Remyelination Pharmacotherapy Investigations Highlight Diverse Mechanisms Underlying Multiple Sclerosis Progression

Abstract

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system characterized by a complex lesion microenvironment. Although much progress has been made in developing immunomodulatory treatments to reduce myelin damage and delay the progression of MS, there is a paucity in treatment options that address the multiple pathophysiological aspects of the disease. Currently available immune-centered therapies are able to reduce the immune-mediated damage exhibited in MS patients, however, they cannot rescue the eventual failure of remyelination or permanent neuronal damage that occurs as MS progresses. Recent advances have provided a better understanding of remyelination processes, specifically oligodendrocyte lineage cell progression following demyelination. Further there have been new findings highlighting various components of the lesion microenvironment that contribute to myelin repair and restored axonal health. In this review we discuss the complexities of myelin repair following immune-mediated damage in the CNS, the contribution of animal models of MS in providing insight on OL progression and myelin repair, and current and potential remyelination-centered therapeutic targets. As remyelination therapies continue to progress into clinical trials, we consider a dual approach targeting the inflammatory microenvironment and intrinsic remyelination mechanisms to be optimal in aiding MS patients.

Multiple Sclerosis Pathogenesis

Multiple sclerosis (MS) is a debilitating chronic inflammatory disease of the central nervous system (CNS), in which oligodendrocytes (OL), the myelinating glia of the CNS, are attacked by inflammatory cells, resulting in myelin degradation, OL death, axonal dysfunction, and neurodegeneration.¹ MS affects nearly 1 million people in the United States alone, and 2.3 million worldwide.² It is primarily diagnosed in adults between the ages of 20–40, and affects more females than males.³ Furthermore, a combination of genetic and nongenetic factors such as environment, metabolism, and viral infections are believed to significantly contribute to disease risk.⁴ Despite disease heterogeneity and an unknown combination of factors influencing etiology, the most prevalent presentation of MS (relapsing-remitting MS) involves days to weeks of enhanced inflammation in regionalized areas of the CNS, causing white matter lesions characterized by myelin loss.¹ These white matter lesions can then lead to muscular, somatosensory, or neurocognitive disabilities depending on their location in the CNS.⁴ Following demyelination, inflammation may be resolved in some lesions, contributing to remyelination and partial functional recovery in patients. Relapsing-remitting MS (RRMS) can last for decades with repeated cycles of enhanced inflammatory periods followed by resolution and partial recovery. However, most patients later transition to the secondary, progressive form of MS (SPMS) which is marked by prolonged CNS inflammation, glial cell death, and subsequent and widespread neurodegeneration. Although in the relapsing-remitting phase of MS, remyelination is possible—the typical, secondary phase of MS is marked by a slow expansion of lesions, characterized by chronic macrophage activity at the lesion edge, an inability to remyelinate, and progressive neuronal degradation.^5,6 The underlying cause of this transition from a disease state in which disability recovery is possible, to a state of unrelenting health decline, remains poorly understood. Therefore, the development of therapeutics targeting both CNS inflammation and remyelination would be beneficial in promoting CNS repair and potentially preventing disease progression.

Animal Models of MS

Although no singular animal model completely encompasses the pathophysiological hallmarks of MS, they each provide crucial information on varying, sometimes overlapping, components of MS that have and continue to inform therapeutic development. Each model is a tractable system that can be used to investigate cellular and molecular factors either contributing to demyelination, inflammation, oligodendrocyte death, remyelination and/or axonal damage for the testing of potential therapeutics for immunomodulation or remyelination (Table 1). Due to model heterogeneity it is imperative that pathophysiological aspects of MS and hypotheses are matched to the appropriate animal model. Below we summarize key animal models used for preclinical studies in MS research.

Table 1. Animal Models Used in Multiple Sclerosis Investigations.

animal models of MS
model	overview	advantages	limitations
Lysolecithin	focal lipotoxin-induced demyelination via breakdown of lipid-rich myelin membranes	highly tractable	does not accurately capture MS disease state (toxin-based demyelination, rather than immune-mediated demyelination)
		allows for clear analysis of remyelination stages
		useful for pro-remyelination and lineage cell progression investigations	no behavioral phenotype that can be reversed by therapeutics
Ethidium bromide	focal DNA intercalating agent-induced cell death of oligodendrocytes, OPCs and astrocytes	tractable	heavy Schwann cell-mediated remyelination
		unique opportunity to investigate astrocyte contributions to remyelination	does not accurately captue MS disease state (toxin-based demyelination, rather than immune-mediated demyelination)
Cuprizone	copper chelating agent is fed to rodents over several weeks, resulting in progressive demyelination in the corpus callosum. Returning to normal diet facilitates remyelination.	can be used to model chronic demyelination	etiology not fully understood
		useful for pro-remyelination investigations	does not accurately capture MS disease state (toxin-based demyelination, rather than immune-mediated demyelination)
			difficult to distinguish remyelinated myelin sheaths from undamaged myelin
Experimental autoimmune encephalomyelitis (EAE)	Immune-mediated demyelination: either active immunization with myelin-derived proteins or passive transfer of activated CD4+ T lymphocytes	resembles many pathological aspects of MS	differences between MS and EAE has hampered the development of many effective drugs
		has led to the development of FDA-approved therapies	manifests mainly in the spinal cord, not the brain
		useful for immunomodulation investigations	difficult to track and characterize true remyelination

