oligodendrocyte maturation without affecting on OPC numbers (Shimizu et al. 2005).

A whole-genome screening study for transcription factors (TFs) expressed during remyelination, especially those expressed with the oligodendrocyte lineage, revealed specific expression of Tcf4 (also called TCF7L2), a critical intranuclear component of canonical Wnt signaling within remyelinating lesions—but not normal adult white matter (Fancy et al. 2009). Moreover, in a gene microarray analysis, MS lesions have been shown to express multiple Wnt signaling genes, including Wnt2, Wnt7a, β- catenin, Tcf4, and GSK3β, and their expression levels in active plaques were higher than in chronic silent plaques and normal-appearing white matter (Lock et al. 2002). Moreover, a proteomic analysis revealed the same phenomenon showing an up-regulated expression of Wnt signaling-related proteins (including Wnt3a, APC, and β-catenin) in MS lesions; the highest expression being observed in chronic active plaques (Han et al. 2008). Results from a blood RNA profiling study in a large cohort of MS patients also supported consideration of the Wnt pathway as a player in MS pathogenesis (Nickles et al. 2013). An explanation about activation of this pathway in remyelination is that in the initial stages of remyelination, a large number of OPCs are generated for myelin repair. It is necessary that OPCs do not exit cell cycle too early and differentiate before a sufficient numbers of cells have been generated (Casaccia-Bonnefil and Liu 2003). In order to ensure this, it seems probable that inhibitory regulators of differentiation such as Wnt are activated in OPCs. When these cells reached the appropriate number, these inhibitory pathways need to be switched off allowing the cells to then become responsive to inducers of differentiation and completion of remyelination. This model, however, requires very precise timing, whose dysregulation might be involved in differentiation failure. Altogether, these evidences of Wnt pathway activity in human MS lesions suggest that its dysregulation might contribute to inefficient myelin repair in MS patients.

RXR-γ Pathway

Several lines of evidence suggested that RXR-γ signaling might be involved in remyelination. RXR-γ is a nuclear receptor that dimerizes with other receptors, including retinoic acid receptors, thyroid hormone receptors, vitamin D receptors, and peroxisome proliferator activator receptors (PPARs) to modulate cell differentiation, proliferation, and apoptosis (Germain et al. 2006). In the healthy mammalian CNS, it is expressed at low levels by all glial cell types (Moreno et al. 2004). However, its expression is increased by activated microglia or macrophages, reactive astrocytes, and oligodendrocytes after CNS myelin injury (Huang et al. 2011b). The results of microarray analysis from demyelinated lesions of rodent brain at different stages of remyelination indicated RXR-γ as a positive regulator of endogenous remyelination (Huang et al. 2011b). OPCs express this receptor, and treatment of OPCs with an RXR-γ antagonist in vitro has led to impaired OPC maturation. In contrast, incubation with a RXR agonist, 9-cis-retinoic acid stimulated OPC differentiation and induction of remyelination in culture. Moreover, focal demyelinating lesions in RXR-γ knockout mice were associated with accumulation of immature OPCs, and treatment of rats with the RXR-γ agonist improves remyelination. In MS, RXR-γ expression increases in the nuclear component of OPCs in active lesion borders but is decreased in chronic inactive lesions, suggesting RXR-γ plays a positive role in remyelination (Huang et al. 2011b).

