Remyelinating strategies in multiple sclerosis

Review

Remyelinating strategies in multiple sclerosis Expert Review of Neurotherapeutics Downloaded from informahealthcare.com by UB Mainz on 10/21/14 For personal use only.

Expert Rev. Neurother. 14(11), 1315–1334 (2014)

Felix Luessi*1, Tanja Kuhlmann2 and Frauke Zipp1 1 Department of Neurology, Focus Program Translational Neuroscience (FTN), Rhine Main Neuroscience Network (rmn2), University Medical Center of the Johannes GutenbergUniversity of Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany 2 Institute of Neuropathology, University Hospital Mu¨nster, Pottkamp 2, 48149 Mu¨nster, Germany *Author for correspondence: Tel.: +49 613 117 7156 Fax: +49 613 117 5697 [email protected]

Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disorder of the CNS characterized by infiltration of immune cells and progressive damage to myelin sheaths and neurons. In recent years, the importance of the neuronal compartment in the early pathology of multiple sclerosis has become increasingly clear. Direct axonal damage within the early stages of inflammation as well as neuronal injury as a result of chronic demyelination are essential factors for the development of long-term disability in patients. Viewing MS as both inflammatory and neurodegenerative has significant implications for treatment, with remyelination of denuded axons to protect neurons from damage being necessary in addition to controlling inflammation. Here, we review recent molecular insights into key molecules and pathways controlling the differentiation of oligodendrocyte progenitor cells and the regenerative process of remyelination in MS and discuss the resulting options regarding remyelinating treatment strategies. KEYWORDS: drug target • multiple sclerosis • myelin repair • neurodegeneration • neuroprotection • oligodendrocytes • remyelination • treatment

Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disorder of the CNS, and the leading cause of nontraumatic neurological disability in young adults, which affects 0.1% of the general population in Western countries [1]. About 85% of patients experience a relapsing remitting disease course (RRMS) at first, with remyelination and functional recovery following on from acute demyelinating episodes of neurological deficits [2]. Remission is not always complete, and after a variable number of years the majority of these patients develop a secondary progressive disease course characterized by axonal loss [3]. In 15% of patients, MS is progressive from the beginning without intermediate relapses, referred to as primary progressive MS [4]. The etiology of this chronic disease has not been entirely understood, but association and epidemiological studies strongly suggest the interplay of susceptibility genes and environmental factors [5,6]. These factors induce the infiltration of circulating myelin-specific autoreactive lymphocytes into the CNS followed by inflammation, demyelination, and neuronal injury. Several lines of evidence link axonal loss to the failure of remyelination, which occurs as the disease progresses. For example, areas of remyelination informahealthcare.com

10.1586/14737175.2014.969241

exhibit reduced axonal loss compared to areas of chronic demyelination in brain tissue of multiple sclerosis patients [7], and experimental models of myelin-interfering gene mutations or toxic myelin damage result in subsequent axonal loss [8,9]. Therefore, it is essential to recognize MS as both inflammatory and neurodegenerative, with myelin repair and CNS protection being required in addition to controlling inflammation [10]. Here, we review recently elucidated molecular insights into differentiation of oligodendrocyte progenitor cells (OPC) and the regenerative process of remyelination in MS and discuss the resulting options regarding treatment strategies to restore lost myelin. Recent insights into inflammatory neuronal injury in multiple sclerosis

Although MS was traditionally viewed as an inflammatory demyelinating disease of the CNS, which leaves the axons mostly intact at least at onset of the disease [11], recent studies have demonstrated that neurodegenerative processes also play a major role even early in the pathogenesis of MS. Interestingly, MS research had already focused on axonal damage between 1880 and 1930 [12]. State-of-the-art histopathological analyses of brain tissue and

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Expert Review of Neurotherapeutics Downloaded from informahealthcare.com by UB Mainz on 10/21/14 For personal use only.

Review

Luessi, Kuhlmann & Zipp

neuroimaging studies showed considerable damage to neuronal structures with axonal loss and neurodegeneration occurring at early stages of the disease and likely resulting in permanent neurological impairment [4,13,14]. Axonal pathology particularly occurs in active and chronic active demyelinating lesions throughout the disease course and is closely associated with the presence of immune cells [14–16]. In addition to axonal damage, either immediate or subsequent to acute inflammatory infiltration, neurodegeneration goes on in the progressive phase of the disease [10]. Potential contributors to progression in MS with neuronal dysfunction include mitochondrial dysfunction [17,18], neuronal loss [19–21], synaptic alterations [22–24], and altered expression of neuronal microRNAs [25]. Imitola et al. suggested a model of the steps leading to disease progression [26]. In this model, the initial attack by immune cells and repeated inflammatory waves result in the establishment of an abnormal microenvironment, leading to regional compromise and dysfunction. Thereby, activated glia mediate the sustained inflammation outside the plaques [27]. Neuronal dysfunction develops at an early stage, followed by the establishment of a new adaptive abnormal steady state in the neurons. Repeated events in proximity of the original site of pathologic abnormality exacerbate inflammation and exceed the adaptive capacity of neurons, resulting in axonal loss [28]. Cell death in a dysregulated environment may contribute to ongoing inflammation by activated glial cells, leading to a vicious cycle. In parallel, Wallerian degeneration and axonal loss may account for distant areas of pathology [29]. Recently, data from genome-wide association studies demonstrated that polymorphisms of genes involved in immunological processes are associated with the susceptibility to MS [5,6]. However, the absence of neuronal and oligodendroglial signals in these susceptibility studies does not imply that genetics in MS is purely immune. It is likely that modifier genes are associated with oligodendroglial and neuronal vulnerability to inflammation-induced neurodegeneration in MS [30]. Few genome-wide association studies have focused on MS severity, and have revealed no association with any of the genes relevant in susceptibility to MS [31]. Current efforts are underway by the International MS Genetics Consortium to try to identify genes that may be associated with rate of atrophy. In order to evaluate whether treatments for MS have neuroprotective effects, it is essential to quantify neuronal injury in MS patients. MRI techniques have been extensively explored in this respect for use in clinical studies. MRI has been adopted in clinical trials to monitor contrast-enhancing lesions as a sign of acute inflammatory lesions and numbers of T2-hypointense lesions as a marker of lesion accumulation over time [32]. However, the number of contrast-enhancing lesions is scarcely associated and the T2 lesion load is only weakly to moderately associated with later disability progression [33]. More promising alternative outcome measures to quantitatively assess progressive neuronal loss over time include change in brain volume, evolution of persistent hypointense lesions on T1-weighted scans and magnetic resonance spectroscopy [34]. Interestingly, spinal 1316

cord atrophy is at least as important as brain atrophy in MS. Imaging studies have shown that cord atrophy measures correlate with clinical disability [35]. Histopathological studies suggest that axonal degeneration is responsible for spinal cord atrophy, rather than tissue loss within lesions [36]. Axonal degeneration as a consequence of demyelination