Toxin-Induced Demyelination Models

A frequently used model for MS research involves experimentally induced focal demyelination through injections of toxins, notably lysophosphatidylcholine (LPC) or lysolecithin into CNS white matter such as in the spinal cord or corpus callosum. LPC is a lipotoxin that induces the breakdown of lipid-rich myelin membranes, and when injected focally into the white matter, results in the formation of a well-characterized lesion that repairs itself over time through spontaneous remyelination.⁷ Typically after LPC mediated demyelination, oligodendrocyte progenitor cell (OPC) migration into the focal lesion is apparent by 3–5 days post lesion (dpl). This is then followed by OPC differentiation into oligodendrocytes by 10 dpl, and prominent remyelination by 20–30 dpl. The inflammatory environment is also known to shift within the focal lesion from a pro-inflammatory state to an anti-inflammatory, pro-regenerative state over the course of remyelination.^8,9 Therefore, the highly tractable nature of this model allows for the analysis of oligodendrocyte lineage cell progression, as well as immunomodulation within the focal lesion during the myelin repair process. Moreover, regenerated myelin is highly distinguishable from undamaged myelin in LPC-induced lesions owing to the fact that regenerated myelin is visibly thinner than normal myelin. This particular feature is critical for the analysis of molecular signals or pharmacotherapeutic efficacy underlying true remyelination. Although this is an informative primary demyelination/remyelination model, some of the caveats include a lack of a clear functional deficit in animals that could be potentially reversed by therapeutics,¹⁰ a lack of immune-mediated induction of demyelination,¹¹ and the involvement of peripheral Schwann cells in remyelination.¹²

Another experimentally induced focal demyelination model involves the injection of ethidium bromide (EB) in white matter tracts of the CNS. EB is a DNA intercalating agent that causes the death of oligodendrocytes, OPCs, and astrocytes, forming a well-characterized lesion that also repairs over time.^1,10,13 EB focal lesion injections are primarily conducted in rats, and have a slower remyelination timeline than LPC-induced lesions.¹⁰ Importantly, the death of astrocytes in this model causes widespread Schwann cell-mediated remyelination.^10,13 While the model uniquely allows for investigations into astrocyte-oligodendrocyte remyelination mechanisms, the heavy Schwann cell involvement might complicate studies pertaining to oligodendrocyte-driven remyelination.

Contrasting focal injection models, the cuprizone (CPZ) model is a widely used, yet not fully understood model of systemic toxin-induced demyelination. As a copper (Cu) chelator, the introduction of CPZ in the rodent diet results in low serum Cu levels and corpus callosum demyelination in mice over time.¹⁴ Characterization of the CPZ-mouse model features acute and chronic demyelination, prominent steady-state levels of astrogliosis, microgliosis, megamitochondria formation, and oligodendrocyte lineage cell death.¹⁵ The CPZ model is predominantly used on the mouse C57BL/6 background, as this strain produces reproducible de- and remyelination as well as gliosis.^16,17 The standard protocol involves feeding mice with 0.2% CPZ-supplemented diet for 4–6 weeks, which leads to corpus callosum demyelination with minimal clinical toxicity.¹⁵ Following this period, the replacement of oligodendrocytes and myelin in the corpus callosum can be achieved in 2–4 weeks with a normal diet. Moreover, chronic demyelination can also be reached with prolonged CPZ diet treatment for up to 12 weeks. The etiology of CPZ-induced pathology is not fully understood, but the molecular makeup of OLs are thought to make them particularly susceptible to CPZ toxicity through a combination of mitochondrial dysfunction, ATP shortage, increased oxidative stress, and ER stress.¹⁷ Importantly, the CPZ model does not involve a direct immune-mediated attack against myelin proteins but more so encompasses systemic intoxication. Furthermore, in the CPZ model the blood-brain barrier (BBB) appears to remain intact, with no obvious peripheral immune cell involvement, such as T or B lymphocytes. Therefore, the CPZ model enables isolated investigations into possible underlying mechanisms of OL death and the inherent ability to recover and remyelinate areas of damage without immune cell infiltration. However, a caveat to this model is that myelin membranes are generally thinner in the corpus callosum due to the relatively smaller diameter of axons compared to that in the spinal cord, so distinguishing regenerated vs undamaged myelin may be challenging. Moreover, the corpus callosum is only partially myelinated in young adult mice, with less than 20% of axons myelinated, and continue to myelinate into late adulthood.¹⁸ As such, the ability to distinguish true remyelination from de novo myelination in the corpus callosum after CPZ treatment would need to be considered.

Autoimmune Driven Demyelination Model

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease model of the CNS, which resembles many pathological aspects of MS, including inflammation, demyelination, axonal loss, and gliosis.¹⁹ This autoimmune model is primarily induced in various strains of rodents and nonhuman primates, and can be used to capture different aspects of MS pathology, depending on the immunization method and antigen utilized.^1,19 Many of the current disease modifying treatments (DMTs) prescribed to MS patients or in clinical trials have been developed and validated in EAE models. For example, there are convincing correlations between EAE and MS in the beneficial role of IFN-β, glatiramer acetate, and anti-VLA-4 across various EAE models.²⁰⁻²²

Generally, EAE is induced following administration of CNS extract or myelin peptides in complete Freund’s adjuvant with supplementation of pertussis toxin (active immunization) or via the transfer of pathogenic, myelin-specific CD4+ T lymphocytes generated in a donor animal (passive immunization). Disease onset typically occurs after 9–12 days, in which immune-mediated CNS lesions appear, limb paralysis ensues, and depending on the experimental condition, symptoms worsen or recover. The immunological aspects of EAE models have led to a better understanding of the immune makeup and function in MS, and continue to be an important preclinical model. As a further expansion, the more recent marmoset model of EAE more accurately reflects the human immune response (utilizing incomplete Freund’s adjuvant in the immunization).²³ A limitation to EAE is that the typical presentation in rodents manifests mainly in the spinal cord, and not in the brain as in MS patients. However, the recent development of cortical or callosal EAE with a higher dose of pertussis toxin administration may be useful for studying inflammatory demyelination in the brain.²⁴ Additionally, pertussis toxin injections are needed to facilitate BBB breakdown, which is a prominent feature in MS. A further limitation is that EAE induces widespread stochastic lesions, with varying degrees of inflammation and demyelination throughout the CNS. Therefore, despite its major strength as a model for studying immune-mediated demyelination, the ability to reliably track or characterize the extent of CNS remyelination and repair in EAE tissues remains a challenge. G-ratios are typically a standard determinant of remyelination, considering the diameter of the myelin sheath in comparison with the diameter of the axon. The g-ratio therein allows for an assessment of thinner myelin sheaths—indicative of remyelination. However, the heterogeneity of axons in the spinal cord and the difficulty in tracking lesion age and stage²⁵ make it difficult to utilize g-ratios as indicators of true remyelination in EAE models. Further, the clinical scoring system in EAE is impacted by multiple pathological features including demyelination, inflammation, and axonal dysfunction. Therefore, the efficacy of remyelination therapies to selectively enhance remyelination cannot be inferred from reductions in clinical score.²⁶ This makes it particularly challenging to utilize the EAE model for evaluating potential drugs that promote remyelination, but implementing genetic approaches to enhance remyelination in EAE might allow for selective investigations into pharmacological remyelination targets.²⁶

