Repair strategies for multiple sclerosis

Repair strategies for multiple sclerosis: challenges, achievements and perspectives

Bruno Stankoff; Janusz Joachim Jadasze; Hans-Peter Hartunge; Patrick Ku ̈rye; Bernard Zalc and Catherine Lubetzki

Current Opinion in Neurology

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Purpose of review: Despite major progress in multiple sclerosis (MS) treatment, to date, accumulation of irreversible clinical disability is not sufficiently prevented with immunotherapies. In this context, repair strategies aimed at reducing axonal damage are becoming a very active field of preclinical and clinical research.

 

Recent findings: Improved understanding of the cellular and molecular mechanisms of myelin repair, together with the emergence of new therapeutic candidates are paving the way for novel therapeutic strategies in MS. In parallel, there is a very active development of imaging methods to assess lesions ongoing remyelination that are crucially needed to evaluate therapeutic efficacy.

 

Summary: The current development of a very dynamic and multidisciplinary research on remyelination should accelerate the development of myelin repair strategies in MS, to prevent disability progression.

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INTRODUCTION

Major progress has been achieved in the treatment of multiple sclerosis (MS) through the development of therapies targeting the immune system. Acting on different mechanisms/steps of the inflammatory component of the disease, these currently approved treatments reduce the relapse rate, with different efficiency and safety profiles [1–3]. However, there is little if any evidence on the efficacy of these drugs during the progressive phase of the disease [4–6]. Accumulation of irreversible clinical disability during this progressive phase mostly relates to irreversible neuronal/axonal damage and loss [7–10], which occurs early in disease evolution, before becoming clinically eloquent years later, after reaching clinical threshold.

 

Preventing neurodegeneration is therefore a major challenge in MS, to prevent disability progression. This can be achieved through the development of neuroprotective drugs, which currently is an active, even though to date disappointing, area of research in different neurodegenerative diseases. Alternatively, promoting remyelination might favor neuronal protection, as suggested by studies in different experimental models, on the one hand, and by analysis of demyelinated and remyelinated lesion from postmortem MS brain, on the other [11–13].

 

Recent years have been fruitful for our better understanding of cellular and molecular mechanisms of the remyelination process, using a wide variety of demyelination/remyelination models, and this has resulted in the identification of several therapeutic targets and recent development of different preclinical and, very recently, to early phases of clinical remyelinating strategies. Although these recent translational developments are highly interesting, repair strategies are facing one major obstacle which is the lack of suitable markers of neuroprotection (or damage) and remyelination (or persisting demyelination) to evaluate therapeutic efficacy. In this respect, the current emergence of promising imaging markers represents a crucial advance that will impact the evaluation of repair strategies and design of therapeutic trials.

 

In this article, we will first review some recent and promising findings on the mechanisms of myelination and endogenous myelin repair, which illuminate our understanding of the repair process and might impact the development of therapeutic strategies. Then we will make an overview of the current and very active development of new screening tools and resulting early clinical trials. For the sake of space, we will focus on strategies aimed at promoting endogenous myelin repair, without addressing the potential value of exogenous remyelination strategies through grafts. Finally, we will address the usefulness of new imaging methods to assess de/remyelination and how these new methods might influence therapeutic trials design in MS.

 

MECHANISMS OF ENDOGENOUS REPAIR

We will focus on two types of results which modify our understanding of the repair process and might impact therapeutic strategies.

 

Is the active brain able to stimulate repair?

