Achievements and obstacles in the design of remyelinating therapies for multiple sclerosis

Martin Stangel 1, Tanja Kuhlmann 2, Paul M. Matthews 3, Trevor J. Kilpatrick 4

1 Clinical Neuroimmunology and Neurochemistry, Department of Neurology, Hannover Medical School, Hannover, Germany

2 Dept. of Neuropathology, University of Münster, Münster, Germany

3 Division of Brain Sciences, Dept. of Medicine, Imperial College, London, United Kingdom

4 Melbourne Neuroscience Institute, Melbourne University, Parkville, VIC, Australia

Corrspondence to:

Dr. Martin Stangel

Dept. of Neurology

Hannover Medical School

Carl-Neuberg-Str. 1

30625 Hannover

Germany

Ph. +49 511 5326676

F. +49 511 5323115

e-mail: stangel.martin@mh-hannover.de

Abstract

Remyelination in the central nervous system (CNS) is the inborn program to repair the damage in demyelinating diseases such as multiple sclerosis (MS). This program, however, becomes inadequate in many people with MS, leading to axonal degeneration and clinical disabililty. Enhancement of remyelination is a logical therapeutic goal. However, all licensed MS therapies are immunomodulatory and do not support remyelination directly. Several molecular pathways have been identified as potential therapeutic targets to induce remyelination. Some of these have now been assessed in proof-of-concept clinical trials. However, there are several obstacles in trial design. Optimal clinical or paraclinical outcome measures to assess remyelination remain ill-defined. The timing of application of these therapies is also a critical issue. In addition, realistic expectations concerning the likely benefit to be achieved by such therapies need to be appreciated. Nevertheless, enhanced remyelination is likely to be protective for the axon and thus may prevent long-term neurodegeneration. Future MS treatment paradigms are thus likely to involve a combinatorial approach involving both immunomodulatory and regenerative treatments.

Key points

· Remyelination in the central nervous system is the inborn program that serves to limit and repair the damage in demyelinating diseases such as multiple sclerosis.

· Several intrinsic molecular pathways that execute endogenous remyelination have been identified and are potential therapeutic targets.

· The first clinical proof-of-concept trials to enhance remyelination in MS have been conducted recently.

· The optimal clinical and paraclinical outcome measures for assessing remyelination are not known, but neurophysiological measures, magnetic resonance imaging (MRI), myelin-targeted positron emision tomography (PET) radiotracers, and optical coherence tomography (OCT) all are possible adjuncts to clinical outcomes in proof-of-concept studies.

· The timing of application of these therapies is a critical issue.

· The future of MS therapy is likely to involve a rational combination of both immunomodulatory and regenerative treatments.

Introduction

Since the licensing of the first interferon-beta (IFN-) preparation for the treatment of Multiple Sclerosis (MS) in 1995 there has been a revolution in the development of MS-specific therapies (Fig. 1). The currently licensed MS therapeutics in the USA and Europe are based on 9 different mechanisms of action. It is anticipated that there will be two more available in the course of 2017. All these drugs target the immune system, either via immunosuppression or via immunomodulation, based on the assumption that MS is caused by autoimmune mediated demyelination of the central nervous system (CNS)1,2. After a demyelinating attack, remyelination, an innate program invoked to repair tissue damage is commonly triggered. However, this program fails or is insufficient in many patients with MS, resulting in downstream consequences, the most important of which are later vulnerability to axonal degeneration and consequent progression of clinical disabililty. Thus, a remyelination promoting treatment is desirable.

Remyelination in acute experimental animal models has been shown to be reliable and efficient3,4 and several molecular pathways by which it can be invoked have been defined5,6. However, it is not clear if these experimental data are directly transferable to the human disease MS. Several putative molecular targets for enhanced remyelination have been described in mouse experiments and several promising therapeutic candidates have been identified (for review see5,6). The monoclonal antibody opicinumab, directed against LINGO-1, the anti-muscarinegic drug, clemastine, and erythropoietin all have been tested in randomised controlled clinical trials (Tab. 1). Unfortunately, none of the results of these early phase trials has been overwhelming, although some suggestive benefits were reported that could stimulate further investigations. In addition, there are several other trials registered at ClinicalTrials.gov (Table 2). It is thus timely to review our current knowledge of remyelination in MS, what we have learned from a clinical perspective, how future trials involving remyelinating strategies should be designed, and what expectations we can have from this therapeutic approach.

Why do we need remyelination?

In the physiological state, both myelinated and unmyelinated axons are found in the CNS. Classical teaching was that myelin thickness is dependent simply on axonal diameter (maintaining a fixed ratio between axonal diameter and myelin thickness, the g-ratio) and that myelin thickness influences internodal length and, in consequence, the speed of saltatory conduction. Recent evidence has shown that these principles do not always hold. For example, in the auditory pathway, axons transmitting signals in response to low frequency sound have thicker myelin sheaths, but shorter internodes, than the axons that are stimulated in response to high frequency sound7. This example serves to demonstrate that myelin almost certainly assists in the efficient temporal processing of axonal signals and suggests that there must be precise regulatory mechanisms governing myelin sheath formation. Other important functions of myelinating oligodendrocytes include the supply of trophic factors for the axon and protection against inflammatory attack8,9.

It is well known that after damage new myelin segments arising as part of a remyelinating program are thinner and shorter, resulting in reduced distances between adjacent nodes of Ranvier, than the original myelin. In addition, a recent study in an animal model of toxic demyelination suggests that network functions may not recover completely when there is cortical demyelination10. These observations raise the question of what remyelination can achieve. A perfect repair process would entail reconstitution of identical myelin thickness, internodal segmental length and distribution of ion channels to what was present prior to injury. It is unlikely that this is achievable even when remyelination is supported by a treatment. Thus, we have to adapt our expectations for what remyelination can achieve in a disease like MS. Nevertheless, even an imperfect myelin sheath will improve conduction velocity and thus may improve functional recovery. Theoretically, this benefit could encompass reduced fatigue and resolution of Uhthoff's phenomenon. In the long term, an intact axon-myelin unit could protect the axon from further degeneration or at least slow neurodegeneration down. Thus the primary logic for therapeutic enhancement of remyelination is to diminish future clinical disability.

