Demyelination and Remyelination: From Mechanism to Therapy

Journal Articles

The challenges of reversing myelin loss

Bung MB, Bunge RP, Ris H. Ultrastructural study of remyelination in an experimental lesion in adult cat spinal cord. J Biophys Biochem Cytol. 1961;10(1):67-94.

Franklin RJ. Why does remyelination fail in multiple sclerosis? Nat Rev Neurosci. 2002;(9):705-14.

Franklin RJ, Ffrench-Constant C. Remyelination in the CNS: from biology to therapy. Nat Rev Neurosci. 2008;9(11):839-55.

Huang JK, Jarjour AA, Nait Oumesmar B, et al. Retinoid X receptor gamma signaling accelerates CNS remyelination. Nat Neurosci. 2011;14(1):45-53.

Ruckh JM, Zhao JW, Shadrach JL, et al. Rejuvenation of regeneration in the aging central nervous system. Cell Stem Cell. 2012;10(1):96-103.

Smith PM, Jeffery ND. Histological and ultrastructural analysis of white matter damage after naturally-occurring spinal cord injury. Brain Pathol. 2006;16(2):99-109.

Stacpoole SR, Spitzer S, Bilican B, et al. High yields of oligodendrocyte lineage cells from human embryonic stem cells at physiological oxygen tensions for evaluation of translational biology. Stem Cell Reports. 2013;1(5):437-50.

Woodruff RH, Fruttiger M, Richardson WD, Franklin RJ. Platelet-derived growth factor regulates oligodendrocyte progenitor numbers in adult CNS and their response following CNS demyelination. Mol Cell Neurosci. 2004;25(2):252-62.

Zawadzka M, Rivers LE, Fancy SP, et al. CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination. Cell Stem Cell. 2010;(6):578-90.

Mechanisms of remyelination in the CNS

Jarjour AA, Manitt C, Moore SW, et al. Netrin-1 is a chemorepellent for oligodendrocyte precursor cells in the embryonic spinal cord. J Neurosci. 2003;23(9):3735-44.

Piaton G, Aigrot MS, Williams A, et al. Class 3 semaphorins influence oligodendrocyte precursor recruitment and remyelination in adult central nervous system. Brain. 2011;134(Pt 4):1156-67.

Spassky N, de Castro F, Le Bras B, et al. Directional guidance of oligodendroglial migration by class 3 semaphorins and netrin-1. J Neurosci. 2002;22(14):5992-6004.

Molecular mechanisms of myelination and repair

Belkina AC, Denis GV. BET domain co-regulators in obesity, inflammation and cancer. Nat Rev Cancer. 2012;12(7):465-77.

Casaccia-Bonnefil P, Tikoo R, Kiyokawa H, et al. Oligodendrocyte precursor differentiation is perturbed in the absence of the cyclin-dependent kinase inhibitor p27Kip1. Genes Dev. 1997;11(18):2335-46.

Dugas JC, Ibrahim A, Barres BA. A crucial role for p57(Kip2) in the intracellular timer that controls oligodendrocyte differentiation. J Neurosci. 2007;27(23):6185-96.

Filippakopoulos P, Picaud S, Mangos M, et al. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell. 2012;149(1):214-31.

Gacias M, Gerona-Navarro G, Plotnikov AN, et al. Selective chemical modulation of gene transcription favors oligodendrocyte lineage progression. Chem Biol. 2014;21(7):841-54.

He Y, Dupree J, Wang J, et al. The transcription factor Yin Yang 1 is essential for oligodendrocyte progenitor differentiation. Neuron. 2007;55(2):217-30.

Lee PR, Fields RD. Regulation of myelin genes implicated in psychiatric disorders by functional activity in axons. Front Neuroanat. 2009;3:4. [eCollection]

Magri L, Swiss VA, Jablonska B, Lei L, et al. E2F1 coregulates cell cycle genes and chromatin components during the transition of oligodendrocyte progenitors from proliferation to differentiation. J Neurosci. 2014;34(4):1481-93.

Pedre X, Mastronardi F, Bruck W, et al. Changed histone acetylation patterns in normal-appearing white matter and early multiple sclerosis lesions. J Neurosci. 2011;31(9):3435-45.