Whether neuronal activity impacts repair capacity in the central nervous system (CNS) regained major interest recently. Indeed, the influence of electrical activity on myelination capacity was addressed long ago [14], using different neurotoxins acting on voltage dependent sodium channels and either blocking, like tetrodotoxin, or stimulating, like alpha scorpion toxin, neuronal electrical activity. By showing that silencing electrical activity reduces myelination without affecting oligodendroglial numbers, and that increased activity results in increased myelination, this study demonstrated for the first time the key role of electrical activity in the myelination process. It added new insight into the observations made decades ago on the influence of light on optic nerve myelination, showing retarded myelination in mice reared in darkness, and accelerated myelination induced by artificial eye opening in the rabbit [15,16]. After several years of ‘electrical silence’, these results were further confirmed and extended with the identification of adenosine, released from astrocytes [17], then glutamate, released along axons as molecular mediators of activity dependent myelination [18,19& ]. Using the zebrafish model, it was demonstrated that neuronal activity regulates myelin sheath production by individual oligodendrocytes in a synaptic vesicle release dependent manner [20& ]. Alternatively, the influence of neuronal activity for biased axon selection was suggested [21]. The influence of electrical activity was further addressed in vivo, using optogenetic stimulation of the murine unilateral premotor cortex, allowing us to demonstrate not only that blue light-induced neuronal activity promotes oligodendrogenesis and increases myelination, but also that premotor cortex stimulation results in behavioural improvement, quantified on swing speed of the correlate forelimb during normal gait [22&&]. This result opens new perspective on the potential functional benefit of brain activity. Finally, the relevance of this mechanism was addressed in a remyelination model in the rat, showing that blockade of electrical activity results in reduced remyelination, an effect which was attributed to neuronal activity-induced glutamate release at the neuron/oligodendrocyte precursor cells (OPCs) synapse, acting on OPCs differentiation [23].

 

Taken together, and although the models and interpretation differ (notably the debate on synaptic versus extra synaptic mechanism), these very important findings extend recent discoveries that social experiences and experimentally altered neuronal activity can influence myelin plasticity [24,25], hence have the potential to influence a functional outcome. Even if the clinical translation is still far, these data, which bring a novel insight into the mechanisms of myelination plasticity, are paving the way for new therapeutic development in which neuronal activity is a target to promote disability improvement.

 

What are the cells achieving remyelination in the adult brain?

Remyelination in the CNS has been shown several years ago to be achieved by parenchymal oligodendrocyte precursor cells (pOPCs), which persist in the adult CNS, representing up to 5% of the total cells in the brain. These cells are defined by the expression of platelet-derived growth factor receptor alpha (PDGF-Ra) and neural/glial antigen 2 proteoglycan. Fate mapping studies have demonstrated that pOPCs are able to generate new oligodendrocytes that contribute to remyelination following demyelinating insults [26]. To gain an insight into their specific characteristics, our group recently showed, on pOPCs purified from a transgenic mouse line in which PDGF-Ra expressing cells are green fluorescent protein (GFP) positive, that the transcriptomic profile of adult OPCs is more similar to that of adult mature oligodendrocytes, that is, the oligodendroglial differentiated cells, than from neonatal OPCs [27]. Interestingly however, these pOPCs are not the only remyelinating cells of the adult brain. Previous reports in experimental models in which focal demyelination occurs adjacent to the subventricular zone (SVZ) and in postmortem tissue obtained from MS patients [28], had already suggested that after a demyelinating insult, oligodendrocytes might be generated from subventricular neural progenitor cells (NPCs). This has been further explored in a recent study in which fate mapping was used to trace NPCs and pOPCs in a model of cuprizone-induced demyelination. This study provides strong evidence that demyelination induces the migration of large numbers of NPCs and that NPC-derived oligodendrocytes are the major contributors of remyelination in the corpus callosum, notably in its rostral part. Interestingly, myelin sheaths produced by these NPC-derived oligodendrocytes are thicker than those produced by pOPCs, regardless of axon calibre, hence indistinguishable from untreated controls [29& ].

 

In this search for the identification of remyelinating cells, the potential role of mature oligodendrocytes, addressed long ago with conflicting results [30,31] was recently revisited. Using inducible transgenic mouse strains in which mature oligodendrocytes were prelabeled by the expression of GFP and a spinal cord focal demyelinating model, it was shown that mature oligodendrocytes did not proliferate nor migrate into the damaged area. This genetic fate mapping study therefore firmly establishes that mature oligodendrocytes do not normally contribute to remyelination, hence are not a promising target for regenerative therapy [32& ].

 

Taken together, these findings have important implications for our understanding of the repair process and the development of myelin repair therapies. In relation with this improved knowledge of the repair process, identification of compounds with remyelinating activity using newly developed screening tools recently became a highly active area of research.