The course of remyelination in MS

Although debated for a long time, the presence of remyelination has been demonstrated by electron microscopy in MS lesions11-13. In MS, lesions can be either completely or only partly remyelinated and the remyelinated areas are frequently but not exclusively located at the lesion border. However, a histological differentiation between partly demyelinated and remyelinated axons in human MS samples may be difficult as there is no marker that can unequivocally distinguish newly formed myelin. Using light microscopy, the resolution is usually not high enough to measure internodal length or axon diameter. With routine histology of MS tissue, the intensity of the myelin staining and the thickness of the myelin thickness are used to identify remyelination, although these are imperfect measures (Fig. 2).

Axons are remyelinated by adult oligodendroglial progenitors that differentiate into myelinating oligodendrocytes in response to a demyelinating stimulus. The extent of remyelination can differ from lesion to lesion, even within the same patient, depending on lesion location14-16. However, in a histological study, sub-groups of MS patients with either extensive or limited remyelination were identified, suggesting that individual (possibly genetic) factors also influence the ability of lesions to remyelinate 14. The reasons for variable remyelination capacity depending on lesion location are unclear. Intrinsic differences within the OPC population or the extracellular milieu could contribute to this phenomenon. Interestingly, a recent study was able to identify transcriptionally heterogeneous oligodendroglial subtypes in the rodent CNS with an enrichment of individual populations in certain brain regions17. It also has been recently reported that rodent neural precursor cells within the subventricular zone have the capacity to differentiate into oligodendrocytes with superior remyelination capacity than oligodendrocytes emanating from parenchymal oligodendrocyte progenitors18.

In inflammatory active and demyelinating lesions, the initial phenotype of established MS lesions, remyelination occurs frequently. Remyelination is also a common phenomenon in MS tissue samples from patients with short disease durations, but is less frequent in samples from patients with more chronic disease, in whom only approximately 20% of the lesions are completely remyelinated (so-called “shadow plaques”)4-16. As indicated above, the histological identification of ongoing remyelination is difficult, since it is so far impossible to determine when the new myelin sheath was formed. One cogent approach to identify ongoing remyelination is to use markers that label oligodendroglia at different stages of maturation. Results from such an approach suggest that remyelination is initiated immediately after onset of demyelination but is rare or absent in long-lasting MS lesions19. Similar dynamics have been identified, in demyelinating rodent models of remyelination. In zebrafish, individual oligodendrocytes have an even narrower time window of five hours in which to complete the formation of internodes20. On the other hand, using MRI techniques like voxel based MTR (see below), Chen and colleagues observed significant increases in MTR, consistent with remyelination, for approximately 7 months after initial formation of a MS lesion21. Experimental studies in a rodent model of spinal cord injury also suggest that, although the length of the myelin sheaths is short in the early remyelinative phase, the sheaths length and thickness are both adjusted during the following 6 months to approach control levels, suggesting that some refinement of remyelination can occur in the subacute phase22. Synthesis of the available data suggests that while there is variation between species, remyelination is initiated promptly and is subject to later refinement, but is likely to stop within weeks to months of its commencement within a given region and is unlikely to occur in long-standing, chronic lesions.

Mechanisms governing remyelination and its failure

Remyelination is a complex biological process that requires the orchestrated interaction of both intrinsic and extrinsic signaling molecules and pathways. A prerequisite for remyelination is the presence of (functionally intact) axons, as well as oligodendroglial progenitor cells (OPCs) that differentiate, establish contact with and ensheath axons as well as to form compact myelin. Especially in long-lasting MS lesions, oligodendroglial differentiation and migration is impaired, resulting in insufficient remyelination23-25. Although oligodendrocytes are able to wrap around nanofibers that have comparable physical properties to axons26-28, they do not form compact myelin sheaths around these inert structures, suggesting that axonal activity or signaling is required for the final steps of myelination. Electrically active axons are preferentially myelinated29,30. The interaction between axons and oligodendrocytes party depends on synaptic and non-synaptic vesicular release of glutamate20,29,31. Axonal glutamate release promotes remyelination via increased differentiation of OPCs32. Axonal vesicles also have been shown to contain growth factors such as NRG-1 and BDNF which have promyelinating capacity. Furthermore, NRG-1 and BDNF stimulation of oligodendrocytes has been shown to induce activity-dependency to their myelinative capacity, demonstrating interdependency between these extrinsic stimuli. These findings from in vitro and animal studies demonstrate the close interaction of axons and oligodendrocytes during (re-)myelination and also stress the fundamental importance of functionally intact axons in guiding outcome. This is especially important in the context of MS where axons are frequently injured and little is known about the functionality of demyelinated axons. In addition to functional, intact axons, the timely migration and differentiation of OPCs into myelinating oligodendrocytes is a prerequisite for successful remyelination. OPCs accumulate at the borders and within the body of MS lesions, where the OPCs are unevenly distributed, suggesting a disturbed migration of OPCs23. During development, migration is regulated by short-range molecules expressed predominantly in the local extracellular milieu, such as laminin, fibronectin, vitronectin, and tenascin-C (for review see33), and diffusible molecules such as the netrins, semaphorins, and cMet/HGF. Histological studies suggest that a migration-promoting environment in active demyelinating lesions shifts to a less favourable milieu in long-lasting lesions23,34-39.