Additionally, a combination cuprizone+EAE model has been developed that exhibits multiple focal demyelinated and inflammatory forebrain lesions.²⁷ The model involves a three week course of cuprizone diet, followed by active EAE induction. The model has great potential for investigations into immune cell recruitment and forebrain lesion development, correcting the primary spinal cord limitation of normal EAE. However, the forebrain lesions present in the CPZ+EAE model spontaneously resolve while the spinal cord lesions do not,²⁷ potentially complicating analyses and interpretations following intervention studies. As the model continues to be characterized it could serve to test developing pharmacotherapies in MS.

Current Therapeutic Approaches: Targeting Neuroinflammation

As an immune-mediated disease, the onset of MS pathology is characterized by the infiltration of peripheral immune cells into the CNS parenchyma. Although the exact molecular mechanisms underlying the complex activation of the immune system against CNS myelin are not fully understood, various immune components in MS pathology are well-known.⁵ Notably, MS pathology consists of a compromised BBB, lymphocyte-mediated infiltration and myelin degradation, and peripheral macrophage/microglia-mediated debris clearance.¹ Increasing evidence suggests that CNS glia and neurons, along with peripheral immune cells, mediate cytokine and chemokine-induced pro-inflammatory profiles responsible for myelin degradation and axonal injury, and participate in anti-inflammatory immune resolution during the remyelination process.²⁸ Indeed, as MS progresses, macrophage and microglia activity is closely associated with OL death and myelin degradation, gliosis, and axonal damage. To counteract this effect, current disease interventions seek to modulate various immune system components implicated in MS pathogenesis.

Since 1993, when interferon β-1b was approved for MS treatment, most pharmacological therapeutic research has focused on immunomodulation to combat and reduce neuroinflammatory outbreaks that induce demyelination.²⁹ With current FDA-approved treatments, the duration of relapses is shortened and the progression to the secondary phase of MS is delayed.⁶ These disease modifying drugs include the aforementioned anti-inflammatory class of β-interferon drugs, immunomodulators such as glatiramer acetate and fingolimod, and therapeutics that prevent immune cell infiltration into the CNS parenchyma such as natalizumab (anti-VLA-4).²¹ Although available FDA-approved MS therapies have been crucial to MS patient care, they are predominantly immuno-modulatory and lack the ability to promote CNS repair.²⁰ Moreover, immune therapies generally fail to prevent progression of the disease, as these therapies are unable to stop or reverse the failure of remyelination.³⁰ Therein, a large bulk of current MS research is centered on understanding the mechanisms underlying demyelination and remyelination in order to address remyelination failure.

New Therapeutic Approaches: Targeting Remyelination Efficacy

Following demyelination in MS, the oligodendrocyte lineage cell population in the CNS, driven by oligodendrocyte progenitor cells (OPCs), is capable of endogenous remyelination. OPCs migrate to the site of damage, expand their population, differentiate into OLs, and subsequently mature into myelinating OLs, capable of remyelinating previously lost axonal internodes.³¹ The successful progression of lineage cells through these many steps and subsequent remyelination is closely regulated by intrinsic OPC mechanisms as well as microenvironmental inflammatory conditions (Figure 1).⁹ However, spontaneous remyelination is incomplete or fails in secondary, progressive MS despite immunomodulation.³² Therefore, an intrinsic mechanism or other unknown aspect of the lesion environment contributes significantly to remyelination capabilities. Several studies indicate that OPC recruitment is not a major issue, as many OPCs remain present in chronically demyelinated lesions.³³⁻³⁵ This suggests that the inability for OPCs to differentiate into oligodendrocytes in lesions may be the key to remyelination failure. Additionally, immune system dysfunction and white matter modifications that occur with normal aging have also been suggested to contribute to disease progression.^36,37 Therein, understanding how the lesion microenvironment regulates remyelination dynamically over time as well as determining the necessary components required for endogenous oligodendrocyte lineage cell progression are both necessary avenues to advance MS therapeutic potential. Below we describe several research efforts aimed at understanding and therapeutically aiding intrinsic remyelination capabilities that have led to a number of promising pharmacotherapeutic targets in MS to prevent disease progression (Figure 2).

Figure 1.

Schematic representation of the lesion microenvironment characterized in successful and failed remyelination. (A) In a typically demyelinated or early, acute lesion, T-cells, neutrophils, and monocytes release cytokines that damage myelin and axons. This inflammation recruits OPCs to the lesioned environment. (B) Downregulation of proinflammatory cytokines plus adequate phagocytosis of myelin debris provides a permissive environment for endogenous OPC differentiation into early, nonmyelinating OLs. (C) Upon maturity, new myelinating OLs will wrap denuded axonal internodes with a thin myelin sheath. (D) In the case of remyelination failure, while OPCs are able to undergo proliferation and expansion, they generally fail to differentiate into early, mature OLs. Additionally, myelin debris is also not cleared efficiently by macrophages and microglia. (E) Of the differentiated OLs generated, many do not mature to myelinate axonal internodes and therefore prominent areas of demyelination are prevalent. Created with Biorender.

Figure 2.

Phases of OL lineage progression at which remyelination therapies target. Following demyelination, there are no apparent problems with OPC recruitment or proliferation. However, there is dysfunction with regards to OPC differentiation. Ongoing investigations are centered on drug targets for remyelination aimed at enhancing OPC differentiation, OL maturation, or OL viability. Created with Biorender.