Prinjha RK, Witherington J, Lee K. Place your BETs: the therapeutic potential of bromodomains. Trends Pharmacol Sci. 2012;33(3):146-53.

Shen S, Sandoval J, Swiss VA, et al. Age-dependent epigenetic control of differentiation inhibitors is critical for remyelination efficiency. Nat Neurosci. 2008;11(9):1024-34.

Tokumoto YM, Apperly JA, Gao FB, Raff MC. Posttranscriptional regulation of p18 and p27 Cdk inhibitor proteins and the timing of oligodendrocyte differentiation. Dev Biol. 2002;245(1):224-34.

Ye F, Chen Y, Hoang T, et al. HDAC1 and HDAC2 regulate oligodendrocyte differentiation by disrupting the beta-catenin-TCF interaction. Nat Neurosci. 2009;12(7):829-38.

Zezula J, Casaccia-Bonnefil P, Ezhevsky SA, et al. p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal. EMBO Rep. 2001;2(1):27-34.

Reversing myelin loss after preterm hypoxia

Aguirre A, Rubio ME, Gallo V. Notch and EGFR pathway interaction regulates neural stem cell number and self-renewal. Nature. 2010;467(7313):323-7.

Bi B, Salmaso N, Komitova M, et al. Cortical glial fibrillary acidic protein-positive cells generate neurons after perinatal hypoxic injury. J Neurosci. 2011;31(25):9205-21.

Fagel DM, Ganat Y, Silbereis J, et al. Cortical neurogenesis enhanced by chronic perinatal hypoxia. Exp Neurol. 2006;199(1):77-91.

Jablonska B, Scafidi J, Aguirre A, et al. Oligodendrocyte regeneration after neonatal hypoxia requires FoxO1-mediated p27Kip1 expression. J Neurosci. 2012;32(42):14775-93.

Salmaso N, Jablonska B, Scafidi J, et al. Neurobiology of premature brain injury. Nat Neurosci. 2014;17(3):341-6.

Scafidi J, Hammond TR, Scafidi S, et al. Intranasal epidermal growth factor treatment rescues neonatal brain injury. Nature. 2014;506(7487):230-4.

Skranes J, Vangberg TR, Kulseng S, et al. Clinical findings and white matter abnormalities seen on diffusion tensor imaging in adolescents with very low birth weight. Brain. 2007;130(Pt 3):654-66.

Woodward LJ, Moor S, Hood KM, et al. Very preterm children show impairments across multiple neurodevelopmental domains by age 4 years. Arch Dis Child Fetal Neonatal Ed. 2009;94(5):F339-44.

Identification of small molecule modulators of remyelination

Deshmukh VA, Tardif V, Lyssiotis CA, et al. A regenerative approach to the treatment of multiple sclerosis. Nature. 2013;502(7471):327-32.

High-throughput screening of therapeutic agents for myelin repair using micropillar arrays

Lee S, Leach MK, Redmond SA, et al. A culture system to study oligodendrocyte myelination processes using engineered nanofibers. Nat Methods. 2012;9(9):917-22.

Rosenberg SS, Kelland EE, Tokar E, et al. The geometric and spatial constraints of the microenvironment induce oligodendrocyte differentiation. Proc Natl Acad Sci U S A. 2008;105(38):14662-7.

In vivo target discovery using a zebrafish model

Bertrand S, Thisse B, Tavares R, et al. Unexpected novel relational links uncovered by extensive developmental profiling of nuclear receptor expression. PLoS Genet. 2007;3:e188.

Curado S, Stainier DY, Anderson RM. Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat Protoc. 2008;3:948-54.

Jung SH, Kim S, Chung AY, et al. Visualization of myelination in GFP-transgenic zebrafish. Dev Dyn. 2010;239:592-7.

Münzel EJ, Schaefer K, Obirei B, et al. Claudin k is specifically expressed in cells that form myelin during development of the nervous system and regeneration of the optic nerve in adult zebrafish. Glia. 2012;60:253-70.