Although irreversible neurological disability in MS patients is a consequence of axonal degeneration [37,38], the understanding of the mechanisms by which demyelinated axons degenerate is far from complete. It is assumed that the concentric layers of compact myelin surrounding axons exist to provide trophic support for the axon [39]. Aerobic glycolysis products derived from oligodendrocytes have been reported to be rapidly metabolized within the white matter tracts, indicating an axon–glia metabolic coupling [40]. It has also been shown that oligodendrocytes harbor peroxisomes whose function is crucial for maintaining white matter tracts throughout adult life [41]. Furthermore, it was demonstrated that the function of glia in supporting axonal integrity can be uncoupled from its function in maintaining compact myelin [42], the latter being required to facilitate the fast conduction of nerve impulses throughout the nervous system [43]. Segments of adjacent myelin internodes cluster sodium channels into exposed nodes of Ranvier, where saltatory conduction speeds signal transduction in an energy efficient manner. The ‘virtual hypoxia hypothesis’ postulates that demyelination elevates the energy demand in denuded axons [44]. Since the number of voltage-gated Na+ channels that are usually concentrated in axons is decreased upon incomplete myelination, in order to safeguard nerve conduction, higher numbers of Na+ channels are required to compensate for the loss of saltatory axon potential propagation [45,46]. However, higher numbers of Na+ channels need an increased energy supply to restore trans-axolemmal Na+ and K+ gradients. Additionally, reduced axoplasmatic adenosine triphosphate production in chronically demyelinated axons as a result of mitochondrial dysfunction has been reported [17]. The function of mitochondrial respiratory chain complex I and II was impaired by 40–50% in mitochondrial-enriched preparations from the motor cortex of MS patients [47]. Furthermore, defects of mitochondrial respiratory chain complex IV have been described [18,48], have been associated with hypoxia-like tissue injury [49], and resulted in reductions in brain N-acetyl aspirate concentration [50]. Remyelination is the generation of new myelin sheaths around denuded axons in the adult CNS. An immediate consequence of remyelination is adequate redistribution of ion channels at the nodes of Ranvier and restoration of salutatory conduction [43], partially resolving the increased energy demand that is observable by reduced axonal mitochondrial content [51], and resulting in the functional recovery of deficits caused by experimental demyelination [52]. In addition, evidence suggests that the protection of demyelinated axons from subsequent injury is better when they become Expert Rev. Neurother. 14(11), (2014)


Remyelinating strategies in multiple sclerosis

remyelinated [7,53], perhaps by restoring structural as well as trophic support to axons provided by myelin and oligodendrocytes [54]. However, a recent study in the cuprizone-induced demyelination model reported ongoing axonal degeneration after complete remyelination [55]. This finding might be also explained by a direct toxic effect of cuprizone on neurons. In vitro studies indicate that oligodendrocytes produce trophic factors such as insulin-like growth factor-type 1 and neuregulin, which promote normal axonal function and survival [56,57]. Moreover, a late onset, slowly progressing axonopathy was observed in mice, lacking structural components of compact myelin such as proteolipid protein [9]. Further evidence that the symbiotic relationship between the axon and oligodendrocytes/myelin sheaths is active and is not simply insulating, is that axons become extensively damaged when oligodendrocyte cell bodies are targeted for ablation, even in the absence of any observable demyelination [58]. Remyelinated axons seem to regain normal function, although there are detectable differences in myelin architecture. Most evident is the thickness of remyelinated sheaths and length of remyelinated internodal segments. Myelin sheath thickness usually rises with axonal diameter. However, remyelinated fibers tend to have invariantly thinner sheaths around the axons of all caliber [59]. Remyelinated internodes also tend to be shorter than developmentally myelinated nodes [60]. Indeed, the ratio between the inner axonal diameter and the outer myelin diameter has become a gold standard for assessment of demyelination and remyelination in experimental models. Remyelination is a true regenerative process. Chronic demyelinated MS lesions show hardly any mature oligodendrocytes, although they contain significant numbers of OPC [61–64]. It is hypothesized that remyelination is mediated through OPC in the adult CNS, identified by the expression of neuron-glial antigen 2 and platelet-derived growth factor receptor a [65–67]. There are several lines of indirect evidence that OPC are the major source of remyelinating oligodendrocytes: Retroviral and autoradiographic tracking studies demonstrate that dividing cells in normal adult white matter originate from remyelinating oligodendrocytes [63,68]. In addition, transplanted OPCs are capable of remyelinating demyelinated areas with great efficiency [69,70]. Focal demyelinated areas in which cell death of oligodendrocytes and OPCs occur, are repopulated by OPC before the appearance of new oligodendrocytes [71,72]. As a result of that, the temporal and spatial repopulation pattern suggests that OPC gives rise to the remyelinating cells. Finally, transitional expression OPC and oligodendrocyte markers can be detected at the beginning of remyelination [73]. In vitro & in vivo models of myelination & OPC differentiation

A large body of knowledge about the process of OPC differentiation and myelination has been derived from a number of in vitro and in vivo models. Many of these models of immunedriven demyelination have become standard tools for the preclinical screening of drug candidates in MS. informahealthcare.com

Review

The most commonly used animal model in the field of MS is experimental autoimmune encephalomyelitis (EAE), in which fragments of myelin peptides or myelin-sensitized cells are injected into animals, leading to the invasion of autoreactive T cells and macrophages in the CNS, which attack the myelin sheaths [74]. This results in a characteristic ascending paralysis. Despite much of the current knowledge concerning the molecular mechanisms of autoimmune neuroinflammation in multiple sclerosis being derived from the EAE animal model, this model provides only limited insights into the process of remyelination [75]. A major drawback of EAE is the inability to follow the evolution of the diffusely distributed lesions longitudinally in order to demonstrate that remyelination occurs over the disease course of once demyelinated lesions. As a workaround, it has been attempted to correlate oligodendrogenesis in CNS lesions with myelination of regenerating axons [76]. Using double staining for proliferating cell marker bromodeoxyuridine (BrdU) and mature oligodendrocyte marker 2’,3’-cyclic-nucleotide 3’phosphodiesterase (CNPase), the BrdU±CNPase+ cells present in EAE CNS were concluded to derive from proliferating OPC differentiation [77], since the mature oligodendrocytes are postmitotic and unable to proliferate [78]. Furthermore, functional recovery from EAE has been suggested to reflect newly formed myelin sheaths, as determined by electron microscopy [79]. However, these approaches to assess regeneration are purely correlative and do not sufficiently demonstrate prior demyelination. Furthermore, the marked axonal injury has to be considered as a confounding factor of the EAE model in this context. In addition, it is challenging to differentiate whether an agent has a direct effect on myelin repair or if assumed remyelination is the consequence of an anti-inflammatory effect in EAE. Given this ambiguity, demyelinating models with minimal inflammation have been developed. These models typically involve the local injection of toxins that either kill the oligodendrocytes or lead to selective myelin loss. The detergent lysolecithin and the DNA chelating agent ethidium bromide are the typical toxins used [80,81]. The advantage of these models lies in the good reproducibility of demyelination and the predefined area where demyelination occurs. However, the disadvantage is that the injection of a toxin results in a minor traumatic injury of the brain associated with a partial breakdown of the blood-brain-barrier with recruitment of immune cells. An alternative method to cause demyelination, especially in the corpus callosum, is the use of the copper chelator cuprizone, which is fed for several weeks to induce demyelination [82]. Demyelination becomes evident after 4 weeks of cuprizone feeding and reaches its maximum extent after 5–6 weeks [83]. A longer cuprizone treatment results in a slower rate of remyelination. OPC recruitment already occurs during cuprizone treatment and formation of myelin sheaths starts after switching to a normal diet. A combination of ethidium bromide injection and subsequent irradiation results in the death of proliferating cells and thereby inhibits endogenous remyelination, which is useful for 1317