The differentiation of OPCs into mature myelinating oligodendrocytes is also frequently impaired, especially in long-lasting MS lesions19,40,41. The differentiation of these cells is regulated by intracellular signaling cascades and transcription factors, such as Olig1/2, Sox10 and Myrf (for review see42). Using a combination of in vitro and animal experiments, several intracellular signaling cascades have been identified which contribute to the regulation of oligodendroglial differentiation and remyelination, e.g. wnt signaling, Lingo1 or RXR signaling43-47 (see also Table 3). Other pathways stimulating oligodendroglial differentiation have emerged in middle or high-throughput compound screens using rodent OPCs. For example, benztropine and clemastine promoted oligodendroglial differentiation via muscarinic receptor antagonism and miconazole via ERK phosphorylation48-50. In addition to enhancing remyelination by direct modulation of pathways within oligodendrocytes, the process is also modulated by paracrine factors. It is well established that the removal of myelin debris by phagocytes is required for initiation of remyelination51. The capacity of macrophages and microglia to phagocytose myelin decreases with ageing and, in consequence, the remyelination capacity decreases as well, at least in rodents. Accordingly, impaired myelin phagocytosis in aged animals can be circumvented by heterochronic parabiosis resulting in the “rejuvenation” of remyelination52.

Possibilities and caveats concerning the enhancement of remyelination

There are several strategies that can be implemented and a number of putative targets that can be tested in attempts to enhance remyelination with in the CNS. As described above, myelin debris and impaired myelin phagocytosis delays remyelination. Thus, modulation of phagocytosing cells could, in principle, enhance remyelination by creating a receptive microenvironment. Although this has been shown experimentally53, a drug that specifically modifies either resident microglia or infiltrating macrophages for therapeutic benefit is not currently available. Clearly, care needs to be taken in adopting such an approach since overactive phagocytosing cells could potentially cause further damage.

A direct target for remyelination is the OPC itself. Enhanced remyelination can be achieved by either improving the migration of these cells in order to recruit more OPCs to sites of demyelination or by enhancing their subsequent proliferation and maturation to increase the number of oligodendrocytes available to myelinate axons. Targeting one or more of these steps would appear feasible and there have been experimental studies to provide proof of principle. However, since it is not exactly known why remyelination is insufficient in MS, it is not clear which of these steps is rate limiting. Furthermore, the rate limiting step could vary both between individuals and between lesions in any given patient. Interrogation of MS lesions suggests that both insufficient migration of OPCs into the lesion and a differentiation block can occur19. It is unknown whether these deficits are due to an error within the OPC differentiation program or, alternatively, due to either the lack of external signals or the presence of inhibitory factors within the lesions. These signals can, in principle, be derived from any of the other cell types within a lesion. It has already been mentioned that microglia are important for the phagocytosis of myelin debris, but, in addition, microglia and also astrocytes produce a number of important factors required for successful remyelination. However, to date we do not have specific substances that can selectively target these cells.

Besides the promotion of endogenous repair mechanisms, it has been also suggested that remyelination may be achieved by administration of exogenous cells. An initial suite of experiments suggested that these cells could directly myelinate demyelinated axons. Recent considerations rather favour the concept that precursor cells create a remyelination promoting environment54. There remains an active debate concerning which stem cells (neural, mesenchymal, etc.) would be preferable to use and how to administer them (e.g., intravenous, intrathecal, or into the lesion). However, the transplantation of remyelinating cells directly into lesions is probably not practical in a complex disease like MS as this potentially requires stereotactic injections at numerous sites. When stem cells are administered via the intravenous route the effect could well be mediated via modulation of the immune system without direct effects on remyelination55.

Timing of treatment

As mentioned above, remyelination starts immediately after demyelination has occured and potentially lasts up to 6-7 months thereafter. Nevertheless, the most important molecular switches that establish the remyelination program are probably set within the first few weeks. Thus, it seems appropriate to initiate a remyelination promoting therapy immediately after the immune damage. Consistent with this view, most trial designs currently apply investigational drugs after a relapse. Optic neuritis has emerged as a favoured clinical presentation to assess remyelinative therapies since its presenting features are fairly stereotyped and there are several good clinical outcome parameters, as well as robust neurophysiological tests and optical coherence tomography (OCT), by which to assess outcome.

Although most investigators would agree that a remyelinating treatment should start as soon as possible after a demyelinative event, it remains uncertain as to whether confining the utilization of such treatment to immediately after clinical relapses will be sufficient. It is well known that many new lesions develop without clinical signs and symptoms. In additon, for unknown reasons, as the disease progresses, the CNS pathology that ensues becomes less obviously a consequence of peripheral immune attack. Therefore, continuous application of a treatment would have the advantage that the clinically silent lesions would be treated, as well.

Besides directly influencing remyelination, other neuroprotective treatments could be explored as effective therapies for MS. Since the first clinical symptoms of MS usually occur early in the disease course when there is only limited neuronal damage, this is theoretically of great advantage from a therapeutic perspective when compared to classical neurodegenerative diseases such as Parkinson's or Alzheimer's disease where the initial clinical symptoms occur after a considerable number of neurons have already degenerated. Thus, MS could prove to be an important test-bed to assess the efficacy of neuroprotective therapies applicable to the treatment of neurodegerative disease, in general.

It will be also important to determine whether it is possible to repair lesions that have been demyelinated for a long time. The clemastine trial, where patients with long standing optic neuritis have been treated (see table 1), suggests that we may be able to achieve at least a small functional improvement in such patients. However, it is not entirely clear if the improvement in this trial was actually mediated via remyelination or whether it was dependent upon other mechanisms.