Schebesta M, Serluca FC. olig1 Expression identifies developing oligodendrocytes in zebrafish and requires hedgehog and notch signaling. Dev Dyn. 2009;238:887-98.

Stern HM, Zon LI. Cancer genetics and drug discovery in the zebrafish. Nat Rev Cancer. 2003;3:533-9.

Yoshida M, Macklin WB. Oligodendrocyte development and myelination in GFP-transgenic zebrafish. J Neurosci Res. 2005;81(1):1-8.

Identifying targets for remyelination: from cultures to animal models

Humbert PO, Dow LE, Russell SM. The Scribble and Par complexes in polarity and migration: friends or foes? Trends Cell Biol. 2006;16:622-30.

Huang JK, Jarjour AA, Nait Oumesmar B, et al. Retinoid X receptor gamma signaling accelerates CNS remyelination. Nat Neurosci. 2011;14:45-53.

Lee S, Leach MK, Redmond SA, et al. A culture system to study oligodendrocyte myelination processes using engineered nanofibers. Nat Methods. 2012;9:917-22.

Miron VE, Boyd A, Zhao JW, et al. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nat Neurosci. 2013;16:1211-8.

Tracking remyelination using MR techniques

Barkhof F, Bruck W, De Groot CJ, et al. Remyelinated lesions in multiple sclerosis: magnetic resonance image appearance. Arch Neurol. 2003;60:1073-81.

Brown RA, Narayanan S, Arnold DL. Segmentation of magnetization transfer ratio lesions for longitudinal analysis of demyelination and remyelination in multiple sclerosis. Neuroimage. 2012;66C:103-9.

Chen JT, Kuhlmann T, Jansen GH, et al. Voxel-based analysis of the evolution of magnetization transfer ratio to quantify remyelination and demyelination with histopathological validation in a multiple sclerosis lesion. Neuroimage. 2007;36:1152-8.

Tracking remyelination by PET: rodents to humans

Dong GC, Chuang PH, Chang KC, et al. Blocking effect of an immuno-suppressive agent, cynarin, on CD28 of T-cell receptor. Pharm Res. 2009;26:375-81.

Paula Faria D de, de Vries EF, Sijbesma JW, et al. PET imaging of demyelination and remyelination in the cuprizone mouse model for multiple sclerosis: a comparison between [11C]CIC and [11C]MeDAS. Neuroimage. 2014;87:395-402.

Stankoff B, Freeman L, Aigrot MS, et al. Imaging central nervous system myelin by positron emission tomography in multiple sclerosis using [methyl-¹¹C]-2-(4'-methylaminophenyl)- 6-hydroxybenzothiazole. Ann Neurol. 2011;69:673-80.

Stankoff B, Wang Y, Bottlaender M, et al. Imaging of CNS myelin by positron-emission tomography. Proc Natl Acad Sci U S A. 2006;103:9304-9.

Wang C, Wu C, Popescu DC, et al. Longitudinal near-infrared imaging of myelination. J Neurosci. 2011;31:2382-90.

Wu C, Zhu J, Baeslack J, et al. Longitudinal positron emission tomography imaging for monitoring myelin repair in the spinal cord. Ann Neurol. 2013;74:688-98.

Human glial progenitor cell-based treatment and modeling of myelin disease

Goldman SA, Nedergaard M, Windrem MS. Glial progenitor cell-based treatment and modeling of neurological disease. Science. 2012;338:491-5.

Sim FJ, McClain CR, Schanz SJ, et al. CD140a identifies a population of highly myelinogenic, migration-competent and efficiently engrafting human oligodendrocyte progenitor cells. Nat Biotechnol. 2011;29:934-41.

Wang S, Bates J, Li X, et al. Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell. 2013;12:252-64.

Windrem MS, Nunes MC, Rashbaum WK, et al. Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain. Nat Med. 2004;10:93-7.

Windrem MS, Schanz SJ, Guo M, et al. Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell. 2008;2:553-65.

Myelin: development, disease, and cell-based therapy

Chang A, Tourtellotte WW, Rudick R, Trapp BD. Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis. N Engl J Med. 2002;346:165-73.