Review


the evaluation of cell-transplantation experiments. The downside of this model is the injury to astrocytes and the alteration of their reaction. All of these models rely on pathological examination of myelin content in the CNS to demonstrate demyelination, which makes animal sacrifice necessary for assessment. Although these toxin-based models are well suited for the study of remyelination, they do not mimic the inflammatory reaction in MS pathology. Furthermore, the extent of damage is more widespread in comparison to the typical focal lesion nature of MS, and as a consequence, the behavioral phenotype of these models can be quite pronounced. To overcome this limited comparability to the human disease, the Cup-EAE animal model with MS-like lesions in the brain has recently been established by immunizing cuprizone-fed mice with myelin oligodendrocyte glycoprotein (MOG)35-55 [84]. Through this approach, a remarkable number of T cells were recruited into the demyelinated corpus callosum, resulting in significant axonal damage in this area, while the extent of demyelination remained unchanged. However, all current animal models have limitations and none of the existing animal models mimics even closely the pathological and/or pathogenetic characteristics of MS. Of note is the increased use of transgenic mice with conditional knockouts or overexpression of oligodendrocytespecific genes, which has profoundly improved our understanding of the mechanisms underlying OPC differentiation and remyelination. Primarily, OPC respond to injury; they divide and migrate toward the site of demyelinating lesions, attracted by chemotrophic factors secreted by astrocytes or activated microglia [85]. Subsequently, the differentiation of OPC is promoted by transcription factors (FIGURE 1). The transcription factors Sox10, Olig1, Olig2, Ascl1, Nkx2.2, and Tcf4 participate in response of the OPC to demyelination [26,86,87]. It has been shown that Sox10 is required for oligodendrogenesis [88], and Nkx2.2 and Olig2 coexpressing cells proliferate and differentiate into myelinating oligodendrocytes in response to demyelination [73]. A recent study reported the role of Ascl1 as a critical positive regulator for oligodendrogenesis in response to demyelinating insults [89]. Olig1 is present in OPC surrounding demyelinated MS plaques and appears to have a critical role in effective remyelination [90]. The dual role of the Gli1 gene in contributing to oligodendrogenesis in addition to maintaining neural progenitor proliferation has been identified [91]. Interestingly, despite the initial upregulation of the sonic hedgehog (Shh) Gli1 pathway in EAE and active MS lesions, Gli1 was significantly decreased in spinal cord OPC after the onset of EAE and in chronic MS lesions [91]. The suppression of Gli1 expression was attributed to the Th1 cytokine interferong. It has been reported that experimental demyelination in animal models reactivates subventricular zone (SVZ) progenitors, increasing proliferation and inducing to some extent oligodendrogenesis [92]. However, the relevance of oligodendrogenesis in MS remains to be elucidated, and longitudinal MRI studies of 1318

MS patients do not point to an increased capacity of remyelination in periventricular lesions [93]. Mature oligodendrocytes use their processes to establish contact with demyelinated axons and enwrap them with concentric layers of myelin membrane, and ultimately, concentrate these layers into functional myelin sheaths [94]. Thereby, the critical role of the expression of Ptprz, a member of the receptor-type protein-tyrosine phosphatase family, in the survival mature oligodendrocytes in mouse models has been described [95]. It has been further demonstrated that PTPRZ1, the human homolog of Ptprz, is specifically expressed in remyelinating oligodendrocytes in MS lesions. Using models with defects in oligodendrocyte-specific genes, axonal pathology occurs secondary to primary oligodendrocyte death [8]. An extensive overview of the complex genetic programs and regulation of oligodendrocyte differentiation and myelination in response to demyelinating events has been published [96,97]. Current evidence from models of demyelination indicates that remyelination is an efficient, rapid and highly beneficial process, which might be the best opportunity for prolonged protection of demyelinated axons [98,99]. It emphasizes that the promotion of endogenous myelin repair could be a treatment strategy for MS patients. Endogenous remyelination in MS patients

Endogenous remyelination is a spontaneous regenerative process that can occur with considerable efficacy, not only in animal models following experimental demyelination but also in MS patients [100,101]. Histological studies have reported the presence of both demyelination and remyelination at the edges of demyelinating lesions with oligodendrocytes identified as the proliferating cell population within these lesions [102,103]. Interestingly, remyelination occurs not only in RRMS, but also in some patients with progressive disease courses [101]. It is noteworthy that even in reports of cases of extensive remyelination, there is a considerable variability in the degree of remyelination of individual lesions within the same patient [100,104]. The location of the demyelinating lesions seems also to play a key role, as periventricular lesions tend to be less remyelinated than subcortical lesions [101,104]. Furthermore, a study comparing the extent of remyelination in cortical and white matter lesions in the same patients reported that remyelination of cortical lesions was consistently more extensive [105]. Accordingly, another study investigating demyelinating lesions involving cortex and adjacent white matter observed more actively remyelinating oligodendrocytes and fewer reactive astrocytes in cortical lesions [106]. The reasons for these differences in remyelination are not yet clear. Generally, remyelination occurs more prominently in the early phase of the disease. A study comparing acute and chronic lesions reported evidence of remyelination in 80.7% of early lesions, with only 60% in chronic lesions [104]. In accordance, the number of OPC observed immediately outside of early lesions is significantly greater than outside of chronic lesions [107]. However, a recent study emphasized the Expert Rev. Neurother. 14(11), (2014)


Neuroepithelial stem cells

Pre-oligodendrocytes

OPCs

Review

Oligodendrocytes

miR-219 miR-338


Shh (Gli2) Olig2 Hes5, Id2, Id4 Sox5/6 Ascl1 Sox10 ZFP191 Nkx2.2 Olig1 (nuclear) Tcf4 Olig1 (cytoplasmic) MRF YY1