Assessing demyelination and remyelination in MS patients

There are several possible approaches for non-invasive assessment of demyelination and remyelination. Evoked potentials have been used for the evaluation of patients with MS for decades, although it has been introduced only recently that measures have been interpreted quantitatively with respect to myelination56. Now classical observations of slowing and subsequent recovery of the visual evoked potential latency after optic neuritis are interpreted as evidence for demyelination and subsequent remyelination57. While measures of latency generally are stable in asymptomatic patients, differences over time correspond with behavioural changes in visual contrast sensitivity58. However, interpretation of the changes in latency as evidence of demyelination is not unequivocal, e.g. changes also may reflect redistribution of ion channels59. Complementary information concerning demyelination associated axonal loss is provided by the amplitude of the evoked response. Visual evoked potential measurements are highly reproducible in healthy volunteers, although more data are needed in people with MS and in routine clinical use outside of study or trial protocols60.

More recently OCT measures of retinal nerve fibre layer thickness have provided a more direct measure of axonal loss associated with inflammatory demyelination in the anterior visual tract61. The methods are quantitative, reliable in test-retest studies and the methods already are widely diffused62. The low cost and ease of OCT measurements makes them attractive as outcome measures in clinical trials, although they limit trial designs to assessment of demyelination-remyelination in the anterior visual pathways. The clinical usefulness of the measures other than as paraclinical tests to support diagnosis is unclear.

MRI and PET imaging are sensitive to demyelination-remyelination across the whole brain and spinal cord63,64. MRI supports a range of approaches for evaluation of myelin content indirectly through its effects on imaging characteristics of brain water. All of these approaches are non-specific and rely on empirical relationships established with animal models and post mortem studies ex vivo. Nonetheless, they have the advantages of providing images of conventional brain imaging resolution, full brain and spinal cord coverage, and are able to be acquired on current clinical high-field MRI systems.

Myelin is highly enriched in intrinsic membrane proteins. Water associates reversibly with the exposed polar amino acids of myelin proteins to create a loosely bound compartment in exchange with free tissue water. Although imprecise and insensitive, conventional T2 imaging signal intensity reflects myelin density because of this „bound“ water component65. The bound water shortens the T2 relaxation time of bound relative to intra- or extracellular or free water. The proportion of the total brain water that is in the rapidly relaxing component of bound water can be estimated from analysis of serially acquired images using a range of methods, although all share use of different delays for T2 relaxation66. Validation experiments ex vivo have shown strong correlations between the bound water fraction and myelin content67. Longitudinal studies in vivo have demonstrated variable recovery of myelin water content in individual lesions after an acute demyelinating episode in MS68. New fast imaging methods now allow whole brain imaging in a clinically practical time frame69. Combining information on the myelin bound water pool with measures of total tissue water from MRI proton density imaging allows estimates of the g-ratio in highly oriented white matter, such as the spinal cord70.

Diffusion MRI is sensitive to the relative directional diffusion of water molecules. In bulk water (e.g. the ventricular cerebrospinal fluid), rates of diffusion are equal in all directions (isotropic diffusion). However, water molecules associated with myelin proteins are constrained in their diffusion. Axons and myelin particularly restrict diffusion transverse to their axis (anisotropic diffusion). Measures of mean diffusivity increase and measures of directionality (fractional anisotropy) decrease with demyelination, although these changes also can reflect oedema or axon loss, depending on the context71. Increases in fractional anisotropy also can be seen over time which have been interpreted to reflect possible remyelination72.

The specificity of diffusion imaging for myelin can be increased by using more realistic models for analyses. A recently proposed variant to this approach, called q-space imaging, which assesses the kurtosis of the diffusion measure, has been validated with respect to histological measures in myelin-deficient mice and after chemical spinal cord injury in a non-human primate, where it demonstrated sensitivity to remyelination73. A pilot application has confirmed that q-space imaging is sensitive to variation between healthy volunteers and patients with MS74. 

The final and currently most widely used method is magnetization transfer imaging. This relies on differences in the resonance frequency of water molecules associated with myelin proteins and “memory” of water protons for short-term magnetization introduced by the imaging sequence. To do this, the difference in chemical shift relative to bulk water allows selective “tagging” of water molecules to reduce the signal in the image. As these “tagged” molecules exchange with free water, the overall MRI signal (which arises overwhelmingly from free- or non-bound water) is reduced. The greater the myelin content, the greater the exchange of “tagged” molecules and the greater the decrease of the free water signal immediately after the selective “tagging”. By subtracting an image made with the tagging sequence ‘on’ from that with it ‘off’, ultimately, it is possible to create an image of the relative distribution of exchanging water pools75.

Empirical correlations with post mortem brains and preclinical studies have validated the relative magnetization transfer (the magnetization transfer ratio or MTR) as an index of myelin76. The precision of the measurement and low test-retest variance on a given scanner suggest that well powered studies may demand only modest numbers of subjects77. While measuring MTR in a reproducible way across sites and scanners is difficult, current technology has been demonstrated in a Phase II trial78. However, like the other MRI measures of myelin, MTR is an indirect index. Absolute myelin content cannot be inferred from it, as it is also influenced by water content, inflammation and axonal loss68,76. Perhaps most troubling is that information provided by myelin water, diffusion and magnetization, while sharing common general relationship with myelin content, are not directly comparable79.