Gupta N, Henry RG, Strober J, et al. Neural stem cell engraftment and myelination in the human brain. Sci Transl Med. 2012;4(155):155ra137.

Kuhlmann T, Miron V, Cui Q, et al. Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in achronic multiple sclerosis. Brain. 2008;131(Pt 7):1749-58.

Mudhar HS, Pollock RA, Wang C, et al. PDGF and its receptors in the developing rodent retina and optic nerve. Development. 1993;118:539-52.

Najm FJ, Lager AM, Zaremba A, et al. Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells. Nat Biotechnol. 2013;31:426-33.

Uchida N, Chen K, Dohse M, et al. Human neural stem cells induce functional myelination in mice with severe dysmyelination. Sci Transl Med. 2012;4:155ra136.

Windrem MS, Schanz SJ, Guo M, et al. Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell. 2008;2:553-65.

Yang N, Zuchero JB, Ahlenius H, et al. Generation of oligodendroglial cells by direct lineage conversion. Nat Biotechnol. 2013;31(5):434-9.

Clinical investigation of rHIgM22: a potential remyelinating agent

Mitsunaga Y, Ciric B, Van Keulen V, et al. Direct evidence that a human antibody derived from patient serum can promote myelin repair in a mouse model of chronic-progressive demyelinating disease. FASEB J. 2002;16:1325-7.

Pirko I, Ciric B, Gamez J, et al. A human antibody that promotes remyelination enters the CNS and decreases lesion load as detected by T2-weighted spinal cord MRI in a virus-induced murine model of MS. FASEB J. 2004;18:1577-9.

Rodgers JM, Robinson AP, Miller SD. Strategies for protecting oligodendrocytes and enhancing remyelination in multiple sclerosis. Discov Med. 2013;16:53-63.

Anti-LINGO-1 to target myelin repair

Mi S, Lee X, Shao Z, et al. LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex. Nat Neurosci. 2004;7:221-8.

Mi S, Miller RH, Lee X, et al. LINGO-1 negatively regulates myelination by oligodendrocytes. Nat Neurosci. 2005;8:745-51.

Mi S, Miller RH, Tang W, et al. Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells. Ann Neurol. 2009;65:304-15.

Mi S, Pepinsky RB, Cadavid D. Blocking LINGO-1 as a therapy to promote CNS repair: from concept to the clinic. CNS Drugs. 2013;27:493-503.

Miller RH, Mi S. Dissecting demyelination. Nat Neurosci. 2007;10:1351-4.

Pepinsky RB, Arndt JW, Quan C, et al. Structure of the LINGO-1-anti-LINGO-1 Li81 antibody complex provides insights into the biology of LINGO-1 and the mechanism of action of the antibody therapy. J Pharmacol Exp Ther. 2014;350:110-23.

Sponsors

Supported by

Note: The statements and views expressed in these conference materials or the publications or presentations made by conference speakers or moderators are their own and do not reflect the position or policy of the corporate sponsors or supporters of the conference nor does mention of trade names, commercial practices, or organizations imply endorsement by any of the corporate sponsors or supporters of the conference.

Keynote Speaker:
Robin Franklin, Wellcome Trust – MRC Cambridge Stem Cell Institute, University of Cambridge, UK

Highlights

• Remyelination is a spontaneous process that occurs in response to myelin injury.
• The age-related decline in the efficiency of remyelination can be partially reversed by manipulating environmental signals.
• Identifying signaling pathways in spontaneous remyelination will point researchers to drug targets for remyelination.

Introduction

Myelin disorders represent a debilitating and relatively frequent pathology. Multiple sclerosis (MS), one of the most common disabling neurological diseases in young adults, affects approximately 2.3 million people worldwide, according to the National MS Society; other demyelinating conditions include genetic and inflammatory disorders as well as injuries such as white matter injury caused by neonatal hypoxia. Myelin disorders are characterized by the deterioration or the defective development of myelin, the fatty coating extending from glial cells called oligodendrocytes that ensheathes neurons and ensures proper neuronal activity. The destruction of myelin in turn causes degeneration of the neuron underneath it, leading to both physical and cognitive disability.