Figure 1. The figure shows the different stages of the oligodendrocyte lineage. The temporal expression pattern of transcription factors is illustrated by gradient bars. The role of miRNAs in the posttranscriptional control of myelination is represented in a simplistic schematic.

persistence of low numbers of OPC in chronic lesions [108] that do not mature to myelinating oligodendrocytes [64,107]. A recent study in active MS lesions reported an increased susceptibility of human OPC to cell death compared to mature oligodendrocytes [109]. In vitro culture experiments revealed that OPC show reduced process extension under stress condition compared to oligodendrocytes. It is assumed that reduced numbers of lesion-associated OPC, whether through increased susceptibility to cell death, less migration, decreased proliferation or impaired differentiation to myelinating oligodendrocytes, contributes to remyelination failure in chronic MS. The role of other cell types involved in this deficiency is currently under investigation. It appears that remyelination occurs in MS to a varying degree, but further insights into the mechanisms of remyelination failure will be necessary to further identify molecular targets for the development of remyelinating strategies. Evidence of remyelination with current therapeutics

At present, eight disease-modifying drugs have been approved for MS therapy (TABLE 1). Approved in February 2014 in Europe, dimethyl fumarate follows fingolimod and teriflunomide as the third oral agent for MS. Prior to the emergence of oral therapies, IFN-b preparations and glatiramer acetate (GA) were established as first-line disease-modifying immunomodulatory treatments. All currently available MS therapeutics primarily target ongoing inflammation. However, it has been speculated that some of informahealthcare.com

these agents may additionally act on the CNS via promotion of myelin repair to prevent chronic disability. The existing data on these potential effects will be subsequently discussed. Through binding to a specific receptor, IFN-b exerts a variety of immunological effects. Presumed mechanisms of action include inhibition of T-cell activation and co-stimulation, modulation of anti-inflammatory and proinflammatory cytokines, and downregulation of T-cell migration [110,111]. It has been reported that IFN-b stimulates the production of NGF in early stages of the disease and inhibits microglia and gliosis [112,113]. In MRI-based studies, treatment with IFN-b reduced the development of persistent ‘black holes’ and decreased the brain atrophy rate [114,115]. GA is a synthetic peptide composed of a random mix of four amino acids resembling myelin basic protein (MBP) and acts by a shift in immune response from Th1 to a more antiinflammatory Th2-profile [116]. GA also takes effect by limiting T cells through downregulating proliferation, activation and induction of apoptosis [117,118]. GA-specific T cells showed an increased secretion of brain-derived neurotrophic factor, which supports neuronal survival [119]. Furthermore, GA treatment was associated with a reduction of persistent ‘black holes’ in patients [120], and increased the N-acetyl aspirate concentration in magnetic resonance spectroscopy [121], which implies that this treatment may reduce axonal injury in developing lesions and maintains axonal metabolic function [3]. Whether these 1319


Compound

Proposed mechanisms

Indication

Clinical outcome

MRI outcome

Filippi et al. (2004), Jacobs et al. (2000), Jacobs et al. (1996), Kappos et al. (2007)

Interferon IFN-b1a and -b1b

• Inhibition of T cell activation and co-stimulation • Modulation of anti-inflammatory and proinflammatory cytokines • Downregulation of T cell migration • Suppression of Th17 cell differentiation • Stimulates the production of NGF in early stages of the disease

CIS RR-MS SP-MS (IFN-b1b)

Delay to Poser MS in CIS patients Reduces relapse rate Increased time to confirmed progression in SP-MS

Reduces gadolinium enhancing lesions Reduces T2 lesions Reduces the mean T2 lesion volume Reduces development of permanent black holes (IFN-b1b) Slowing progressive loss of brain tissue in CIS patients (IFN-b1a)

[115,227–229]

Filippi et al. (2001), Khan et al. (2008), Comi et al. (2001), Johnson et al. (1995)

Glatiramer acetate (GA)

• Secretion of BDNF by GA-reactive T cells • Modulation of T cell activation and proliferation • Augmentation of the ratio of anti-inflammatory to proinflammatory cytokines

CIS RR-MS

Reduces disability rate Reduces relapse rate

Reduces proportion of new lesions evolving to black holes Reduces gadolinium enhancing lesions Increases Nacetylaspartate/ creatine ratio

[120,121,230,231]

Hartung et al. (2002), Edan et al. (2011)

Mitoxantrone

• B and T cell suppression • Eliminates and deactivates monocytes and macrophages • Inhibits T cell migration

Active RR-MS SP-MS

Reduces relapse rate Reduces progression of disability

Reduces the T2 lesion load Reduces gadolinium enhancing lesions

[232,233]

Polman et al. (2006)

Natalizumab

• Inhibits transendothelial migration of leukocytes across blood-brain-barrier

Active RR-MS

Reduces relapse rate Reduces progression of disability

Reduces gadolinium enhancing lesions Reduces T2 lesions

[234]

Kappos et al. (2010)

Fingolimod (FTY720)

• Modulates activation of sphingosine 1-phosphate (S1P) receptors 1, 3-5 • Prevents egress of lymphcytes from secondary lymphoid tissue to sites of inflammation • Differentially retains effector memory cells and Th17 cells • Might promote remyelination by acting on oligodendrocyte S1P5 receptors

Active RR-MS

Reduces relapse rate Reduces risk of disability progression

Reduces the rate of brain atrophy Reduces gadolinium enhancing lesions Reduces number of new or enlarging T2-hyperintense lesions

[128]

MS: Multiple sclerosis; RR-MS: Relapsing-remitting multiple sclerosis; SP-MS: Secondary-progressive multiple sclerosis.

Ref.


Expert Rev. Neurother. 14(11), (2014)

Study (year)

Review

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Table 1. Approved therapies in multiple sclerosis.


informahealthcare.com

Table 1. Approved therapies in multiple sclerosis (cont.). Compound

Proposed mechanisms

Indication

Clinical outcome

MRI outcome

Confavreux et al. (2014), O’Connor et al. (2011), O’Connor et al. (2006)

Teriflunomide

• Active metabolite of leflunomide used for rheumtoid arthritis • Impairs cellular nucleotide metabolism by inhibiting the dihydroorotate dehydrogenase • Suppresses tyrosine kinases involved in signal transduction pathways

RR-MS


Reduces gadolinium enhancing lesions Reduces the total volume of T2-hyperintense lesions

Coles et al.(2008), Coles et al. (2010), Coles et al. (2012), Coles/Twyman et al. (2012)

Alemtuzumab

• mAb to CD52, a surface antigen of unknown function on lymphocytes, monocytes and dendritic cells • Induces a sustained T cell depletion and a transient B cell depletion • Increases levels of BAFF and regulatory T cells • Increases secretion of BDNF by lymphocytes

Active RR-MS

Improves mean disability score Reduces relapse rate

Reduces gadolinium enhancing lesions Reduces brain atrophy rate

Fox et al. (2012), Gold et al. (2012), Kappos et al. (2008)

Dimethyl fumarate (BG00012)

• Activation of transcription factor nuclear factor erythroid 2-related factor 2 • Induction of Th2-like cytokines • Induction of apoptosis in activated T cells • Downregulation of intracellular adhesion molecules and vascular adhesion molecules • Upregulation of anti-oxidant response elements

RR-MS


Reduces gadolinium enhancing lesions Reduces number of new or enlarging T2-hyperintense lesions

MS: Multiple sclerosis; RR-MS: Relapsing-remitting multiple sclerosis; SP-MS: Secondary-progressive multiple sclerosis.