ECM Compounds as Modulators of Remyelination

A hallmark of CNS injury is the activation and proliferation of local glial cells, including microglia, astrocytes, and oligodendrocytes. Reactive glial cells, in particular astrocytes and microglia, deposit extracellular matrix proteins and increase the production of molecules that are often inhibitory to regeneration, the presses that eventually forms glial scar. In MS patients, ECM composition changes occur in both active and chronic lesions, as well as in the normal-appearing white matter. Aberrant deposition of ECM molecules occurs by leakage from the damaged vasculature and BBB disruption and by altered secretion from reactive glial cells, in particular astrocytes and microglia. The aberrant deposition of ECM molecules then creates a lesion environment that is often inhibitory to oligodendrocyte process extension and remyelination of axons, which in turn leads to increasing neuronal damage as the disease progresses (Wheeler and Fuss 2016; Lau et al. 2013). These are dynamic processes and the level of each ECM molecule changes as lesions progress and recover. The degree of remyelination likely results from the net ratios of supportive and inhibitory ECM molecules (e.g., merosin, CSPGs, tenascins, hyaluronan, fibronectin aggregates) within and around the lesion. In the following, we will discuss briefly the ECM compounds with inhibitory effects on remyelination. ECM is mainly secreted by reactive astrocytes; therefore, the removal of astrocytes may improve remyelination. As glial scars are distributed within the CNS in MS setting, removal of gliosis through surgery-like approaches is not feasible. Recently, engineering of these astrocytes into functional cells, such as neurons or oligodendrocytes has provided a new strategy for cellular regeneration in CNS injuries. Currently transcription factors, small molecules, and microRNAs are administrated as mediators of this cell conversion. For instance, forced expression of Oct4 and miR-302/367 converted the transduced astrocytes into oligodendrocyte lineage cells, in vivo (Dehghan et al. 2016; Ghasemi-Kasman et al. 2016). Moreover, previous work showed direct conversion of astrocytes into neuroblasts by miR-302/367, both in vivo and in vitro (Ghasemi-Kasman et al. 2015).

Chondroitin Sulfate Proteoglycans (CSPGs)

CSPGs consist of a protein core and a varying numbers of long-sulfated unbranched negatively charged glycosaminoglycan (GAG) chains made up of repeating disaccharide units. High levels of CSPGs are present in the glial scars formed after many types of CNS insults including MS lesions (Mohan et al. 2010). After injury, reactive astrocytes and macrophages up-regulate CSPGs expression and OPCs themselves become reactive, change morphologically, and increase expression of CSPGs (Chen et al. 2002; Massey et al. 2008). Thus, oligodendrocytes are exposed to very high levels of CSPGs after CNS damage. Direct exposure of OPCs to CSPGs revealed a significant inhibition of process outgrowth, morphological differentiation as well as OPC migration (Lau et al. 2012; Siebert et al. 2011; Kippert et al. 2009; Siebert and Osterhout 2011). Thus, high levels of GSPGs in MS lesions environment contribute to the remyelination aversive.

Protein tyrosine phosphatase sigma (PTPσ), which is expressed by oligodendrocytes, has been identified as a functional receptor for CSPGs in oligodendrocytes. This receptor appears to be a mediator of the inhibitory effects of CSPG on oligodendrocytes process outgrowth and capacity to remyelinate. Knockdown of PTPσ also reversed the inhibition of process outgrowth on CSPGs (Pendleton et al. 2013). PTPσ activation by CSPGs likely impedes myelination by interfering with oligodendrocytes process extension towards axons, rather than by inhibiting OPCs differentiation directly. Since immature OPCs express low levels of CSPG receptors, they are able to migrate into active lesion areas, despite the high CSPG concentration. As OPCs mature, the levels of PTPσ expression are increased. Thus, differentiating oligodendrocytes, PTPσ negatively, regulate Rho signaling pathway that promotes cytoskeletal rearrangements necessary for process extension and maturation (Pendleton et al. 2013). This is in agreement with the studies that report that CSPG-mediated inhibition of oligodendrocyte process outgrowth acts through Rho-associated protein kinase (ROCK) and in vitro studies show that inhibition of ROCK can reverse the inhibitory effects of CSPGs on oligodendrocyte process outgrowth and differentiation (Pendleton et al. 2013; Siebert and Osterhout 2011).

Fibronectin and osteopontin are two ECM molecules increased in demyelinating injury (Zhao et al. 2008; Selvaraju et al. 2004; Stoffels et al. 2013). During the process of demyelination these compounds are leaked from the blood circulation or produced by CNS resident cells including astrocytes. Temporal dimeric fibronectin expression is suggested to play an important role in remyelination at earlier stages of MS. However, increased aggregations of fibronectin within chronically demyelinated MS lesions contribute to remyelination failure predominantly via perturbed OPCs differentiation and subsequent impairment of remyelination (Stoffels et al. 2013). Thus strategies to promote remyelination should predominantly interfere with fibronectin aggregation, without preventing fibronectin deposition.

The production of osteopontin is also unregulated in demyelinated areas that undergo spontaneous and complete remyelination (Woodruff and Franklin 1999). The osteopontin indicated to have capacity to induction of proliferation and maturation of oligodendrocyte-derived cell lines and also enhancement of remyelination (Selvaraju et al. 2004). However, studies on osteopontin^−/− mice showed that osteopontin plays a major role in governing the efficiency of remyelination (Zhao et al. 2008).