Molecular imaging of myelin with positron emission tomography (PET) has been explored more recently in effort to provide a more direct and quantitative measure. PET involves reconstruction of an image of the tissue of interest based on the detection of positron annihilation events localised to the distibution of an injected tracer molecule that has been labeled with a positron emitting isotope (typically 11C or 18F). The PET radiotracers that bind to brain amyloid also have shown binding to myelin. For example, differences consistent with the patterns of relative white matter demyelination in people with MS are apparent across paired MRI scans with the amyloid-targeted radioligand, N-Methyl-[11C]-2-(4′-methylaminophenyl)-6-hydroxybenzothiazole (Pittsburgh Compound B or PIB)80. Despite the relatively low spatial resolution of PET (linear resolution of a few mm, depending on acquisition methods and signal), recent data suggest that the sensitivity could be sufficient to monitor relative myelin changes over short follow up periods (of months)81. In a younger patient population, the PIB signal would not be expected to be confounded by amyloid deposition, although this should be considered if increased cortical uptake is found. While evaluations of other amyloid radiotracers for myelin mapping are limited to date, there is no reason to believe that the commercially manufactured, fluorinated amyloid radiotracers do not share similar characterstics. Myelin PET imaging for MS patients using amyloid radiotracers therefore could build on infrastructure and experience in clinical sites for its use in Alzheimer’s disease. Newer agents such as [11C] N-methyl-4,4′-diaminostilbene ([11C]MeDAS (or a fluorinated derivative of the same structure)82 promise greater sensitivity with higher affinity for myelin.

While PET imaging is of great interest because it provides a measure that directly reflects myelin content, thus far, only maps of relative myelin content have been able to be generated. As noted above, these images are limited by the lower spatial resolution of PET, so that the myelin content in small lesions may not be well estimated. PET myelin imaging is not yet ready for confident application in drug development studies, although the groundwork has been laid for potential rapid progress. Development of improved methods for co-registration of MRI and PET scans will help with localization of the signal and will become easier to address as integrated MRI-PET scanners begin to be used.

The measures available now for clinical applicaiton each reflect different properties of myelin. Although not demonstrated formally, they have different dynamic ranges and different sensitivities to change. There is a strong argument for including multiple measures in clinical trials intending to provide mechanistic proof of concept. With knowledge of outcomes from clinical trials of remyelination therapies, the potential utility of these measures for stratification and monitoring will become clear.

What can we expect from a treatment

In simplistic terms, we expect truly novel therapeutics targeting MS to influence long-term disease outcome. This ultimately relates to the modulation of clinical disability. However, it will be impractical to routinely assess the efficacy of such treatments in RCTs utilising primary clinical endpoints. Instead, our immediate expectation will be that effects of treatment will be evident from differences in paraclinical indicators (e.g. VEP, OCT, MRT or PET), which can, in turn, be extrapolated to subsequent clinical outcome. Such analysis will be applicable not only to clinical trials but also eventually to the monitoring of individual patients in routine clinical practice.

The expectations of a treatment are dependent on its direct and indirect biological effects, the nature of the therapeutic regimen, and the pathological context in which it is applied. Remyelinating therapy can only be expected to be effective when and where demyelinaton is extant. There is emerging evidence that MS pathology is not always accompanied by demyelination83. This indicates that there will be occasions when it is important to implement robust paraclinical measures that can quantitate the degree of demyelination on both a regional and global scale in order to identify in whom a remyelinative approach is likely to be beneficial and therefore applied. It is also possible that our expectations of a remyelinative therapy will only be realised by adopting strategies that target multiple components of the myelination cascade. In a heterogeneous disease such as MS, it is unlikely that a singular deficit will apply to all lesions such that there will be circumstances in which it is appropriate to enhance the recruitment of progenitors, whereas, in other circumstances, enhancement of either the extent or the quality of myelination might be sufficient. Ion channel redistribution also will be a necessary component of the enhancement of functional recovery. This implies that therapies which serve to initiate the myelination cascade rather than those which target specific structural events are most likely to be broadly beneficial.

The fact that trials for remyelinating therapies that have been conducted to date have focused on optic neuritis suggests that this is a practicable approach and one that could be ultimately applicable to routine clinical practice. As such, these therapies could be applied acutely in the context of a clinically significant relapse, aiming to enhance remyelination in the reparative phase. The downstream benefits could be multifaceted, including the direct promotion of functional recovery and a reduction in the risk of secondary axonal degeneration. It is important to note that limiting axonal degeneration may not have direct clinical benefit after a single relapse but, by maintaining axonal reserve, remyelination could provide a protective effect against clinical disability arising from subsequent disease activity affecting the same neural pathway. This would suggest that a remyelination promoting treatment may not necessarily improve the recovery from an individual relapse but could still provide meaningful benefit in the long-term by delaying the onset of the progressive phase of the disease.

The administration of remyelinative therapies in a sporadic way based on clinical activity may not, however, be the most efficient approach to adopt. It is well known that lesional activity within the brain exceeds clinical relapses by up to 10-fold84. Although it could be argued that lesions resulting in clinical disability are more significant based on their severity or their targeting of eloquent pathways, the cumulative pathology resulting from asymptomatic lesions, as well as changes in so called normal appearing white matter, will almost certainly also ultimately contribute significantly to long-term disability. An argument could therefore be made for the ongoing chronic administration of remyelinative therapies, independent of relapse activity. If this approach were adopted, it would be predicated on the relevant agents having a high therapeutic index. There would be a low threshold for cessation of therapy of this nature in the context of a disease such as MS, if significant side-effects were to ensue.

The provision of continuous remyelinative therapy could, however, also have caveats. For example, if the therapy directly enhanced the maturation of oligodendrocyte progenitors it could theoretically result in the depletion of this cellular pool. This caveat could be addressed by administering remyelinative treatments as pulse therapy, thereby allowing the progenitor population sufficient time to re-expand. Such an approach could, itself, generate difficulties if the pulse therapy were not delivered concordant with temporal peaks in demyelinative activity, especially if there is a narrow window of opportunity in terms of enhanced remyelinative potential. As indicated above, the trial of clemastine delivered in the context of chronic optic neuritis suggests that this might be an unnecessary concern given that chroncially demyelinated axons appeared to remain responsive although whether their responsiveness is quantitatively diminished is unknown. A hope for the future would be the ability to routinely assay a cost-effective peripheral marker of the real-time global level of myelin breakdown (e.g., myelin basic protein fragments in the blood) and to deliver remyelinative therapy when the levels rise. However, to date there is no such marker known that reliably predicts demyelination.