Ref. [235–237]

[130,131,238,239]

[240–242]


Study (year)

Review

1321


Review


findings are mediated by direct beneficial effects of GA and IFN-b on neurons, or result from their anti-inflammatory properties remains to be elucidated. It is also debated whether the oral immune-modulatory treatment with fingolimod (FTY720) may have direct effects in the neuronal compartment. Following in vivo phosphorylation, it acts as a modulator of the activity of sphingosine 1-phosphate receptors, thus preventing lymphocyte egress from secondary lymphatic organs and subsequent migration to sites of inflammation [122]. It may also diminish astrogliosis and promote remyelination via sphingosine 1-phosphate receptors on astrocytes and oligodendrocytes [123]. After lysolecithin-induced demyelination, it has been shown that fingolimod enhances remyelination in both organotypic slice culture and cell aggregate models [123,124]. However, in contrast to these findings, treatment with fingolimod in vivo in lysolecithin and cuprizone animal demyelination models did not promote remyelination [125,126] and failed to slow deterioration in secondary progressive EAE [127]. In a recent 2-year Phase III trial, fingolimod-treated patients displayed a reduced rate of disability progression and brain volume loss as well as a smaller increase in T1 hypointense lesion volume than patients who were given a placebo [128]. More studies are required to determine the role of fingolimod, if any, in myelin repair. Targeting mechanisms of the immune system with recombinant antibodies such as alemtuzumab provides additional selectivity in the treatment of MS. Alemtuzumab is a humanized monoclonal antibody targeting the CD52 antigen, which is a protein of unknown function expressed on the surface of T and B cells, NK cells, a majority of monocytes and macrophages and some dendritic cells [129]. The binding of alemtuzumab leads to a rapid and prolonged depletion of targeted cells by complement-dependent and antibody-dependent cellular toxicity. In a recent 3-year Phase II trial, alemtuzumab significantly reduced the risk of relapse, brain volume loss, and accumulation of disability in early RRMS compared to interferon1a [130]. Patients treated with alemtuzumab experienced an improvement in disability at 6 months that was sustained in the 5 year follow-up study [131]. These findings for alemtuzumab treatment might result, in part, from neuroregeneration associated with increased lymphocytic delivery of brain-derived neurotrophic factor to the CNS [132]. Another recombinant antibody used in MS therapy is natalizumab, a monoclonal antibody directed against a-4 integrins. In a magnetization transfer imaging study, treatment with natalizumab in MS patients was associated with increased magnetization transfer ratio suggestive of remyelination [133]. More studies are needed to assess the remyelination capacity of natalizumab. The newly approved oral dimethyl fumarate (BG00012) has anti-inflammatory and neuroprotective properties that are likely to result from the activation of the nuclear factor erythroid 2-related factor 2 transcription factor, which binds to antioxidant response elements [134]. It has been shown that dimethyl fumarate mediates cytoprotective effects by protecting against oxidative stress [135] and reduces the production of proinflammatory 1322

cytokines such as TNF-a, IL-2, and IL-17 from immune cells [136]. However, application of dimethyl fumarate in the cuprizone model did not show a direct effect on remyelination [137]. Laquinimod, a compound currently in advanced clinical trials, showed a modest reduction of the annualized relapse rate and a reduction in the risk of confirmed disability progression in a 24-month Phase III trial on RRMS patients [138]. In this study, treatment with laquinimod was also associated with reduced MRI measures of disease activity. The effect by which laquinimod exerts its anti-inflammatory activity may be due to its impact on the dendritic cell compartment and a Th1-Th2 shift [139,140]. Additionally, laquinimod ameliorated EAE via brain-derived neurotrophic factor-dependent mechanisms which may contribute to neuroprotection [141] and protected from cuprizone-induced demyelination by reduced astrocytic NF-kB activation [142]. Signals that control OPC differentiation & myelination

In the past few years, marked developments in identification of signals regulating OPC differentiation and myelination have been made [143]. These signals can be divided into two categories: intrinsic signals and extrinsic signals/environmental cues (FIGURE 2). Here, we describe examples of the group that are in the focus of potential drug interventions. Intrinsic signals

Transgenic animals and gene expression analysis such as DNA microarrays have allowed the identification of several oligodendrocyte-specific genes that control oligodendrocyte differentiation and myelination, including positive regulators such as myelin gene regulatory factor (MRF), zinc finger protein 191 (Zfp191), retinoid X receptor-g (RXRg), and negative regulators such as G protein-coupled receptor 17 (GPR17), Notch and Wnt [144,145]. Myelin gene regulatory factor

MRF is an important transcriptional regulator required for oligodendrocyte maturation and CNS myelination. MRF is specifically expressed by postmitotic oligodendrocytes and promotes OPC differentiation into MBP-positive and MOG-positive oligodendrocytes [145]. It has been demonstrated that in MRFdeficient mice, premyelinating oligodendrocytes are generated but fail to express many myelin genes, which results in a phenotype with pronounced neurological deficits [145]. Interestingly, the genetic ablation of MRF by the use of an inducible conditional knock-out strategy led to delayed but severe CNS demyelination [146]. This report highlights that ongoing expression of MRF within the adult CNS is necessary for the maintenance of both the myelin sheath and the mature oligodendrocyte identity. Zinc finger protein 191

Zfp191 is another transcription factor recognized as being critical for CNS myelination [147]. The lack of functional Zfp191 is associated with the presence of abundant late-stage oligodendrocytes in the CNS that extend processes to the axons but fail to myelinate them. Expert Rev. Neurother. 14(11), (2014)


Demyelinating lesion

Remyelination:


OPCs

Semaphorin 3A

Promoting factors Inhibiting factors

BMP4 Lingo-1 Semaphorin 3A Hyaluronan PSA-NCAM

Pre-oligodendrocytes

Review

Activin-A L1CAM

Semaphorin 3F

Oligodendrocytes

9-cis-RA Id2/4 Sox5/6 Hes5 Myelin gene promotors

Tcf4

Id2/4

RXRy

Wnt

Myelin gene promotors

Sox10 Olig1 β-catenin MRF Zfp

Notch

Jagged1

Myelin gene promotors

Contactin

GPR17

Figure 2. The figure illustrates intrinsic signals and extrinsic signals as well as environmental cues that influence the remyelination process. OPCs need to be recruited to demyelinated lesions and further undergo maturation to myelin-forming oligodendrocytes for remyelination to occur. Promoting signals compete with inhibitory signals at the different necessary steps of remyelination.