Hyaluronan-TLR2 Pathway

Accumulation of high molecular weight (HMW) hyaluronan (900–1000 kDa), produced by astrocytes, is another factor that has been implicated in the inhibition of OPCs differentiation in chronic MS lesions as well as in experimental demyelination models (Back et al. 2005). Culturing rat oligosphere-derived OPCs in the presence of HMW resulted in a significant reduction in the expression of myelin basic protein (MBP), a marker for mature oligodendrocytes. Furthermore, the addition of HMW hyaluronan to OPC cultures reversibly inhibits progenitor-cell maturation, whereas degrading hyaluronan in astrocyte-OPC co-cultures promotes oligodendrocyte maturation, supporting the notion that astrocyte-derived HMW is able to inhibit the differentiation of OPC (Back et al. 2005). In addition, expression of Toll-like receptor2 (TLR2), a receptor for hyaluronan, by oligodendrocytes was found to be up-regulated in MS lesions. Neutralizing antibodies to TLR2 lead to blockage of the inhibitory effect of HMW hyaluronan on OPC maturation, supporting the role for hyaluronan as a negative regulator for oligodendroglial differentiation via a TLR2 signaling pathway (Back et al. 2005; Sloane et al. 2010).

Tenascins

Besides adding to ECM structural stability, tenascins influence oligodendrocyte survival, proliferation, migration, differentiation, and morphological maturation (Czopka et al. 2009; Kiernan et al. 1996). Tenascin R (Tnr), expressed by oligodendrocytes themselves and by postnatal astrocytes, is necessary for the timely differentiation of oligodendrocytes in vitro. Conversely, Tenascin C (Tnc) expressed by radial glia cells at embryonic and by astrocytes at early postnatal stages exerts a strong inhibitory influence on OPC differentiation (Fuss et al. 1993). However, in contrast to their antagonistic effect on differentiation, both Tnc and Tnr similarly inhibited morphologic maturation by reduction of process elaboration and membrane expansion of OPCs through interfering with the activation of the small GTPase RhoA (Czopka et al. 2009). During the evolution of MS lesions, Tnc and Tnr remodeling occurs. In acute MS lesions, Tnc and Tnr glycoproteins are lost, and the loss extends into the normal-appearing white matter (Gutowski et al. 1999). As lesions become chronic, further reactivation of astrocytes increases the production of Tnc and Tnr, which might lead to the chronic inhibitory situation of glial scar formation.

Contribution of OPC Guidance Factors in Remyelination Failure

Failure of OPC recruitment into areas of demyelination may also arise due to disturbances in the local expression of the OPC migration guidance cues. A number of OPC guidance cues, whether repellant or attractive, have been implicated as regulators of OPC migration in developmental myelination and MS remyelination (Boyd et al. 2013; Tepavcevic et al. 2014). The most important long-range guidance cues include netrins and semaphorins. Chemokines, that are mainly produced by astrocytes, also play important roles in migration and maturation of glial cells.

Semaphorins

Semaphorins, a large family of secreted or transmembrane and glycophosphatidylinositol-anchored proteins, originally described as axonal growth cone guidance molecules, are among the most important OPC guidance cues. Two members of Semaphorins, called Semaphorin 3A (sema 3A) and sema 3F, have been shown to play important roles in the regulation of OPC migration (Spassky et al. 2002; Sugimoto et al. 2001). OPCs express Neuropilin 1 (NP1) and NP2, which are sema 3A and sema 3F receptors, respectively. During development, binding of sema 3A receptors to NP1 respells OPCs migration. Conversely, binding of sema 3F to NP2 leads to attraction of these cells (Cohen et al. 2003; Piaton et al. 2011; Spassky et al. 2002).