 

DEVELOPMENT OF MYELIN REPAIR STRATEGIES

The tools

To test the repair efficiency of potential candidates the most commonly used animal model is experimental autoimmune encephalomyelitis (EAE) developed in rodent. The difficulty to predict the size and localization of the demyelinating lesions are a drawback of EAE. An alternative to EAE is the use of toxic-induced demyelinations. One frequently used model is feeding the animal with cuprizone, a copper chelator. The advantage of the cuprizone model is to limit the localization of the demyelination to the corpus callosum and superior cerebellar peduncle. To better define the localization and the size of the lesions, models using local injections of myelinotoxic agents such as lysolecithin or ethidium bromide are also often used. Despite their advantages and limitations, these rodent models are not adapted to screen large series of compounds, but rather as a semi-final in-vivo test of a handful of candidate molecules that have been preselected by more adapted high-throughput or medium-throughput screening tests.

 

Chemical libraries of large pharmaceutical companies comprise between 0.5 and 3 million compounds [33]. An alternative to screening large libraries of unknown chemicals is reprofiling (repurposing or repositioning) of collections of small molecules with validated biological and pharmacological activities, in which safety and effectiveness have been demonstrated in clinical trials and many of them are Food and Drug Administration (FDA)-approved or European Medicines Agency-approved for other indication than MS.

 

High content screens are being developed based on OPC stable lines (Oli-neu or CG4) engineered to turn on a fluorescent reporter (GFP, or mCherry) when driven towards a more mature oligodendrocyte [[34]; Nait-Oumesmar, personal communication]. Other tests, more tedious, are based on immunolabeling of primary cultures of OPCs with antibodies detecting molecules expressed by mature oligodendrocytes (myelin basic protein or myelinoligodendrocyte glycoprotein being prime marker candidates). A library of 727 FDA-approved drugs has been tested on mouse pluripotent epiblast stem cell-derived OPCs to promote differentiation into mature oligodendrocytes [35].

 

To test final maturation of OPCs into myelinating oligodendrocytes some attempts are aimed at examining the wrapping potential of mature oligodendrocytes. This can be achieved on oligodendrocyte lineage cells co-cultured with neuron [36,37]. Although very powerful such co-cultures are more adequate to test candidate molecules and are not well suited for high-throughput formats [38]. More recently developed is a micropillar array device engineered with conical dimensions, which permits detection of concentric wrapping of oligodendrocyte processes similar to rings of myelin [39& ].

 

The myelination potential of lead compounds identified using these in-vitro tools needs to be confirmed in vivo. Before probing these candidates using the expensive and time-consuming rodent models, simple vertebrate experimental models have been developed, taking advantage of the transparency of their embryo. A novel screening platform for identifying potential remyelination-promoting compounds and for further filtering those that need not be investigated further has been developed using zebrafish larvae [40]. A Xenopus laevis transgenic line has also been developed allowing conditional ablation of myelinating oligodendrocytes. After completion of demyelination spontaneous remyelination occurs. Remyelination can be dramatically accelerated by adding drugs to be tested in the swimming water of the tadpoles. This X. laevis transgenic line constitutes a new and robust medium-throughput screening platform for myelin repair therapeutics in demyelinating diseases [41,42].

 

SOME OF THE CANDIDATES

Several promyelinating candidates have been proposed that could potentially be applied to MS. Here we report few examples (not an exhaustive review) and other therapeutic developments that are described in the article by Jadasz et al. (pp. 205–212) in this issue of Current Opinion in Neurology. Surprisingly enough, among the most efficacious, their mode of action is apparently very different. The most advanced candidate is antiLingo-1, an antibody directed against Lingo-1 protein [43]. The rationale for anti-Lingo-1 therapy is based on the observation that Lingo-1 protein inhibits the production of myelin, therefore antagonizing Lingo-1 should promote remyelination. Anti-Lingo-1 has performed well in animal models and is now in phase 2, as described in the article by Jadasz et al. (pp. 205–212), olesoxime (phase 1) is a cholesterol-oxime compound belonging to the family of mitochondrial pore modulators [44]; benzatropine (no ongoing clinical trial) is a selective M1 muscarinic acetylcholine receptor antagonist [45]; clemastine (phase 2) is a selective histamine H1 antagonist, binding to the histamine H1 receptor [39& ]; siponimod (phase 2 in relapsing–remitting form of MS; phase 3 in secondary progressive MS) is a sphingosine-1-phosphate ligand with an affinity to S1P1 and S1P5 receptors [42]. Using a repurposing strategy, two drugs, miconazole (an antifungic) and clobetasol (a glucocorticoid used in eczema), have been shown to be effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups [35]. So far it is not clear whether the remyelination potency of these candidate drugs is mediated by acting directly on the oligodendrocyte cell lineage or on the microglial environment or both. This remains to be established and is an important issue because it may reorient notably future therapeutic developments aimed at favouring myelin repair in MS.