Current Drugs

LINGO-1 Antagonists

The earliest remyelination therapy was first examined with LINGO-1 (leucine-rich repeat and Ig domain-containing, nogo receptor-interacting protein 1) and is actively being investigated in clinical trials (see below). LINGO-1 negatively regulates OPC maturation and myelination, and is expressed normally in OPCs and neurons, and in activated microglia and astrocytes in MS.^38,39 Mechanistic investigations suggest that LINGO-1 activates the small GTPase, RhoA, in OPCs and this may mediate inhibition of OPC differentiation.⁴⁰ Preclinical studies demonstrated that loss of LINGO-1 enhanced myelin sheath formation and myelination.^41,42 These studies led to the development of a functional inhibitory monoclonal antibody to LINGO-1, opicinumab (BIIB033), which was evaluated in phase II clinical trials (NCT02657915, NCT01864148, NCT01721161, NCT01721161). Unfortunately, the findings did not meet significance, potentially due to the relatively short treatment period, and the study (RENEWed NCT02657915) has been extended.

Muscarinic Antagonists

Intracellular cholinergic signaling pathways in myelinating glia present a thoroughly investigated and promising avenue for mediating endogenous remyelination.⁴³⁻⁴⁶ Interestingly, muscarinic acetylcholine receptor (mAChR) expression is increased in proliferating OPCs, and downregulated in mature OLs.⁴⁵ Further, in vitro studies demonstrate that muscarinic antagonist treatment of primary OPC cultures switched OPC phenotype from a proliferative state to a state of differentiation characterized by the formation of mature, myelinating oligodendrocytes.^{26,45,47−50} Moreover, removing M1 mAChR specifically in oligodendrocytes has been shown to improve global remyelination in EAE models.²⁶ These studies opened a new avenue in remyelination research toward the role of muscarinic antagonists, although the mechanism of this action continues to remain elusive. Through small compound library screens for OPC differentiation, several muscarinic antagonists, including clemastine, benztropine, and quetiapine, have been found to promote oligodendrocyte differentiation and remyelination in animal models of MS.

Clemastine is a widely available first-generation antihistamine that exhibits M1 antimuscarinic properties.²⁶ Initially identified in a high throughput drug screen, clemastine has been validated in vitro and in vivo in several rodent models of demyelination to promote OPC differentiation, thus implicating promising potential for remyelination therapies.^47,48 In the lysolecithin mouse model, clemastine treatment prior to demyelination injections resulted in more differentiated oligodendrocytes when compared to littermate controls.⁴⁸ Additionally, in cuprizone⁵⁰ and EAE models,²⁶ clemastine intervention promoted myelin repair in demyelinated regions. Further, the enhancement of OL differentiation and myelination in the prefrontal cortex rescued behavioral changes in socially isolated mice.⁵¹ The robust therapeutic effects of clemastine in vitro and in vivo along with its favorable safety profile, rendered it an attractive target to advance onto the phase II clinical trial, ReBUILD (NCT02040298), a 150 day, double-blinded, randomized, controlled, crossover study. Owing to the commonality of MS patients diagnosed with optic neuropathies,^52,53 this study assessed the visual pathway using visual evoked potentials (VEPs), a sensitive electrophysiology method to predict remyelination. Clemastine treatment reduced VEP P100 latency; however, the MRI imaging methods utilized in the study were unable to detect remyelination differences. Nevertheless, the results suggest a potential for OPC populations to be medically induced to remyelinate in MS patients, and therefore the ongoing phase II clinical trial, ReCOVER (NCT02521311), is reconsidering the parameters of the experimental design. The ReBUILD trial represents a stepping stone in the search for remyelination therapies, as one of the first clinical trials to shift away from immunomodulatory treatments and examine OL lineage progression, along with LINGO-1,⁴¹ for MS therapeutic development.

Similar to the discovery of clemastine through high throughput screens, other FDA-approved muscarinic antagonists have been examined in the context of remyelination. Benztropine, a muscarinic antagonist, is an FDA-approved adjunct treatment for Parkinson’s disease. Through muscarinic antagonism, coupled with inhibition of dopamine transporters⁵⁴ and histamine receptors⁵⁵ benztropine balances the relative tone of cholinergic and dopaminergic signaling.⁵⁶ High throughput screens have revealed that benztropine acts through M1 antagonism to promote OPC differentiation. Treatment in both the cuprizone and EAE models of demyelination resulted in decreased severity of the acute disease and relapse phases, and functional recovery was better than or comparable to immunosuppressants such as FTY720 or Interferon-β.⁴⁷Quetiapine, another M1 antagonist, is currently used as an atypical antipsychotic drug.^47,48 It has been shown to stimulate OPC differentiation through the ERK1/2 pathway.⁵⁷⁻⁵⁹ Further, quetiapine treatment was shown to reduce demyelination of the corpus callosum in the cuprizone model, seen with histological analysis⁵⁹⁻⁶¹ and MRI,⁶¹ along with a reduction of activated microglia and astrocytes in demyelinated sites,⁶⁰ and improved cognition when compared to nontreated, cuprizone-induced mice.⁵⁹ Preclinical studies deem quetiapine as an attractive remyelination target, and it is currently being examined for safety and tolerability in an open level phase I/II dose-determining study (NCT02087631) in patients with RRMS and SPMS (clinicaltrials.gov).

Though preclinical studies of these muscarinic antagonists provide a clearer understanding of the role of mAChRs in OL lineage cell progression, further studies are necessary to validate the off-target effects and efficacy of these drugs in humans, as the cellular environment in humans may differ from animals to promote remyelination, especially in progressive MS. Overall, research on muscarinic antagonists have strongly expanded and helped shift the field toward an OL lineage progression-focused approach for future MS therapies.