The dozen or so available treatments for MS target the inflammatory component of the disease; no treatments promote remyelination, in MS or other conditions. Recent discoveries have improved our understanding of the white matter microenvironment and of oligodendrocyte precursor development, and new screening approaches are yielding ever more promising targets. These advances will help researchers develop treatments for such disorders. On June 26, 2014, scientists convened at the New York Academy of Sciences for a symposium that explored the biology of myelin formation and therapeutic prospects for remyelination. It focused on the genetics, epigenetics, and signaling pathways of myelination and highlighted new imaging modalities for tracking myelin and clinical trials for remyelinating therapies.

Despite the recent identification of promising targets for drugs promoting remyelination, and advances in clinical imaging that allow for easier and more accurate assessment of demyelination, much remains unknown about the mechanisms underlying myelination, demyelination, and remyelination, and many more targets will be needed to develop new therapies.

The keynote address focused on recently identified signaling pathways involved in spontaneous myelin regeneration that occurs in response to injury, exploring how these pathways might be harnessed in drug development. The first two sessions described molecular, genetic, and epigenetic mechanisms of myelin regeneration, as well as screening efforts to identify novel drug targets for remyelination. Later in the meeting, speakers presented novel clinical imaging techniques and data from clinical trials of experimental drugs. Finally, in the panel session, speakers discussed whether techniques for imaging myelin content in the central nervous system (CNS) are ready for widespread use in clinical trials.

The challenges of reversing myelin loss

Robin Franklin of the Wellcome Trust – MRC Cambridge Stem Cell Institute began his keynote address by defining demyelination as a pathological process in which myelin—not the underlying axon—is the primary target of injury. Remyelination therapy is not always necessary for primary demyelination (resulting from oligodendrocyte injury) in the CNS because remyelination often occurs spontaneously if the underlying axon is present.

Remyelination occurs spontaneously after primary demyelination, which results from oligodendrocyte injury (left). However, when demyelination is secondary to axon injury, no spontaneous regeneration occurs (right). (Image courtesy of Robin Franklin)

But some conditions, including the chronic demyelination seen in MS, do require therapy, in large part because differentiation of oligodendrocytes from oligodendrocyte precursor cells (OPCs) becomes inefficient with age. This inefficiency results from both environmental cues and cell-intrinsic changes.

In genetic demyelinating diseases, OPCs are inherently damaged and cell-based approaches that transplant myelin-producing cells from a healthy person into the patient make most sense. But for MS, pharmaceutical approaches are preferable because age-associated changes appear to be reversible. Heterochronic parabiosis animal experiments showed that surgically joining the circulatory systems of aged mice to those of younger animals rejuvenated the ability of OPCs in aged animals to remyelinate. This result suggests that pharmacological approaches that manipulate environmental cues may be sufficient to increase remyelination.

Franklin and others have therefore focused on identifying regulators of remyelination and designing drugs to increase the efficiency of this endogenous process. Drug targets on positive regulatory pathways include retinoid X receptor gamma (RXRG), endothelin-2, and activin-A; those on negative regulatory pathways include Wnt signaling molecules and phosphodiesterase-4 (PDE4).

Franklin's team identified the first of these molecules in a screen for genes associated with oligodendrocyte differentiation. Further studies showed that RXRs accelerate remyelination in an animal model and in human cultured OPCs and that the molecules were present in human MS lesions. The team is now testing an RXR agonist called bexarotene in a small clinical trial. Franklin cautioned that researchers will need to add more candidates to the list to identify effective therapies.

Franklin also pointed to two challenges for developing treatments for demyelination disorders. First, although poor oligodendrocyte differentiation is usually the reason for impaired remyelination, sometimes remyelination fails because of flawed OPC recruitment mechanisms. It is likely that researchers will need to develop specific drugs for each type of remyelination problem. Second, while the quantity of myelination is key to the proper functioning of neurons, the quality of remyelination is also important and must be considered in treatment regimes.