Another theoretical risk is that an adverse outcome could ensue if inappropriate axons are myelinated, in effect, resulting in a “dysmyelinating” phenotype, that could compromise functional recovery by interfering with coordinated axonal transmission within neural networks. This caveat could be addressed by combining the remyelinative therapy with physiotherapy or transmagnetic stimualtion to promote activity dependent myelination.

Finally, the stimulation of OPC or other progenitor cells could theoretically lead to the development of uncontrolled growth and tumours. Fortunately, the animal studies investigating the various pathways so far have not indicated that this is likely to be pertinent, but it is important to note that safety data from the long-term administration of these treatments are not, as yet, available.

Future directions

Since there is such a large number of MS immunomodulatory therapies with different modes of action already available, many experts believe that the scope to develop truly novel immune therapies is limited. Despite this armamentarium of treatments, none is currently able to silence the disease completely and there are treatment failures with all the available drugs. Thus, MS therapeutics needs to advance with development of neuroprotective and regenerative treatments. Although remyelination is the most obvious approach by which to promote regeneration in MS, we also have to bear in mind what could be achieved with such treatment. It seems unlikely that remyelinating therapies will consistently lead to a better outcome after an acute relapse, since many relapses recover well clinically and, where functional impairment does ensue, the deficits are probably not exclusively due to demyelination, but also to other factors (e.g., axonal degeneration or dysfunction). Remyelination is most effective immediately after damage ensues and thus it seems reasonable to begin a remyelinating treatment immediately after a relapse or at least early in the course of the disease in patients who are experiencing clinical or paraclinical signs of incomplete repair. Another fundamental question that has so far not been addressed is whether the molecular processes governing remyelination are the same in all patients. Since the immunological process leading to demyelination may vary between patients85 it is conceivable that there may also be differences in how remyelination can be enhanced.

Currently, it is not clear how best to measure remyelination in a clinical trial and new imaging methods need to be developed and applied for this purpose. At the same time, we need to better define clinical endpoints for clinical trials. The favoured site of interrogation is currently the visual system, focusing on the clinical presentation of optic neuritis since we can define outcome relatively accurately both from clinical and paraclinical perspectives. One caveat in focusing on optic neuritis is that MRI is difficult in the optic nerve, recognizing that MRI outcome measures have become an essential component of judging the efficacy of immunomodulatory therapies amongst MS trialists.

Spinal cord lesions after myelitis would, in principle, be another good region to focus on for clinical trials of remyelinative therapies. Again, clinical symptoms can be easily localised to the spinal cord, there are neurophysiological measurements available, and the region is accessible for MRI. However, to effectively interrogate therapeutic responsiveness utilizing the spinal cord as a region of interest we need to acquire more information concerning how lesions develop within the spinal cord, since most of the MRI studies investigating remyelination have been performed in the brain. It is also important to acknowledge that remyelinaitng therapies are alos being tested in other contexts, for example in children with Pelizaeus Merzbarcher disease86. Success in these contexts would provide a rationale for fast tracking of follow-up clinical trials in people with MS.

It will be also necessary to assess the potential enduring clinical benefits of remyelination beyond the short-term paraclinically-based benefits identified in short-term proof of principle studies. The definitive identification of such benefit would require long-term trials that may not be practical. Whether other approaches such as registry-based, long-term observational studies using propensity matching will provide meaningful insights in this regard remains uncertain87.

In summary, aiming for a remyelinating treatment to enhance the natural repair process in MS is a rational goal. Several important questions including the best molecular target, the best method to measure short-term responsiveness, and how to interrogate likely long-term clinical benefit remain. Nevertheless, there is cause for optimism that we will solve these questions in the years to come. Once available, remyelinating treatments will complement immunomodulation by inducing repair and potentiating neuroprotection to improve long-term outcome in MS.

Acknowledgements

PMM is in receipt of generous personal and research support from the Edmond J Safra Foundation and Lily Safra, the Imperial College NIHR Biomedical Research Centre, the NIHR Senior Investigator Programme, the UK Medical Research Council, Biogen and the Engineering and Physics Science Research Council.

Author contributions

All authors performed literature searches, selected important literature to be cited, wrote various sections of the manuscript, and revised the entire manuscript.

Figure legends:

Fig. 1:

Timeline of the licensing of MS-specific therapeutics in the EU. Cladribine and Ocrelizumab are currently being assessed by the regulatory authorities and are expected to be licensed in 2017.

Fig. 2:

Remyelination in MS. (a and b): A MS lesion in which the total lesion area is remyelinated (shadow plaque). Due to the thinner myelin sheaths and reduced numbers of axons the staining intensity is pallor within the lesion compared to the adjacent normal appearing white matter (NAWM) ((a) Luxol-fast blue/PAS staining, (b) immunohistochemistry for MBP). (c) Higher magnification of black boxed area in A. Within the lesion the numbers of oligodendrocytes (arrows) is lower than in the NAWM. (d) Higher magnification of the white boxed area in A. In the NAWM numerous oligodendrocytes are present (arrows). Oligodendrocytes are characterized by round nuclei and white perinuclear halos. (e) Demyelinated white matter lesion; remyelination is limited to the lesion border. (f)Higher magnification of the boxed area in e. shows thin and irregularly formed myelin sheaths.

Scale bars: a, b, e: 2mm, c and d: 200 µm, f: 100 µm.