Retinoid X Receptor (RXR) Agonists

Early transcriptomic profiling to interrogate intrinsic mechanisms underlying remyelination resulted in a number of potential therapeutic avenues. Utilizing laser capture microdissection, analysis of differentially expressed genes during remyelination in a focal toxin model led to the identification of retinoid x receptor gamma (RXRγ), a nuclear receptor which plays important roles in immune regulation and oligodendrocyte lineage progression.⁶² Following injury, RXRγ was observed to translocate from the cytosol of immature OPCs to the nucleus of mature oligodendrocytes, suggesting that RXRs influence OPC differentiation.⁶² Moreover, RXRγ knockdown or antagonism in OPC cultures impairs OPC differentiation, but has no effect on OPC survival/viability, further indicating its selective importance for OPC differentiation and maturation.^63,64 The role of RXR in OPC differentiation was supported in animal models of demyelination, such that lysolecithin-induced lesions in RXRγ knockout mice resulted in a reduced number of oligodendrocytes in the lesion area, and that treatment with a pan-RXR agonist, 9-cis-retinoic acid (9cRA), in rodents after experimental demyelination resulted in increased remyelination.⁶² Moreover, RXRγ was shown to form heterodimeric complexes with other nuclear receptors, such as vitamin D receptor (VDR), and pharmacological inhibition of VDR also impaired the pro-differentiating effects of RXRs.⁶⁵ These preclinical studies provide compelling evidence of RXR’s role in promoting remyelination, and have led to the initiation of MS remyelination clinical trials using RXR agonists, such as bexarotene, a pan-RXR agonist currently approved for cutaneous T cell lymphoma⁶⁶ and in clinical trial for Alzheimer’s disease.⁶⁷ Additionally, IRX4204, a second-generation RXR agonist that has been shown to attenuate disease severity in EAE,⁶⁸ has undergone clinical testing for human safety for cancer treatment and can potentially be transitioned for safety and efficacy in MS patients.

Experimental Drugs

Additional drug screens have led to the identification of several molecular pathways capable of facilitating OPC differentiation. Clobetasol, a synthetic glucocorticoid (GC), has recently been identified in such a screen as a potent stimulator of OPC differentiation.³⁰ GCs have been suggested to negatively regulate OPC proliferation⁶⁹ and are known to exhibit robust immunomodulatory activity, mediated through glucocorticoid receptor (GR) signaling. Indeed, impaired glucocorticoid signaling is associated with several autoimmune diseases and MS. However, clobetasol has been found to affect OL lineage cell progression in the absence of immune cell activity. In lysolecithin focal lesions, clobetasol treatment has been shown to increase oligodendrocyte differentiation and remyelination.³⁰ Further, in EAE models, clobetasol treatment was found to reduce disease severity, the extent of demyelination in the spinal cord, and clinical disability scores. Importantly, clobetasol was efficacious in a MOG EAE model which has controlled immune activity, thus suggesting a mechanistic role specific for OLs outside of immunomodulation. Whether acting through canonical GR signaling or as an agonist of the Smoothened (Smo) transmembrane receptor, clobetasol has been linked to multiple nuclear hormone receptors (including RXRγ).^70,71 Combining this preclinical evidence with the well-supported implication that GCs act as inhibitors to OPC proliferation⁶⁹ and that Hedgehog signaling (Hh) has been shown to reactivate in OPCs during remyelination,⁷¹ suggests a specific role for clobetasol in the temporal regulation of OPC differentiation.⁶⁹

Another drug that was shown to stimulate OPC differentiation is miconazole, a synthetic derivative of imidazole. Original pathway analysis indicated that miconazole’s OL-specific mechanism of action worked through MEK-dependent phosphorylation of ERK1/2,³⁰ important kinase components of pro-growth pathways implicated in myelination.⁷² Additionally, miconazole targets and inhibits cytochrome p450, family 51 (CYP51). This inhibition has also been linked to increased oligodendrocyte formation and increased myelination.⁷³ Miconazole treatment was shown to enhance OPC differentiation and remyelination in lysolecithin focal lesions.³⁰ In the MOG_35–55 EAE model, miconazole treatment was further found to reduce disease severity and improve motor functionality.³⁰ However, in the PLP EAE model (characterized by active inflammation), miconazole treatment did not significantly affect remyelination, indicating that immunomodulation is necessary for miconazole to enhance OPC differentiation. These findings highlight the importance of immunomodulation in facilitating effective OL lineage cell progression and remyelination.

Future Drug Targets

Potential Drugs Targeting Oligodendrocyte Bioenergetics and Myelination

Global metabolomics studies have recently provided indications of important metabolites utilized in OL lineage cell progression, OL viability, and subsequent remyelination. Taurine, an endogenous amino acid, exists in robust concentrations in the brain and is a general cytoprotective agent linked to osmoregulation and intracellular calcium modulation.⁷⁴ In a global metabolomics study of OL lineage cell progression, taurine was increased 20-fold in differentiated OLs and mature OLs.⁷⁵ The late-stage gene expression enrichment implicates a role for taurine in the maturation of premyelinating OLs. Further, supplementation of taurine to OPC cultures in OPC differentiation drug-containing media significantly increased levels of OPC differentiation.⁷⁵ The underlying mechanism is thought to be largely due to exogenous taurine reversing metabolic pathway flux, thus increasing pools of metabolites (notably serine). As the building block of glycosphingolipid (GSH), increases in serine pools would have profound effects on myelination,⁷⁵ as GSH, along with cholesterol, make up over half of the proteins that constitute myelin. Directly modulating the GSH biosynthesis pathway could explain taurine-induced increases in MBP+ cells when coupled with known OPC differentiation drugs (miconazole and benztropine). The efficacy of taurine treatment remains to be tested in various animal models of MS, but noting that it passes the BBB and exists at millimolar concentrations in the brain makes it a promising treatment unlikely to have adverse side effects in potential clinical trials.

Creatine is an endogenous nitrogenous organic acid, generally associated with ATP modulation and mitochondrial function.⁷⁶ Creatine is synthesized in a two-step process from arginine and glycine, requiring glycine amidinotransferase (GATM) to form the intermediary, guanidinoacetate, and guanidinoacetate N-methyltransferase (GAMT) to directly convert the intermediary to creatine. Although not uniquely expressed or utilized by oligodendrocyte lineage cells, several studies have suggested an OL-specific metabolic, survival role for creatine.^62,75,77 Creatine expression is higher in myelinating and premyelinating OLs in the healthy adult brain, compared to all other cell types.⁷⁷ Interestingly, OLs express high levels of both required enzymes for creatine synthesis (GATM and GAMT), but then upregulate the expression of GAMT in the lysolecithin model of MS.⁶² Specifically, the upregulation of GAMT expression occurs in the later stages of recovery, both at 14 and 28dpl, which are associated with premyelinating and myelinating OLs, respectively. Moreover, in vitro and in vivo studies indicate that creatine directly affects OL viability without affecting other components of remyelination, such as OPC recruitment, proliferation, and differentiation.⁷⁸ Creatine treatment is effective in promoting OL viability when administered at early time points in lesion models of MS, as well as in promoting viability of newly differentiated OLs during recovery. Accordingly, GAMT^–/– mice experience lowered levels of OL viability and remyelination compared to wildtype mice, which was then shown to be rescued with creatine treatment.⁷⁸ It remains unclear exactly how creatine regulates OL survival, but it may directly influence the amount of ATP produced, thereby promoting mitochondrial function. Further studies are needed to determine the extent to which creatine can influence remyelination and examine possible routes of administration—given that peripheral creatine passes the BBB with limited capability.⁷⁶ However, since creatine is safe and tolerable in other clinical trials^79,80 it serves as a promising candidate therapeutic for remyelination studies.