Retinoid X receptor-g

RXRg is a nuclear receptor that is critical in the regulation of OPC differentiation. Upon treatment of RXR agonists in culture, OPC differentiates and forms myelin membrane-like sheets. Knockdown of RXRg by RNA interference strongly inhibited OPC differentiation in culture [148]. In contrast, the RXR agonist 9-cis-retinoic acid improved the extent of remyelination in demyelinated cerebellar slice cultures and in demyelinated aged rats [148]. RXR agonists are widely available and show promising results in cancer cell differentiation therapy and in the treatment of metabolic diseases [149]. The RXR agonist bexarotene has been approved for all stages of cutaneous T-cell lymphoma [150]. Furthermore, the administration of bexarotene in a mouse model of Alzheimer’s disease enhanced the clearance of soluble b-amyloid, leading to a significant reduction of b-amyloid plaques [151], pointing to the capacity of the drug to mediate enhanced phagocytic clearance of remyelination-inhibitory myelin debris [152]. These findings show that RXR agonists can modulate inflammation and informahealthcare.com

stimulate oligodendrocyte differentiation and remyelination in the injured CNS, thereby pointing to an additional role as potential drugs for regenerative therapy in demyelinating disorders. G protein-coupled receptor 17

The expression of G protein-coupled receptor GPR17, a recently deorphanized receptor for both uracil nucleotides and cysLTs, is restricted to the early differentiation stages of oligodendrocyte lineage cells but downregulated during the peak period of myelination and in adulthood [153]. In vitro, GPR17 overexpression inhibits terminal differentiation of primary OPCs, whereas in transgenic mice, sustained overexpression of GRP17 in oligodendrocytes lead to oligodendrocyte loss and myelination arrest [144]. In contrast, another in vitro study reported that in vitro exposure to the endogenous GPR17 ligand UDP-glucose promoted the differentiation of OPCs into MBP-positive oligodendrocytes [154]. Further investigations revealed a restriction of GPR17 expression to O4- and 1323

Review


neuron-glial antigen 2-positive OPC and immature oligodendrocytes. Stimulation of GPR17 with its natural ligands results in potent inhibition of intracellular cAMP production [155]. This effect can be counteracted by receptor silencing with siRNAs and GPR17 antagonists [155]. It has been suggested that GPR17 is an intrinsic timer that controls OPC maturation by inhibiting cAMP formation early in differentiation, and, when a critical stage of OPC differentiation is reached, allowing the reversal of cAMP to sufficient levels for terminal differentiation [155,156].


Notch

The Notch–Jagged signaling pathway is critical for the timing of myelination in the developing nervous system [157]. The Notch ligand Jagged1 has been identified to be upregulated in human astrocytes activated by the cytokine TGF-b1 [158]. Notch1 and its effector Hes5 are expressed in immature oligodendrocytes of MS lesions [158] and following CNS demyelination with ethidium bromide in rodents [159]. In vitro experiments demonstrated that Jagged1 signaling inhibited maturation and process outgrowth of OPCs [158]. However, there is conflicting evidence regarding the role of Notch–Jagged signaling on OPC differentiation and remyelination. First, in MOG-induced EAE, remyelination occurs with great efficiency in spite of the expression of Notch1 in OPCs and the expression of Jagged1 in the demyelinated environment [159,160]. Second, OPC-targeted Notch1 inactivation in adult mice accelerated remyelination of demyelinating lesions at the expense of proliferation within the progenitor population [77]. Third, treatment with the g-secretase inhibitor MW167, which blocks Notch signaling, improved myelin repair and axonal survival [161]. However, since Notch is also expressed in immune cells, an indirect immunomodulatory effect of MW167 on remyelination remains possible [162]. Fourth, the noncanonical Notch ligand contactin enhances OPC differentiation and the wrapping of contactin-expressing cells [163]. Therefore, the role of Notch signaling in the regulation of myelination is more complicated than first anticipated, and its value as a target for remyelinating strategies remains uncertain. Wnt signaling

The Wnt pathway negatively regulates developmental myelination. A genome-wide screen for transcription factors associated with CNS remyelination in lysolecithin demyelinated mice resulted in the discovery of Tcf4 expression in oligodendrocyte lineage cells and deeper insights into the role of the canonical Wnt pathway as a negative regulator of OPC differentiation [86]. While Tcf4 was postnatally expressed in the white matter of developing mice, it was barely found in the white matter of adult mice [86,87]. Following adult white matter injury, Tcf4 is re-expressed and upregulated in OPCs recruited to the lesion. As Tcf4 is also highly expressed in MS lesions, Tcf4 may also play a role in remyelination through active Wnt signaling [86]. Upon Wnt pathway activation, Tcf4 is thought to interact with 1324

its binding partner b-catenin in the nucleus to regulate transcription of Wnt-b-catenin pathway target genes [164]. In transgenic mice overexpressing b-catenin in oligodendrocyte lineage, there is no impairment of embryonic development of OPC but the subsequent OPC differentiation is affected, leading to hypomyelination with fewer oligodendrocytes expressing mature myelin markers proteolipid protein in the white matter [86]. Accordingly, in electron microscopy, these mice display hypomyelination of axons at postnatal day 15 (P15), which catches up with normal wild-type levels by P50, indicating that Wnt signaling delays, and in fact, blocks OPC maturation [86]. Experimental toxin-induced demyelination in these transgenic mice resulted in a delayed OPC differentiation and impaired remyelination, without affecting OPC recruitment to lesions [86]. Moreover, two histone modifying enzymes, HDAC1 and HDAC2 have been identified to antagonize the inhibition of Wnt signaling on OPC differentiation and that Tcf4 mediates this crosstalk [87]. Taken together, these studies provide evidence that the Wnt pathway is an antagonist of myelination and remyelination in vivo. Recently, it has been shown that delivery of XAV939, a small-molecule Wnt antagonist, into spinal-cord demyelinating lesions in mice accelerated OPC differentiation and enhanced efficiency of remyelination in vivo [94], highlighting the relevance of the Wnt pathway for future remyelinating therapies. Extrinsic signals for OPC differentiation & myelination