In adulthood, the mRNA expression of sema 3A and 3F in white matter disappears. However, the results obtained from toxin-induced demyelination model, showed that they are re-expressed and regulate OPC migration in demyelinating lesions. In the case of MS lesions, both Semaphorins are re-expressed as well. However, their expressions differ with regard to the degree of inflammatory activity and type of lesion, with active lesions (more inflammatory with ongoing remyelination) containing higher expression of the chemoattractant sema 3F than sema 3A, and chronic active lesions (less inflammatory and less likely to remyelinate) with higher expression of the chemorepellent sema 3A than sema 3F (Williams et al. 2007). Interestingly, the expression of their receptors is restricted to the cells available in the plaque and periplaque areas and, in contrast, neuropilin-1/2-positive cells seem to be absent in normal-appearing white matter (Syed et al. 2011). It is noteworthy that sema 3A also induces a reversible, dose-dependent inhibition of OPC differentiation. Thus, upregulation of sema 3A potentially prevents OPCs recruited into demyelinated lesions from differentiating into myelin-forming oligodendrocytes, which is in agreement with the findings that the presence of sema 3A in demyelinated lesion is associated with a strong impairment of remyelination (Syed et al. 2011). These findings may, at least partially, explain why most chronic active MS lesions are less likely to remyelinate, whereas majority of active MS lesions are likely to remyelinate.

Netrin-1

Netrin-1 is a secreted protein that directs both axonal extension (Manitt and Kennedy 2002) and OPC migration during CNS development (Spassky et al. 2002). Netrin-1 acts as chemorepellent for OPCs during embryonic development, and its effect is mediated by RhoA/ROCK signaling (Rajasekharan et al. 2010). Upregulation of astrocytic netrin-1 expression has been reported in mouse model of demyelination (Tepavcevic et al. 2014). Recently full-length netrin-1 protein and fragments of netrin-1 were found in chronic MS lesions (Tepavcevic et al. 2014; Bin et al. 2013). Besides, netrin-1 receptors were indicated to be expressed by OPCs in the brains of MS patients, and astrocytes in active and chronic MS lesions are netrin-1 + , whereas this expression is low in the normal-appearing white matter (NAWM) and shadow plaques of MS lesions. These data, combined with the function of netrin-1 as a chemorepellent for adult OPCs in vitro, suggested that netrin-1 prevents periplaque OPC recruitment, and thereby would affect myelin repair. However, netrin-1 promotes OPCs differentiation in vitro and increases the acquisition of mature phenotype by oligodendrocytes. The presence of netrin-1 detected in actively demyelinating MS lesions is associated primarily with OPC differentiation and low OPC proliferation. It therefore seems that the timing of netrin-1 expression within areas of demyelination may be an important determinant of myelin repair meaning that early absence of this OPC chemorepellent facilitates OPC recruitment, whereas later expression of netrin-1 may act to switch OPC recruitment to differentiation. Thus, netrin-1 expression prior to OPC recruitment might be detrimental to myelin repair. A possible hypothesis is that during active MS demyelination, netrin-1 stimulates local OPC differentiation and prevents periplaque OPC recruitment (Tepavcevic et al. 2014). Repeated episodes of demyelination exhaust the pool of OPCs in the lesion, leaving only a few quiescent cells, corresponding to either surviving dysfunctional OPCs or recruited OPCs that only arrive at the lesion once the acute inflammatory stimulus has terminated and thus remain undifferentiated.

Chemokine OPC Guidance

Many chemokines play roles in migration and maturation of neural precursor cells during normal development (Stumm et al. 2007; Tsai et al. 2002). Local chemokine released by astrocytes and epithelial cells in MS setting also contribute to the modulation of OPC biology. For instance, chemokine (C-X-C motif) ligand 1 (CXCL1) -expressing astrocytes (a chemokine that provides a migratory stop signal for OPCs) (Tsai et al. 2002), were detected at high levels around active MS lesions being totally absent in normal samples (Omari et al. 2005). The presence of CXCL1-producing astrocytes has been indicated to be associated with CXCL1 receptor (CXCR2)-expressing oligodendrocytes at these lesions. Signaling through CXCR2, CXCL1 is known to inhibit oligodendrocyte precursor migration (Tsai et al. 2002). In contrast to active lesions, in silent lesions, astrocytes displayed diminished expression of CXCL1. This proposed a functional role for CXCL1 in remyelination failure, potentially by inhibition of OPCs maturation. Supporting evidences were obtained from demyelinated CNS slice cultures, which showed enhanced myelin repair when CXCR2 was blocked with either genetic deletion or neutralizing antibodies.