Cholesterol and Lipid Homeostasis

Cholesterol is not only a constitutive lipid component of plasma membranes but is also a major component of myelin, important in the formation and maintenance of myelin sheaths. Cholesterol-rich myelin makes the CNS a major hub of de novo cholesterol synthesis, transport, and efflux, as no peripheral cholesterol enters through the BBB.⁸¹ Transcription factors such as peroxisome proliferator-activated receptor (PPAR), and liver X receptor (LXR), participate in lipid homeostasis and along with major cholesterol transporters such as ApoE, Abca1, and Abcg1, have been implicated in MS.⁸² As all CNS cells are capable of synthesizing cholesterol and participating in efflux, they too suffer from cholesterol dysregulation. The maintenance of cholesterol homeostasis is crucial to physiological CNS function, and has also been shown to be important to the regenerative capability of the CNS following demyelinating injury and other neurodegenerative diseases.⁸¹ Moreover, cholesterol itself has been shown to be essential for oligodendrocyte maturation. Cholesterol is a crucial factor in myelin gene expression, axon ensheathment, and myelin sheath growth.⁸³ The connection between OL maturation processes and cholesterol homeostasis indicates that cholesterol homeostasis is an important aspect of OL lineage cell progression. This is further supported by the finding that a deficiency of cholesterol reduces the levels of myelin proteins in the brain,⁶⁷ while exogenous, dietary supplementation of cholesterol—via a compromised BBB—promotes repair after demyelinating episodes.⁸⁴ Moreover, several of the currently investigated OPC differentiation drugs including clemastine, benztropine, clobetasol, and miconazole have been linked to the cholesterol biosynthesis pathway,⁷¹ providing evidence that it is a potential target for the development of drugs that promote remyelination.

PPAR Agonists Promote OL Differentiation and Immunomodulation

Peroxisome proliferator-activated receptors (PPARs) are a group of ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. PPARs (PPARα, −β/δ,−γ) function through heterodimerization with retinoid X receptors (RXRs), are found in all cells of the adult mouse and human brain, and are variably expressed in immune cells.⁸² These receptors have major regulatory roles in energy homeostasis and lipid metabolism, linking them to cholesterol maintenance in oligodendrocytes.^82,85,86 However, PPARs were initially of interest in neurodegenerative and demyelinating diseases as activation of PPARs was shown to reduce inflammation and glial activation.^82,86−89 Moreover, several studies have shown that PPAR agonists ameliorate clinical disability and delay the onset of disease in varying EAE models^82,85 by initiating an anti-inflammatory phenotype in macrophages and reducing immune cell infiltration or T-lymphocyte restimulation potential.⁸² These findings led to clinical trials in MS patients centering on PPARγ and the synthetic agonist, pioglitazone. These small-scale trials revealed relatively small but positive therapeutic results with pioglitazone treatment, which merit further investigations.⁹⁰

In addition to PPARs role in immunomodulation and glial activation, the receptors have been shown to play a critical role in OL lineage cell progression. Oligodendrocytes express all three isoforms of PPAR (−α, −β/δ, −γ), with PPARγ activation showing the most relevance for OPC differentiation and OL maturation. Several investigations utilizing endogenous and synthetic PPARγ agonist treatments have indicated an acceleration of OPC differentiation into mature oligodendrocytes,^85,91−94 and an increase in myelin gene expression.^85,92,93 Furthermore, inhibition of PPARγ with the potent antagonist, GW9662, was shown to block OPC differentiation.⁹⁵ Taken together these findings support a potential role of PPARγ activation in promoting remyelination. Further evidence suggests cholesterol homeostasis is an important connection between PPARγ activation and the progression of OPC differentiation.^85,93 PPARγ is a crucial regulator of cholesterol uptake and efflux, suggesting the existence of a regulatory loop, linking cholesterol levels and PPARγ expression, by which low levels of cholesterol promote PPARγ transcription to normalize cholesterol levels.⁸⁵ Additionally, investigations have supported the role of mitochondrial respiratory chain activity and intracellular calcium oscillatory behavior as additional downstream targets of PPARγ that facilitate OPC differentiation.⁹³ In addition to PPARγ activation, prior in vitro investigations utilizing treatment with the known PPARα agonist, gemfibrozil, indicated an increase in myelin gene expression in a PPARβ/δ-dependent manner.⁹⁶ Further research in remyelination models is crucial for understanding how PPARs are implicated in myelin maintenance and repair.

Liver X Receptors (LXRs)

Liver X receptors (LXRα, and -β) are another member of the nuclear receptor superfamily. Like PPARs, LXRs are transcription factors that bind to DNA as a heterodimer with retinoid X receptors (RXRs).⁹⁷⁻⁹⁹ As other nuclear receptors, activated LXRs are known to have anti-inflammatory effects.⁹⁹ Additionally, LXRs are known to regulate lipid homeostasis in the periphery,^100,101 and more recently have been suggested to participate in cholesterol homeostasis in oligodendrocytes.¹⁰² LXRs act as cholesterol sensors and modify expression of genes in pathways that govern transport, catabolism, and elimination of cholesterol.¹⁰¹ Both LXR isoforms and LXR-target gene expression is abundantly present in oligodendrocytes in the CNS.¹⁰² As a crucial component of cholesterol homeostasis, knockout studies have shown that loss of LXRs results in thinner myelin sheaths, reduced myelin gene expression, and behavioral consequences.⁹⁸ Importantly, following treatment with TO901317 (TO9), a synthetic agonist, myelin gene expression and protein levels were elevated in primary mixed-glial cultures.⁹⁸ Moreover, LXR activation was shown to increase OL maturation and remyelination in cerebellar slice cultures after lysolecithin-induced demyelination.⁹⁸