Fig. 3:

Dynamic myelin changes in relapsing remitting MS detected by MRI. (A) shows an MTR image at baseline with a region of interest defined by the T2 hyperintense lesion borders labelled in black. (B) is a followup image performed 2 months later. (C) is a difference image in which the first image is subtracted from the second. Outlined regions show changes in signal intensity relative to baseline, highlighting decreases in the magnetisation transfer ratio (MTR), suggesting relative loss of myelin (blue, see colour scale to right), or a lack of change (images courtesy of Dr. R. Brown, McGill University). Methods are described in88

26

Table 1: Completed randomised controlled clinical trials aimed to induce remyelination in MS patients. Trials were either reported at international meetings or published

Title/compound

No. of patients/trial design/patient characteristics

Compound/

dosage

Primary outcome

Secondary outcome

Results

Published/presented

RENEW, Opicinumab (anti-LINGO1) in optic neuritis

Approx. 80;

Randomised, doubleblind, placebo controlled;

patients with first ever optic neuritis

Opicinumab 100 mg/kg i.v. every 4 weeks for 20 weeks

vs. placebo;

add-on to standard i.v. steroids

VEP latency after 24 weeks

Change in RNFL thickness at 24 weeks (OCT), low contrast visual acuity at 24 weeks,

Significant improvement of VEP latency in per protocol patients (normalisation in 53% vs. 26% in controls).

Clinical outcome measures not significant

Presented at AAN 2015,

Cadavid et al.

SYNEGY, Opicinumab (anti-LINGO1) MS

396,

RRMS and SPMS, EDSS 2-6;

randomised, doubleblind, placebo controlled;

Opicinumab i.v. 3 mg/kg (n=44), 10 mg/kg (n=88), 30 mg/kg (n=88), 100 mg/kg (n=88) vs. placebo (n=88) for 72 weeks;

add-on to IFN-1a once weekly

Improvement in either of EDSS, T25FW, 9HPT, PASAT

No significant differences.

Trend in the 10 and 30 mg/kg groups.

Presentetd at ECTRIMS 2016, Cadavid et al.

ReBUILD, Clemastine

50 MS patients with disease duration of at least 5 years, last optic neuritis >6 month ago;

placebo controlled, crossover

5 mg clemastine bid for 3 month vs. placebo, then cross-over

Reduction of P100 latencies on full field transient pattern reversal VEP measured at baseline, 1 month, 3 month, 5 month.

Improvement in VEP by 2 ms, Portion of patients with improvement of >6 ms higher in clemastine group.

Presented at AAN 2016

Erythropoietin

40 patients with acute optic neuritis;

randomised, doublbind, placebo controlled

33000 U rhEPO i.v for 3 days (n=21) vs. placebo (n=19); add-on to standard i.v. steroids

Reduction of retinal nerve fiber layer (RNFL) thickness from baseline to week 16 measured by OCT

Change in optic nerve diameter from baseline to week 16 (MRI), visual acuity, VEP

Statistically significant less reduction in RNFL thickness in treated group.

Decrease in optic nerve diameter was smaller in EPO group (significant).

VEP latencies after 16 weeks were shorter in EPO group (significant).

Only a trend in visual functions.

Published 89

Table 2: Clinical trials with a focus on remyelination in MS registered at ClinicalTrials.gov

Title

Sponsor/

ClinTrials identifier

Drug/

compound

Phase

(no. of patients)

/design

Primary outcome

Start/End

Thyroid Hormone for Remyelination in Multiple Sclerosis (MS): A Safety and Dose Finding Study (MST3K)

Oregon Health and Science University

NCT02760056

Liothyronine sodium vs. placebo

Phase 1

(24)

parallel, double-blind

Determine the maximum tolerated dose (MTD) of oral L-T3 in subjects with MS using the hyperthyroid symptom scale, electrocardiogram (ECG), and blood pressure day 1 and day 8


July 2016

March 2017

Study to Assess Whether GSK239512 Can Remyelinate Lesions in Subjects With Relapsing Remitting Multiple Sclerosis

GlaxoSmithKline

NCT01772199

GSK239512 vs. Placebo

Phase 2

(131)

parallel, double-blind

Mean change in gadolinium (Gd) enhanced (GdE) lesion magnetization transfer ratio (MTR) differences (calibrated to reference scan) from before enhancement to stable recovery (>=3 months post new GdE lesion)

Time Frame: Up to Week 48: Baseline MRI prior to randomization and at week 6, 12, 18, 24, 30, 36, 42, and 48.





Feb. 2013

Sept. 2014

Published78

Assessment of Clemastine Fumarate as a Remyelinating Agent in Acute Optic Neuritis (ReCOVER)

University of California, San Francisco

NCT02521311

Clemastine vs. Placebo

Phase 2

(30)

Parallel, Double-blind

Measure the effectiveness of clemastine relative to placebo at preventing the loss of retinal nerve fiber assessed via optical coherence tomography (OCT). Measure will be reported as difference in nerve fiber thickness between baseline and 9 months.


The second primary outcome is to measure the effectiveness of clemastine relative to placebo at improving patient performance on ETDRS low contrast visual acuity chart testing (2.5% black on white) during the recovery from an acute optic neuritis. Measure will be reported as difference in ETDRS score from baseline to 9 months.

Aug 2016

Jan 2018

Safety and Tolerability of Quetiapine in Multiple Sclerosis

University of Calgary

NCT02087631

Extended-release quetiapine fumarate

Phase 1/2

(36)

Single group, open label

The primary outcome is the occurrence of dose-limiting toxicity (DLT). Dose-limiting toxicity for any patient in this study is defined as early discontinuation of quetiapine XR due to an adverse event (AE) that is possibly, probably or definitely due to use of study drug. The dose-limiting toxicity will be determined for each group of patients: RRMS and progressive MS by the week 4 visit.