However, the available reports are contradictory, with some reporting a protective role for CXCR2 during demyelination. In transgenic mice during EAE, overexpression of CXCL1 in astrocytes led to a decrease in clinical severity, a decrease in demyelination, and an increase of remyelination through CXCL1/CXCR2 pathway (Omari et al. 2009). Moreover, systemic injection of a small inhibitor molecule for CXCR2 at the onset of EAE decreased numbers of demyelinated lesions (Liu et al. 2010). It is suggested that the protective and pro-apoptotic roles of CXCR2 with regards to oligodendrocytes may be context-dependent (Hosking et al. 2010). In vivo studies on models of demyelination revealed that expression of CXCL12 by activated astrocytes is crucial for the maturation of OPCs that express CXCR4 during remyelination in adult mice (Patel et al. 2010, 2012). It has been showed inhibition of CXCR4 signaling, either via pharmacologic antagonism with AMD3100 or via in vivo RNA silencing, prevented OPC maturation and remyelination, proposing that CXCL12-CXCR4 signaling is required for OPCs maturation and myelin repair following CNS injury (Patel et al. 2010). However, the function of CXCL12-CXCR4 pathway is disrupted by upregulation of CXCR7 in the context of demyelination. CXCR7 is an alternative scavenger receptor, which works to sequester and degrades CXCL12 (Naumann et al. 2010; Boldajipour et al. 2008) and regulates activation of CXCR4. High level of CXCR7 was showed to regulate CXCL12 expression during demyelination, which leads to decreased expression of CXCL12 and a down-regulation of activated CXCR4 in OPCs. Since CXCR7 regulates CXCL12-CXCR4-mediated CNS myelin repair, it may, therefore, serve as a therapeutic target to promote OPC differentiation and remyelination in the adult CNS.

PSA-NCAM a Mediator of Myelin Attachment to Axons

PSA‐NCAM, a glycoprotein localized in the plasma membrane of neural and glial cells, acts as an inhibitor of myelination, presumably by preventing myelin‐forming cells from attaching to the axon. It also appears to exert a considerable inhibitory effect on OPCs differentiation (Charles et al. 2002). Disappearance of PSA‐NCAM from the axonal surface during development is coincident with the onset of myelination in human embryonic brain (Fewou et al. 2007). Blocking down-regulation of PSA in oligodendrocytes, in transgenic mice, that exhibit expression of the polysialyltransferase under the control of the PLP promoter led to a reduction in myelin content in the forebrains, both during the period of active myelination and in adult brain (Fewou et al. 2007). Furthermore, antibody‐mediated internalization PSA or removal of PSA sites by treatment with endoneuraminidase N, an enzyme that specifically hydrolyzes PSA, could promote the formation of myelinating internodes in mouse neuron–oligodendrocyte co-cultures (Charles et al. 2000). PSA-NCAM is normally absent from the adult brain but is abundantly re-expressed on demyelinated axons in chronic inactive MS lesions (Charles et al. 2002). In contrast, axons in acute lesions characterized by inflammatory infiltrates and lesions that underwent remyelination did not show evidence of PSA-NCAM expression. It acts as a negative regulator of myelination, presumably by preventing myelin-forming cells from attaching to the axon and blocking the OPCs differentiation (46). However, in addition to its effect on myelination, PSA-NCAM expression in early migratory progenitors is important for migration and recruitment of OPC to the lesion (Franceschini et al. 2004). The second role, therefore, may complicate the design of strategies aimed at removing barrier PSA to promote myelin repair.

Myelin Debris as Inhibitor of Remyelination

The process of primary demyelination generates vast amounts of myelin debris as the myelin sheet unravels and disperses from axons. Myelin removal is a critical step in the remyelination process. Cells of the mononuclear phagocytic system, including monocyte-derived macrophages (MDM) and microglia, are actively implicated in the clearance of myelin debris (Napoli and Neumann 2010). Several lines of evidence reveal the importance of phagocytic removal of myelin debris for efficient remyelination. First, the more rapid remyelination that occurs in young animals is suggested to be associated with efficient removal of myelin debris in contrast to slow remyelination in adult animals as a result of impaired clearance of myelin due delayed activation and recruitment of phagocytic macrophages (Zhao et al. 2006). Second supportive evidence was obtained by impaired differentiation of cultured OPCs plated onto a CNS myelin substrate. (Robinson and Miller 1999). Third, injection of myelin debris in experimentally induced demyelinated animals severely impaired remyelination accompanied with an impairment of OPC differentiation, even after normal recruitment of these precursor cells and macrophages (Kotter et al. 2006). Myelin debris release inhibitory peptides which interact with Nogo-66 receptor and function through LONGO-1 to inhibit remyelination. Two main signaling pathways including Fyn-Rho-ROCK and PKC have been identified as being critical mediators of myelin-mediated inhibition (Baer et al. 2009). However, the concept of inhibitory effects of un-cleared myelin debris on remyelination has been derived exclusively from experimental studies and their role in MS remyelination failure is yet to be demonstrated.