In addition to oligodendrocyte cholesterol homeostasis, LXRs exhibit a wide range of influence on the microenvironment surrounding physiological myelin maintenance and remyelination. LXRs are expressed on CD4+ T lymphocytes, and suppress Th17 differentiation, reducing pro-inflammatory cytokine production.⁹⁷ Treatment with LXR agonists TO901317 or GW3965 has been shown to attenuate inflammation and ameliorate EAE in an LXR-dependent manner.⁹⁷ Further, defective cholesterol clearance by phagocytes has been shown in lysolecithin-demyelinated lesions of aged mice.¹⁰⁰ This ineffective cholesterol efflux from phagocytes results in lysosomal rupture and inflammasome stimulation resulting in an exacerbation of inflammation thus hindering remyelination capabilities.¹⁰⁰ Importantly, in these aged mice, oral administration of GW3965 improved lesion remyelination by reducing the number of phagocytes with myelin debris accumulation, the number of foamy macrophages with lipid droplets, and the amount of cholesterol crystals—all hallmarks of cholesterol overloading—as well as by inducing the expression of genes involved in reverse cholesterol transport.¹⁰⁰ While LXR isoform regional functionality, drug selectivity, and unforeseen differences among animal models and humans provide challenges for LXR drug development,⁹⁹ these findings on LXRs not only highlight the importance of cholesterol efflux but point to a number of roles for microenvironmental cholesterol homeostasis in the maintenance and repair capabilities of myelin following demyelination.

Anti-aging Drugs

Age-related cellular perturbations such as DNA damage, telomere shortening, cell cycle dysregulation, epigenetic alterations, protein aggregation, deficiencies in autophagy, oxidative stress, and activation of stress response and inflammatory pathways have been shown to impair the rate of OPC recruitment and differentiation into remyelinating oligodendrocytes.^{36,37,103−109} The accumulation of disability in MS is invariably an age-related process, as with age progression (not necessarily advanced age), most patients will transition to SPMS, independent of the number of relapses.¹¹⁰ Further, loss of myelin is clinically associated with normal aging, as evidenced by imaging and histopathological studies of human brains indicating 28% white matter volume decreases as well as a 27% decline in OL numbers.¹¹¹ In animal models of MS, aged mice exhibit delayed and incomplete remyelination. This impairment is not linked to OPC availability in aged animals, but rather to factors that influence the rate of OPC recruitment and differentiation.^112,113 A reduced rate of major myelin protein (MBP and PLP) transcripts, and their regulatory factor Gtx has been shown within the lesions of old animals compared to young.¹¹² Additionally, aged animals have delayed mRNA expression of growth factors, TGFβ-1 and IGF-I, which play a role in OPC differentiation.¹¹³ Moreover, aging not only affects oligodendrocyte lineage cells directly, but the entire lesion microenvironment. Macrophage-dependent phagocytosis of myelin debris following demyelination is delayed in aged animals, which results in free myelin debris that contains inhibitors of OPC differentiation.^114−116 Further, an accumulation of senescent cells with distinct phenotypes such as proliferative arrest, and the secretion of pro-inflammatory cytokines with senescence associated secretory phenotypes (SASPs) is seen with age-related diseases.^108,117,118 An additional aspect of the lesion microenvironment is the glial scar which impedes OPC differentiation due to the release of SASPs.^119,120 Moreover, senescent Sox2 progenitor cells in demyelinated lesions have been shown to inhibit OPC differentiation and subsequent remyelination in progressive MS investigations.¹¹⁸ This convergence of evidence indicates an important age-related influence on the OPC maturation process. Indeed, in acute lysolecithin lesions capable of complete remyelination, reactive astrocytes in the microenvironment were shown to produce remyelination associated growth factors, PDGF, FGF-2, and IGF-I.¹²¹ Further, exposure to youthful systemic environments via heterochronic parabiosis has been shown to positively influence remyelination in aged mice.¹¹⁶

These findings highlight age-associated delays in remyelination in acute experimental demyelination models as a potential basis for the consequential evolution of chronic demyelination in MS.^109,122 Importantly, it has been shown that the negative influence of senescence on OPC differentiation and remyelination is potentially reversible by targeting senescent cells.^116,118 Treatment of senescent OPCs in a mouse model of Alzheimer’s disease with a cocktail of FDA-approved senolytic agents, dasatinib and quercetin, was shown to selectively eliminate senescent OPCs from the plaque environment, reduce neuroinflammation, and reverse amyloid-beta clearance and age-related disease processes.¹¹⁷ Additionally, FDA-approved mTOR inhibitors, rapamycin and metformin, have been shown to exhibit anti-aging effects and hold potential for remyelination therapies.^123,124 Treatment of aged OPCs with metformin, a small-molecule fasting mimetic, has been shown to cause aged OPCs to regain responsiveness to pro-differentiation signals, and improve remyelination in aged animals following focal demyelination.¹²⁵ These findings suggest a synergistic effect of anti-aging senolytic agents and pro-differentiation pharmacotherapies to promote remyelination. Further understanding of the role aging plays on OL integrity and remyelination is imperative when considering the complex interactions underlying MS pathology and eventual treatment.

The Future of MS Therapeutics

In this review, we highlighted promising MS pharmacotherapeutics (both in preclinical and clinical stages) targeting oligodendrocyte lineage cell progression in remyelination, with most specifically targeting OPC differentiation (Table 2). These new remyelination pharmacotherapeutics fall into two categories: repurposed FDA-approved drugs or intrinsic substances from OL lineage investigations. In either case, the remyelination drugs have already provided lines of investigations into novel molecular pathways important in enabling remyelination, increasing remyelination efficacy, and/or relieving disability in preclinical models (Figure 3). While undoubtedly beneficial to MS therapeutic research, it is important to note that only a few of the highlighted drugs were discovered from investigations of intrinsic OL lineage cell progression within the lesion microenvironment. This is not to say that any one of the many potential pharmacotherapies discovered through in vitro drug screens are less likely to yield long-term success. However, recent investigations utilizing induced pluripotent stem (iPS) cell-derived neural progenitor cells from primary progressive multiple sclerosis (PPMS) patients suggest that endogenous CNS components are capable of altering the response to in vitro drug screen compounds that should foster OPC differentiation, such as benztropine, clemastine, clobetasol, and miconazole.¹²⁶ Since MS posits a complex pathology involving multiple cell types and an immune-triggered root of damage, more holistic investigations into the lesion microenvironment are necessary.