Dec 2014

Sept. 2016

Adrenocorticotropic Hormone (ACTH) Effects on Myelination in Subjects With MS

Weill Medical College of Cornell University

NCT02446886

Adrenocorticotropic hormone (ACTH) gel (H.P. Acthar®)

Phase 4

(30)

Single group open label

Primary endpoint is the absolute change in myelin water fraction (MWF) within new enhancing lesions over the course of 12 months

April 2016

Aug 2016

Pilot Trial of Domperidone in Relapsing-Remitting Multiple Sclerosis (RRMS)

University of Calgary

NCT02493049

Domperidone

Phase 2

(24)

Single group, open label

Measures of repair within enhancing T1 MRI lesions for up to 32 weeks.

Texture analysis, diffusion tensor imaging (DTI) and magnetization transfer imaging (MTI) will be used. This is a pilot study measuring repair at 2 time points and with 3 imaging methods to aid in the design of future studies.

Aug 2015

Aug 2018

Advanced MRI Measures of Repair in Alemtuzumab Treated Patients (iCAMMS-IST)

University of British Columbia

NCT01307332

MabCampath-1h

Phase 3

(25)

Single group open label

Changes in normal appearing white matter from baseline through month 24.

The MRI study is designed to identify possible mechanisms by which alemtuzumab acts to protect the brain from inflammation and how it may enhance repair through remyelination.

March 2011

Dec 2018

Table 3: Factors and pathways regulating OPC differentiation and remyelination

Pathway/receptor/ligand

Comments

References

Specification of OPCs

FGF

FGF signaling is required for the generation of OPCs from NPC

90

SIRT1

Genetic inactivation of SIRT1, a protein deacetylase implicated in energy metabolism, increases the production of new, normally differentiating OPCs, in part by acting on neural precursor cells. SIRT1 inactivation ameliorates remyelination.

91

Proliferation of OPCs

Notch-jagged

Jagged signaling via Notch receptor on OPCs modulates oligodendroglial differentiation. Lack of Notch1 in Olig1 expressing precursor cells was associated with decreased proliferation of OPCs and accelerated remyelination

92-94

Oligodendroglial differentiation and remyelination

LINGO1

Part of a tripartite receptor that inhibits axonal sprouting and oligodendroglial differentiation

95-97

Wnt

Activation of canonical Wnt signaling inhibits differentiation of OPCs into myelinating oligodendrocytes

98,99

Non-canonical Notch signaling

Binding of axonal contactin to its receptor Notch1 induces oligodendroglial differentiation via translocation of Notch 1 intracellular domain (NICD) to the nucleus. In MS lesions the translocation may be impaired.

100,101

Myelin debris

Myelin debris inhibits oligodendroglial differentiation and remyelination, potentially via myelin associated EphrinB3

102-104

Activin A

Activin A, secreted by anti-inflammatory microglia promotes oligodendroglial differentiation

105

Muscarinic acetylcholine receptor

Antagonism of the muscarinic acetylcholine receptor e.g. by benztropine promotes oligodendroglial differentiation and remyelination

48,50,106

BMP

Inhibitory effect, promotes astrocytic differentiation, blocking increases oligodendroglial differentiation

107,108

PSA-NCAM

Axonal PSA-NCAM is a negative regulator of oligodendroglial differentiation and re-expressed on axons in MS lesions

109,110

RXR

Knockdown or lack of RXR, a nuclear receptor, delays oligodendroglial differentiation and remyelination

45

HGF/cMet

HGF promotes OPC differentiation and remyelination via the receptor cMet

111

FGF receptors

Lack of FGF receptor 1 and 2 results in axons with reduced myelin thickness

112

Oligodendroglial migration

Semaphorin

Sema3A acts as a repulsive signal, whereas Sema3F is an attractive signal. Both signals are upregulated in active MS lesions. In chronic MS lesions expression of Sema3A correlated with lower numbers of OPCs.

23,39,113,114

Netrin

Netrin 1 is a chemorepllent for OPCs. Induction of netrin-1 expression prior to OPC recruitment impairs remyelination. In MS lesions Netrin is up-regulated in astrocytes

38

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Authors biographies

M. Stangel

Martin Stangel is Professor of Clinical Neuroimmunology and Neurochemistry and currently the acting chair of the Department of Neurology at the Hannover Medical School, Germany. He graduated from the University of Göttingen, Germany and has studied at the University of California and the Salk Institute, both in San Diego, USA. He also earned a Bachelor of Medical Sciences from the University of Melbourne, Australia. He received his training as a neurologist at the University of Würzburg and the Free University of Berlin in Germany and as a post-doc at the University of Cambridge in the UK. He has a clinical interest in neuroimmunological diseases with a special focus on Multiple Sclerosis. His laboratory research focuses on glial biology and regenerative aspects of demyelination. He is a member of the advisory board of the National MS Society and a member of the Center for Systems Neuroscience, Hannover, Germany.

T. Kuhlmann

Tanja Kuhlmann is associate professor and senior consultant at the Institute of Neuropathology, University Hospital Münster in Germany. She studied medicine at the University of Göttingen, Germany where she received her medical degree in 1998. She specialized in neuropathology and worked as medical resident and research fellow in different neuropathological departments in Germany and as a post-doc at McGill, Canada in the research groups of Dr. Jack Antel and Dr. Alan Peterson. Her key research interests are oligodendroglial biology and axonal pathology in neurological diseases, especially Multiple sclerosis.

P. Matthews

Paul Matthews, OBE, DPhil, FRCP, FMedSci, is Head of the Division of Brain Sciences at Imperial College, London and Vice President for Integrative Medicines Development in the China Research and Development Unit of GlaxoSmithKline. He also is a Fellow by Special Election of St. Edmund Hall, Oxford and holds a number of other honorary academic appointments. He received his training at Oxford, Stanford and McGill as a neurologist. His research focuses on the relationship between microglial activation, adaptive plasticity and neuroaxonal loss as a mechanism of disability progression in multiple sclerosis.