Remyelination Therapy in Multiple Sclerosis

Given the large numbers of OPCs present in adult brain tissue, there are several potential strategies to enhance the remyelination capacity of endogenous OPCs, such as manipulating intrinsic signaling pathways that govern oligodendrocyte biology to override the inhibition of remyelination or altering the lesion environment to be more permissive of OPC differentiation, migration, and remyelination. In recent decades, small molecules, small interfering RNAs (siRNAs), and monoclonal antibodies that can target specific components of the signaling pathways that underlie myelination have been developed and tested. Among the targeted pathways with impact on OPCs maturation and myelination are Notch (Zhang et al. 2009), Wnt (Fancy et al. 2009), Akt (a serine/threonine kinase), mammalian target of rapamycin (mTOR) (Feigenson et al. 2009; Narayanan et al. 2009), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), mitogen-activated protein kinase (MAPK) (Ishii et al. 2012; Fyffe-Maricich et al. 2011), RXR/PPAR (Huang et al. 2011a), ISR (Cunnea et al. 2011; Mhaille et al. 2008), basic fibroblast growth factor (Dehghan et al. 2012), and LINGO-1 (Mi et al. 2007, 2009; Sun et al. 2015).

For instance, targeting LINGO-1 has been successful in reducing axonal damage, promoting remyelination, and restoring function after EAE (Mi et al. 2007, 2009; Sun et al. 2015). Moreover, blocking LINGO-1 with its antagonist antibody (mAb3B5) has been shown to enhance remyelination and recovery of axonal function in the rat lysolecithin-induced focal spinal cord demyelination model. In our previous study, we indicated that targeting NgR by siRNA leads to a significant remyelination and functional recovery of a focal model of demyelination in the mouse optic nerve. Our findings also showed that NgR inhibition also significantly increased the number of Olig2+ cells recruited in the lesion site and enhanced the numbers of third ventricle progenitor cells produced following chiasm demyelination (Pourabdolhossein et al. 2014). These findings shed a light on potential treatment for remyelination. To date, four randomized, double-blind, placebo-controlled clinical trials to evaluate safety, tolerability, pharmacokinetic profile, and efficacy of an anti-LINGO-1 monoclonal antibody called BIIB033 have been completed (ClinicalTrial.gov identifiers: NCT01864148, NCT01721161, NCT01052506, NCT01244139), and some results have been published (Tran et al. 2014). However, there is a tremendous amount of cross-talk among signaling pathways and manipulation of one pathway often induces alterations in another pathway, making these approaches complicating.

As RXRγ is highly expressed in acute and remyelinating lesions, its agonists have been explored in the pathophysiologic context of MS. The RXR-γ agonist has showed to improve remyelination in both cell culture system and in EAE animal models (Diab et al. 2004). Clinical trials evaluating RXR agonists for chronic MS are anticipated as a licensed RXR agonist, bexarotene, is already in clinical use for the treatment of Alzheimer’s disease and cutaneous T cell lymphoma (Cramer et al. 2012; Rodgers et al. 2013).

Several transcription factors such as Olig1, Olig2 Sox8, Sox10, and Sox17 are known to play important roles in differentiation and maturation of OPCs (Arnett et al. 2004; Ligon et al. 2006; Islam et al. 2009; Stolt et al. 2004; Moll et al. 2013). Olig1 and Olig2 belong to the large family of basic helix–loop–helix (bHLH) transcription factors that function widely in cellular development and differentiation. They play partly overlap roles in developmental myelination and remyelination of CNS (Zhou and Anderson 2002; Dai et al. 2015; Li et al. 2007). Studies on transgenic rodent have showed that both of these transcription factors play essential role in remyelination in the context of white matter injury (Arnett et al. 2004; Maire et al. 2010). Overexpression of Olig1 and Olig2 may provide potential therapeutic target to enhance myelination and remyelination in the CNS. Available studies have showed that inducible Olig2 overexpression is sufficient for enhancing OPC migration and differentiation, leading to significant remyelination (Maire et al. 2010).