OPC differentiation and myelination is also regulated by extrinsic signals in the environment. Bone morphogenetic protein 4 (BMP4) and leucine-rich repeat and Ig-containing 1 (Lingo-1) are examples of negative regulators secreted by neurons. It has been hypothesized that the presence of such negative regulators results in a nonpermissive environment, inhibiting remyelination. In addition, recent reports emphasize the role of macrophages and microglia in the regulation of remyelination. Macrophages & microglia

An important factor in the regenerative process of remyelination is the innate immune system represented by the peripherally derived macrophages and CNS-resident microglia, which can be polarized to distinct functional phenotypes: proinflammatory (M1) and immunoregulatory (M2) [165]. Although macrophages and microglia contribute to CNS autoimmunity via antigen presentation to T cells [166] and secretion of toxic molecules [167], they also exert regenerative functions through the secretion of growth and neurotrophic factors [168] and the phagocytosis of myelin debris [169]. Recently, it has been demonstrated that macrophages and microglia can dynamically switch from an M1 to an M2 phenotype at the initiation of remyelination [170]. OPC differentiation was enhanced in vitro with M2 cell conditioned media and impaired in vivo following intralesional M2 cell depletion. Furthermore, acute active MS lesions and the rim of chronic active lesions exhibited increased M2 cell densities. In Expert Rev. Neurother. 14(11), (2014)


cerebellar slice cultures, blocking M2 cell-derived activin-A inhibited OPC differentiation during remyelination. These findings highlight the relevance of M2 cell polarization for efficient remyelination and identify activin-A as a promising target for regenerative treatment strategies.


Bone morphogenetic protein 4

BMP4 is a polypeptide belonging to the TGF-b superfamily of proteins. OPC and oligodendrocytes derived from the CNS show expression of BMP4 and BMP receptors [171]. Exogenous BMP4 inhibits OPC differentiation and promotes astroglial differentiation [172,173]. Recently, it has been shown that BMP signaling is active in demyelinating lesions in mice [174]. In this study, intraventricular infusion of BMP4 into the brains of mice enhanced the proliferation of endogenous OPC during cuprizoneinduced demyelination. In contrast, infusion of Noggin, which inhibits endogenous BMP signaling during demyelination, promotes oligodendrogenesis and myelin repair. These findings suggest a function for BMP signaling inhibition in enhancing mature oligodendrocyte regeneration and remyelination. Given that BMP4 is expressed in demyelinating lesions in humans [175], it is a potential therapeutic target to enhance myelin repair. Leucine-rich repeat and Ig-containing 1

The transmembrane protein leucine-rich repeat and Ig-containing 1 (Lingo-1) is specifically expressed in the CNS and acts as a negative regulator of OPC differentiation [176]. Mice with Lingo-1 gene knockout or treated with anti-Lingo-1 antibodies demonstrated improved axonal integrity and functional recovery from EAE [79]. Furthermore, in toxin-induced model of demyelination in rats, Lingo-1 antagonists promote CNS remyelination by creating an environment that is more favorable to OPC differentiation [177]. These findings highlight the relevance of Lingo-1 signaling in the regulation of OPC differentiation and remyelination and resulted in a Phase II clinical trial with an anti-Lingo1 monoclonal antibody (BIIB033) for treating MS [178]. Semaphorins

Semaphorins have been implicated as important regulators of several stages of remyelination. Both experimental functional studies and expression analysis of MS lesions support a model in which recruitment is regulated by an appropriate balance of semaphorin 3A, a repulsive guidance cue, which if it becomes the dominant form may prevent precursors entering areas of demyelination, and semaphorin 3F, an attractive guidance cue that facilitates recruitment [108,179,180]. Semaphorin 3A has also been shown to be a potent inhibitor of precursor differentiation, a role which paradoxically might be advantageous to recruitment by preventing cells leaving the cell cycle, and therefore a potential therapeutic target for unblocking remyelination in nonremyelinating MS lesions [108,181]. Hyaluronan

The glycosaminoglycan hyaluronan in the microenvironment of demyelinating lesions has been identified to contribute to OPC informahealthcare.com

Review

maturation and remyelination failure [182]. The high molecular weight form of hyaluronan accumulates concurrently with astrogliosis in demyelinating MS plaques [182] and traumatic spinal cord injuries [183], and inhibits remyelination after lysolecithininduced demyelination. This appears to be mediated by a mechanism where the specific hyaluronidase PH20 degrades hyaluronan into oligomers that block OPC maturation and remyelination through TLR2-MyD88 signaling [184,185]. Inhibition of hyaluronidase activity resulted in increased OPC maturation and increased conduction velocities through lesions [184], indicating that pharmacological inhibition of PH20 might be an effective way to promote remyelination in MS. Cell adhesion molecules

Exact control of cell–cell-interaction is a prerequisite for myelination and several cell adhesion molecules have been shown to be involved in this process. Both neural cell adhesion molecule (NCAM) and L1 cell adhesion molecule (L1CAM) are members of the immunoglobulin gene superfamily. They have different important roles in the CNS: interneuronal and glia-neuronal adhesion phenomena, cell-cell recognition, development of the nervous system, and synaptic plasticity [186,187]. It has been suggested that the polysialylated form of the neural adhesion molecule (PSA-NCAM) may be responsible for the remyelination failure of OPC [188]. During development PSANCAM is expressed on both oligodendrocytes and axons [189,190] and acts as a negative regulator of myelination, presumably by preventing the attachment of OPC and axon. While PSANCAM, that is, normally absent in the adult brain, was reexpressed on demyelinated axons in MS plaques, remyelinated lesions did not exhibit PSA-NCAM expression [188]. These observations suggest that in MS, PSA-NCAM could act as an inhibitor of myelin repair and participate in disease progression. L1CAM has been shown to regulate the initial steps of CNS myelination [191,192]. Its expression is downregulated on myelinated axons [193] and spinal cord transection induced its reexpression on regenerating axons near the lesion site [194]. Treatment with L1CAM has been reported to promote axonal growth and functional recovery after spinal cord injury [195] and to facilitate optic nerve regeneration and remyelination [196]. Furthermore, coexpression of L1CAM with the growthassociated protein GAP-43 acts synergistically to enhance regenerative growth of Purkinje cell axons in vivo [197]. These findings indicate that L1CAM might be beneficial in promoting remyelination by overriding the antiregeneration signals in adult CNS injury. Beyond the previously described signals and pathways, further factors have been reported to regulate OPC differentiation and myelination, such as the transmembrane semaphorin sema 6A [198], the nuclear factor IA [199], and the protein tyrosine phosphatases Dusp15/VHY and PTPRZ [200,201]. Given the multitude of players involved in the regulation of OPC differentiation and myelination, the whole process is complex and far from being completely understood. 1325