 

Taken together, these multiple tools and potential therapeutic hits leading to the onset of clinical trials in MS patients have profoundly modified our views on myelin repair strategy, which now becomes an active field for translation. In this context, developing markers of myelin repair is a crucial need for designing clinical trials assessing drugs with potential remyelinating capacity.

 

ASSESSING REMYELINATING LESIONS IN VIVO BY IMAGING

As the pathophysiology of demyelinating diseases such as MS involves multiple mechanisms such as inflammation, oedema, blood brain barrier leakage, demyelination and neuroaxonal damage, a great specificity towards myelin is required in order to quantify remyelination in vivo. Although some conventional MRI metrics, derived from T1 or proton density sequences, were reported to sensitively capture MS lesion recovery [46], they are generally considered as not sufficiently specific for myelin.

 

Magnetization transfer imaging (MTI), that allows us to assess the proton pool linked to macromolecule, was proposed as a measure of myelin content because of the overwhelming contribution of myelin to the macromolecules involved in the magnetization transfer phenomenon. However magnetization transfer ratio (MTR) changes in brain lesions could be influenced by water content and inflammation, especially in acute and sub-acute lesions associated with a blood brain barrier breakdown [47]. To minimize this limitation a method based on the segmentation of focal areas of MTR decrease was developed to assess longitudinally myelin dynamics in the damaged white matter, and patients with a greater MTR recovery showed a better clinical outcome [48]. In patients with MS the ability of brain white matter lesions MTR to recover trended to a decrease with time, becoming less pronounced during adolescence and adulthood in the case of paediatric MS [49]. However, recovering lesions could also be identified in the brain of patients with progressive MS, whereas they appeared more frequently in previously lesional tissue [50]. MTR has also been shown to be sensitive to cortical lesion and a methodology to assess cortical demyelination at the individual level has been developed [51]. Overall, although being probably the most popular MRI technique to approach the assessment of myelin content, to date the specificity of MTI for myelin is still suboptimal [47,52]. Although still technically challenging, quantitative MTI could provide a better specificity towards myelin, as recently shown in the mouse cuprizone model [53].

 

The multicomponent T2 analysis of spin echo data, by focusing on the short T2 peak, has been proposed to extract the myelin water fraction (MWF), which likely corresponds to water trapped within the myelin bilayer [54]. As the reproducibility of MWF measure has been questioned, using a region of interest analysis strategy a voxel-based analysis is recommended [55]. Few longitudinal data on lesional MWF are available, indicating that some changes compatible with remyelination could be identified in a 6-month time frame [56], but with a greater variability than using MTI [57]. Several adaptations of the concept were recently developed aimed at shortening acquisition times, covering the whole brain and increasing sensitivity, with some promising results in MS [58–61], but their preserved specificity towards myelin still await to be proven.

 

A decrease in radial diffusivity over time on diffusion weighted images compatible with remyelination has been described in MS lesions [62]. However, the measure of radial diffusivity is not yet specific for myelin content as it can be significantly influenced by axonal pathology, inflammation, oedema and crossing fibers at the microstructural level. Recent optimizations of diffusion weighted imaging could provide more specific tools for the myelin compartment. The application of diffusion basis spectral imaging, by modelling diffusion weighted signal as a linear combination of multiple tensors, discriminating between the anisotropic fibre component and a full range of isotropic components, either restricted (and related to cell infiltration) or unrestricted (and related to oedema), was shown to allow differentiation of inflammation and oedema, from axonal and myelin damage in the cuprizone mice model [63& ]. Diffusion kurtosis imaging, an extension of diffusion tensor imaging that provides metrics of diffusional non-Gaussianity, could also generate quantitative measures sensitive to myelin changes in the cuprizone animal models [64,65] with preliminary promising results obtained in MS [66].