The Sox family transcription factors are also play well-established roles in regulation of oligodendrocyte differentiation (Stolt et al. 2004; Li et al. 2007). Based on their roles, a number of available studies have evaluated their potential therapeutic role in myelin repair. For instance, overexpression of Sox17, as inducer of OPC differentiation and suppressor of Wnt/β-catenin signaling, has led to remyelination of white matter lesion (Stolt et al. 2004).

Unquestionably, the components of extracellular environment radically altered in MS lesions to impact a number of functions, including cell migration and differentiation. Thus, more effective remyelination would likely be achieved if the local environment within lesions could be restored. To this end, clearance of myelin debris and ECM or other inhibitory factors produced by astrocytes may enhance remyelination. Therapeutically targeting of CSPGs, achieved through enzymatic degradation and interfering with enzymes involved in CSPG biosynthesis provide potentially effective approaches in promoting OPCs differentiation and migration in the presence of CSPGs (Back et al. 2005). The inhibitory nature of CSPGs has been indicated to become attenuated by the enzyme chondroitinase ABC (ChABC), which degrades inhibitory glycosaminoglycan side chains from the CSPG core protein. 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. In a recent study, administration of a fluorinated analogue of N-acetyl-glucosamine called Fluorosamine, reduces the inhibitory nature of this ECM on oligodendrocyte which reduced the synthesis of CSPGs by astrocytes and subsequently led to the promotion of remyelination both in vivo and in vitro (Keough et al. 2016).

Even though attempts to enzymatic digestion of ECM have generated promising results in the context of spinal cord injuries (SCI) (Starkey et al. 2012; Karimi-Abdolrezaee et al. 2012), this approach is favorable for SCI due to the defined local area of damage which is easily accessed for ECM-degrading enzymes. The challenge for such an approach in MS is currently unpredictable due to the heterogeneous pattern of demyelinated lesions throughout the CNS, which makes local delivery difficult. Therefore, strategies that directly impact cells that produce ECM components, such as astrocytes, or alter oligodendrocyte responses to aberrant ECM molecules may be more successful approaches. Recently, transdifferentiation of astrocytes into neural precursors or OPCs is proposed to potentially provide effective regeneration and myelin repair approach. In this regard, reprogramming astrocytes has been achieved by using different small chemical molecules or microRNAs (Ghasemi-Kasman et al. 2016).

In addition to evaluation of approaches that directly modulate pathways involved in remyelination, high throughput drug screening assay is another course of action to identify new therapeutic agents that accelerate the differentiation of immature OPCs into mature, myelin-producing oligodendrocytes. In this regard, a number of studies have evaluated the therapeutic potentials of different collections of already known drugs (Lariosa-Willingham et al. 2016; Najm et al. 2015). The results of a recent study indicated that two drugs, miconazole and clobetasol, from NIH drug library are able to increase the number of new myelinating oligodendrocytes and enhance remyelination in a lysolecithin-induced mouse model of focal demyelination (Najm et al. 2015). Thus pharmacologically induction of remyelination by enhancing endogenous OPC differentiation may have significant therapeutic potential in MS.

Conclusion

Remyelination of MS lesions often fails as a consequence of failure to OPCs recruitment into the lesions, failure of OPCs to generate mature myelinating oligodendrocytes, and failure of oligodendrocytes to remyelinate axons. Many therapies have been developed to modulate the immune response in MS, but specific remyelinating therapies are not yet a reality. Promoting remyelination is a promising avenue for protecting axons, reversing neurologic disability, and preventing progressive disease in MS. Besides restoring the function of axons by remyelination, remyelination prevents secondary axonal damage caused by long-term demyelination which seems as the main cause of transition of relapsing–remitting MS to progressive type. Thus, it is crucial to pursue a strategy for developing myelin repair therapies. It will therefore be pivotal to further our understanding of the pathways involved in MS lesions remyelination and failure of this process.

Abbreviations

MS

Multiple sclerosis

CNS

Central nervous system

PL

Proteolipid protein,

CXCL1

Chemokine (C-X-C motif) ligand 1

sema3A

Semaphorins 3A

LINGO-1

Leucine-rich repeat and immunoglobulin domain containing NOGO receptor interacting protein 1

RXRs

Retinoid X receptors

EAE

Experimental autoimmune encephalomyelitis

NgR1 complex

Nogo-66 receptor complex