Review


The identification of compounds that selectively induce OPC differentiation at the site of demyelinating lesions and subsequently enhance remyelination remains a major challenge in the development of new regenerative treatments for multiple sclerosis. While some studies are focused on targeting specific identifiable pathways, such as treatment with anti-Lingo-1 antibodies [79], others are essentially ‘black box’ screens, for which the molecular targets may not be known, but the phenotypic target, myelination, can be quantified. Through the latter strategy, high-throughput screening based on the induction of MBP expression in primary rat optic nerve-derived OPC has recently identified benztropine as an effective candidate compound for remyelination [202]. For an in vivo vertebrate analysis and phenotypic characterization, the use of larval zebrafish in a medium-throughput quantitative screen has been described [203]. Future perspectives include the use of human OPC derived from human-induced pluripotent stem cells for screening of remyelinating drugs. Stem cell transplantation

An alternative concept to promoting endogenous remyelination is to transplant exogenous myelinating cells into demyelinated areas in the CNS. This approach has been explored in spinal cord injury-induced demyelination, and transplantation of OPCs following contusion or irradiation of the rat spinal cord leads to increased remyelination and functional recovery [204,205]. Similarly, human neural stem cell transplants have been reported to be capable of inducing robust remyelination with normal compact myelin ultrastructure in mice with severe dysmyelination [206]. However, transplantation of stem cells presents many challenges [207]. MS is a multifocal chronic disease and focally implanted stem cells would be available only to axons within the injected lesions, since data from animal models suggest that transplanted glial progenitor cells may have limited migration potential [208]. Caution must also be taken with respect to the control of proliferation and differentiation process of transplanted cells. The cells proliferate excessively (perhaps forming tumors) and fail to remyelinate. Even if we surmount these hurdles in our animal models, as they may not reflect the exact human pathology, this may lead to different cell behavior and effects in clinical trials of transplants in humans. Patients would need immunosuppression if the source of transplanted OPCs is human embryonic stem cells. However, OPCs can now also be slowly derived from patientspecific induced pluripotent stem cells, and these mature into oligodendrocytes, forming functional myelin around axons at least in the mouse brain. This may provide an alternative to strong immune suppression [209]. However, these techniques are still being developed, and their safety and efficacy remain unknown. Effect of aging on remyelination

The decline in remyelination that occurs with advancing age represents a significant challenge in the treatment of multiple sclerosis [210]. It has been partially attributed to changes in 1326

environmental signals controlling remyelination [211], and epigenetic changes within aging OPC that lose their capacity to differentiate into remyelinating oligodendrocytes [212,213]. These age-dependent changes may reflect the situation in chronically demyelinated MS plaques in which OPC fail to differentiate into myelinating oligodendrocytes [107]. It has been shown that telomerase reactivation reverses ageassociated defects in neural stem cells [214], lending weight to the idea that aged OPC, in theory, keep their capacity for efficient remyelination. Along these lines, a recent study in lysolecithin-induced demyelination demonstrated that young systemic environment through heterochronic parabiosis enhanced the remyelinating functions of endogenous, aged OPC [169]. This study pointed to a cell-based mechanism that involved the recruitment of young blood-derived monocytes from the young parabiotic partner to the demyelinating lesions. Further investigations revealed that parabiotic coupling to a younger mouse increased M2 microglia and macrophages in lesions of aged mice [170]. Another study reported that remyelination of lysolecithin-induced demyelination occurs as extensively but more slowly in old rats compared to young rats [215]. These studies indicate that remyelinating strategies targeting endogenous cells can be in principle effective throughout life, at least in rodents. Expert commentary

We have reviewed the recent and remarkable advances in the understanding of oligodendrocyte differentiation and function from which novel targets related to myelination may emerge. However, selection of a drug target on the basis of these findings is only the first step in the long process of drug development in which the link between basic science of how oligodendrocytes and myelin are developed and the clinical science of how myelination and synaptic transmission are measured will be crucial. For translation into human disease, the novel insights from developmental studies must be validated for conserved roles in the setting of myelin repair and regeneration postinjury. In this regard, data of particular translational relevance has emerged from access to high-quality human brain tissue to analyze histological, protein, mRNA, miRNA, and epigenetic levels. For instance, pathological studies of MS lesions have yielded new insights on the presence of cortical demyelinating lesions [216]. Moreover, subpial and meningeal inflammatory cell aggregates similar to germinal centers have been reported in the proximity of these cortical lesions [217]. It has been hypothesized that these cell aggregates represent sites of chronic inflammation within the blood-brain barrier and contribute to cortical demyelination. Experimental confirmation of this hypothesis would provide a rationale for therapies targeting innate lymphoid cells which are the initial seeds for these cell aggregates [218]. Further research in human brain tissue is necessary to establish conservation of mechanisms across species. Although tissue banks for MS have been set up, access to these resources may still be challenging. Expert Rev. Neurother. 14(11), (2014)



To study successful remyelination in MS patients, reliable imaging measures are an urgent requirement. Several studies have attempted to use novel MRI techniques including magnetization transfer imaging, diffusion tensor imaging, and ultrahigh-field strength MRI in order to quantify myelination [219–221]. However, the current resolution of these imaging techniques is not yet sufficient to differentiate between varying degrees of remyelination and axonal injury. The use of positron emission tomography applying radiolabeled [11C]PIB to quantify myelin loss has been suggested [222]. The outcome of brain atrophy assessed by MRI is closely associated with disease progression [223], but this is downstream of, and possibly in part unrelated to, the remyelination effect and takes time to change. The alternative outcome measures to quantitatively monitor remyelination and progressive axonal loss over time include retinal nerve fiber layer thickness on optical coherence tomography [224,225] and multimodal evoked potentials [226] as non-MRI techniques. Thus, in vivo quantification of remyelination in the human CNS by a robust marker will be a future challenge in MS research. Combining the currently available MRI techniques

Review

with measurements of brain atrophy and retinal nerve fiber layer thickness seems to be the optimal strategy to assess the efficacy of a novel treatment on myelination in MS patients. Five-year view

Remyelination-enhancing therapies for MS patients are not yet reality, but we believe that they will reduce disease progression and disability. Our understanding of how myelination in the CNS is regulated has greatly advanced in the past decade. The combined use of genetics, transcriptome analysis, in vitro, and in vivo models has identified multiple targets affecting the remyelination process and early stage human clinical trials have started. Financial & competing interests disclosure

F Zipp has received research grants from Teva, Novartis, Merck Serona and Bayer. F Zipp has also received consultation funds from Johnson & Johnson, Novartis, Ono and Octapharma. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key issues • Direct axonal damage within the early stages of inflammation as well as neuronal injury as a result of chronic demyelination are essential factors for the development of long-term disability in multiple sclerosis. • Remyelination is a spontaneous regenerative process that can occur not only in animal models following experimental demyelination but also in multiple sclerosis patients. • Spontaneous repair is limited due to an inhibitory microenvironment • Quantification of remyelination by modern imaging techniques is necessary to evaluate novel treatment strategies.

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