 

A specific approach for myelin imaging could be achieved using positron emission tomography (PET). The first myelin radiotracer identified was the stilbene derivative, 1,4-bis(p-aminostyryl)- 2-methoxy benzene, also named BMB [67], that has recently been shown to interact directly with myelin basic protein [68]. A similar affinity for CNS myelin was reported for several other stilbene Congo red derivatives, allowing to monitor myelin dynamics in dysmyelinating and demyelinating models [69–71]. On the basis of a common target expressed in amyloid plaques and myelin, the thioflavin T derivative [11C]-2-(40 -methylaminophenyl)-6-hydroxybenzothiazole ([11C]-PIB) was also shown to stain myelin providing an opportunity to quantify myelin content changes in MS [72]. A pilot [11C]-PIB PET imaging study was therefore performed in MS and an noninvasive quantification method for the dynamic acquisitions was developed and applied [73]. The individual remyelination potential of patients with MS showed a great heterogeneity, and was highly correlated with neurological disability [74&&]. Therefore, monitoring myelin dynamics by PET could be a added value to stratify patients depending on their repair ability, and to specifically quantify myelin repair in early therapeutic trials of promyelinating compounds. There is a need for fluorinated compounds that would allow a more widespread dissemination to this technique, and emerging fluorinated amyloid tracers might be of interest as reported in a preliminary study [75].

 

Sample size estimations have been made to quantify therapeutic effects on brain lesions recovery using either conventional or MTI based metrics, emphasizing that groups of 10–70 patients could allow the detection of therapeutic effects with an acceptable power [46,48,76].

 

CONCLUSION

Favouring remyelination in MS is becoming the next frontier. In this context, our growing understanding of the repair process, combined with identification of promising therapeutic candidates and development of novel imaging tools will result in the onset of a new era of therapeutic trials, aimed at preventing demyelination-related axonal pathology leading to accumulation of disability. Even if optimal timing of these therapeutic interventions is still debatable, the fact that axonal pathology occurs early in disease evolution has to be taken into account. These repair strategies might therefore be better adapted to early MS and combined with anti-inflammatory treatment. They will need innovative trial design with a key role for imaging markers. Of note, two extensive reviews on myelin repair have recently been published [77,78].

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ACKNOWLEDGEMENTS

None.

FINANCIAL SUPPORT AND SPONSORSHIP

Research on the subject of myelin repair in the French laboratory was supported by INSERM, the French MS research foundation ARSEP, the program ‘Investissements d’Avenir’ ANR-10-IAIHU-06, a grant from ‘Investissement d’Avenir – ANR-11-INBS-0011 – NeurATRIS’, and ANR grants STEMIMUS to C.L. and OLGA to B.Z. Research on the subject of myelin repair in the German laboratory is supported by the German Research Council (DFG: SPP1757, KU1934/5-1), the Christiane and Claudia Hempel Foundation for clinical stem cell research and the French societies ARSEP and AFM. The MS Center at the Department of Neurology is supported in part by the Walter and Ilse Rose Foundation and the James and Elisabeth Cloppenburg, Peek and Cloppenburg Du¨sseldorf Foundation.

CONFLICTS OF INTEREST

C.L. has received honoraria from Roche, Biogen, Genzyme, Novartis and participated to Vertex advisory board. B.Z. has received research grants from Novartis and EMD Serono. B.S. received honoraria from Biogen, Teva, Novartis, Genzyme, and research support from Genzyme and Merck-Serono. J.J. reports no conflict of interest. H.-P.H. received compensation for consultancy and speaking from Biogen, GeNeuro, Genzyme, MerckSerono, Novartis, Octapharma, Opexa, Receptos, Roche, Teva, and Sanofis. P.K. received compensation for consultancy and/or speaking from Novartis, Baxter, GeNeuro and Biogen – all with approval by the Rector of Heinrich-Heine-University.

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