Table 2. Current and Experimental Remyelination Therapies Focused on Oligodendrocyte Progenitor Cell Differentiation^a.

oligodendrocyte progenitor cell (OPC) differentiation pharmacotherapies
treatment	target	method of action	discovery method	phase (clinical trial phase)
Opicinumab	LINGO-1 antagonist	antibody produced against LINGO-1	manufactured after in vitro/in vivo studies	4 ongoing phase II trials (NCT02657915,NCT01864148,NCT01721161,NCT01721161): no significant VEP latency differences
Clemastine	M1	antimuscarinic	micropillar drug screen	2 ongoing phase II trials (NCT02521311) ReBUILD: reduced VEP latencies, ReCOVER: ongoing
Benztropine	M1	antimuscarinic	small molecule drug screen	n/a
Quetiapine	M1 or H1/H3	antimuscarinic	small molecule drug screen	Ongoing I/II dose-determining study (NCT02087631)
Bexarotene	RXR agonist	nuclear translocation	in vitro/in vivo studies	n/a
IRX4204	RXR agonist	nuclear translocation	transcriptomic profiling	Recruitment phases in clinical trial for remyelination
Pioglitazone	PPARγ agonist	heterodimer w/RXR and immunomodulation	in vitro/in vivo studies	Several small-scale clinical trials completed: modest results
GW3965	LXR agonist	heterodimer w/RXR, cholesterol homeostasis	in vitro/in vivo studies	n/a
TO901317	LXR agonist	heterodimer w/RXR, cholesterol homeostasis	in vitro/in vivo studies	n/a
Clobetasol	GR/Smo agonist	glucocorticoid/Hedgehog signaling	in vitro drug screen	n/a
Miconazole	ERK1/2,CYP51	pro-growth pathways, inhibition of CYP51	in vitro drug screen	n/a

^aEach potential therapy targets varying molecular signaling components of oligodendrocyte lineage cell progression. Additionally, several therapies influence the oligodendrocyte intrinsic and extrinsic microenvironment, to facilitate OL lineage cell progression.

Figure 3.

Schematic of molecular pathways implicated in remyelination therapies targeting OPC differentiation or OL maturity/viability. Several remyelination therapeutics antagonize muscarinic receptors on oligodendrocytes. Another large group of experimental therapeutics are agonists to nuclear receptors such as PPAR and LXR, which heterodimerize with another target receptor, RXR, to function as transcription factors important for cholesterol homeostasis and myelination. Glucocorticoid receptor is yet another nuclear receptor target that may interact with other nuclear receptors implicated in myelin gene expression. Additionally, LINGO-1-RhoA, Hedgehog (Hh), CYP51, and pro-growth MEK-ERK1/2 signaling have also been linked to OPC differentiation and/or cholesterol homeostasis. With regards to OL maturity and viability, taurine is linked to increased serine pools which supplement GSH synthesis important in myelination. Creatine is associated with ATP levels and mitochondrial function in OLs promoting their viability after demyelination. Created with Biorender.

Investigating and identifying drug targets from within the lesion microenvironment brings a potential to better address the relationships among various cell types in MS and specifically in the capacity and eventual downfall of remyelination. Recent advances in molecular approaches will serve to aid in this purpose. These advances include an improvement in techniques for the isolation of lesions in varying animal models, and the ability to investigate specific cellular responses within the microenvironment at the single cell to single nuclei level. A combination of these methods will equip researchers with an understanding of the mechanisms underlying successful OL lineage cell progression and recovery within the context of MS pathology. Of course, as each model has its advantages and disadvantages, investigators must take caution to match the model to appropriate questions and hypotheses.

Furthermore, current FDA-approved therapies for MS are immunomodulatory drugs. Although these disease-modifying drugs have been instrumental in advancing MS therapies, they are just one piece of the puzzle. It is undoubtedly clear that immunomodulation alone cannot reverse or prevent remyelination failure and the progression of MS to permanent disability. Each phase of remyelination must be carefully considered—including OPC migration and proliferation, differentiation, OL maturation, and OL survival—to develop a valid treatment approach concerning the eventual failure of remyelination. Together, targeting the immune system in conjunction with direct facilitation of OL lineage cell progression provides a potentially ideal prescriptional tactic for MS (Figure 4). Additionally, we considered more recent investigations centering on the influence of cholesterol homeostasis and aging mechanisms in understanding MS pathology. These avenues will serve to provide a better understanding of the complexities of MS and will help to inform therapeutics in the near future. Current advances in MS research present investigators with an exciting, yet challenging road ahead in seeking out an efficacious, safe, and lasting remedy for a complex immune-mediated disease. Although still in its infancy, one thing is clear of this new outlook—as new methods and technologies have arisen, so has an increased knowledge of OL lineage cell progression and remyelination mechanisms. Pharmacotherapies directly targeting oligodendrocyte remyelination and the lesion microenvironment represent the new frontier for MS therapy, and the prospect of such drugs brings with it the possibility of healing.

Figure 4.

A treatment approach for multiple sclerosis that encompasses both immune modulation and the promotion of remyelination. The drugs currently available for treating multiple sclerosis (MS) primarily rely on various means of peripheral immune system modulation (left) to slow disease progression. Several potential drugs that target CNS inflammation and the different aspects and stages of remyelination (right) are now being identified. Remyelination represents a means of rejuvenating the CNS after immune system-induced damage to myelin and oligodendrocytes. Promoting remyelination therefore provides an additional line of defense against the axonal damage that follows the loss of myelin. Created with Biorender.

The authors declare no competing financial interest.