Speakers:
Catherine Lubetzki, University of Pierre & Marie Curie and Salpêtrière Hospital, France
Patrizia Casaccia, Ichan School of Medicine at Mount Sinai
Vittorio Gallo, Children's National Medical Center

Highlights

• Guidance cues used by neonatal OPCs could be reawakened in activated adult OPCs as a therapeutic strategy to increase OPC migration and remyelination.
• Modulating epigenetic regulators of OPC differentiation may offer a strategy for reversing the age-related decline in remyelination capability.
• The growth factor EGF ameliorates the effects of neonatal hypoxia in rodents and can be administered noninvasively.

Mechanisms of myelin regeneration in the adult central nervous system

Catherine Lubetzki of the University of Pierre & Marie Curie and Salpêtrière Hospital in Paris explained that current drugs for MS, which target inflammation and axonal loss, effectively reduce relapse rates but do not treat the progressive phase of the disease, which is marked by increasing demyelination that the CNS is unable to repair.

In the myelin repair process, quiescent OPCs are activated and recruited to a damaged area, where they mature into myelin-producing oligodendrocytes. To study the characteristics distinguishing quiescent from activated adult OPCs, Lubetzki compared transcriptome profiles of adult OPCs to those of neonatal OPCs and differentiated adult oligodendrocytes. Quiescent adult OPCs were more similar to adult oligodendrocytes than to neonatal OPCs. Activated OPCs, however, reverted to an immature gene profile.

Quiescent adult OPCs are more similar to adult oligodendrocytes than to neonatal OPCs. (Image courtesy of Catherine Lubetzki)

After identifying 119 genes differentially expressed in quiescent and activated OPCs, her group focused on two genes, CCL2 and IL-1β, known to code for immune response cues. These genes increased OPC motility, thus aiding OPC recruitment to the site of demyelination.

Further studies examined whether guidance cues that are important in the migration of OPCs during development—specifically, netrin 1 and the semaphorins sema 3A and sema 3F—could be reawakened after demyelination in adults. When strongly upregulated after demyelination, these molecules showed similar effects in adult and embryonic OPCs. Overexpression of sema 3F, an attractant, increased OPC recruitment and myelination, while overexpression of netrin prevented OPC recruitment and delayed remyelination. The group is developing a strategy in preclinical trials to target sema 3F to the lesion site and speed myelination early in the disease, while axonal damage is still reversible.

Molecular mechanisms of myelination and repair

OPCs are kept in a proliferative state by mitogens, molecules that keep cells in the cell cycle and block differentiation. Patrizia Casaccia from Ichan School of Medicine at Mount Sinai is examining developmental epigenetic mechanisms by which OPCs leave the cell cycle and become able to differentiate into myelin-producing oligodendrocytes. The process involves downregulation of mitogen receptor genes followed by upregulation of myelin synthesis genes and genes present in the myelin sheath.

The team identified two transcription factors, E2F1 and YY1, that the regulate mitogenesis. E2F1, highly active in OPCs, strongly acetylates chromatin; YY1 recruits chromatin modifiers histone deacetylases (HDAC) 1 and 2, which decrease the activation of genes that regulate the cell cycle. Silencing HDAC1 prevents chromatin compaction and differentiation in OPCs, and mice lacking HDAC1 and HDAC2 have fewer oligodendrocytes, suggesting that HDACs lift the brakes on OPC differentiation.

When adult myelin is damaged, as in MS, the mechanisms are more complicated. Here, myelin repair must overcome a strong age-dependent block on OPC differentiation, as well as other defective processes such as an increase in acetyl transferase activity at the expense of HDACs. Increasing HDAC activity might therefore be a useful therapeutic strategy, but developing drugs that activate enzymes is very challenging.

Inhibiting HDACs prevents OPC differentiation into mature oligodendrocytes. (Image courtesy of Patrizia Casaccia)

Instead, Casaccia's team focused on a diverse group of protein modules called bromodomains that "read" histone modifications by recognizing acetyl lysine. The researchers identified three bromodomains whose expression is high in OPCs but declines when differentiated cells myelinate. Olinone, an inhibitor the team developed against these bromodomains, increased OPC differentiation in cultured cells. They plan to test olinone in older mice to determine whether it reverses the age-related decline in remyelination. Casaccia proposed that this epigenetic approach could be combined with immunomodulators and other therapies to more effectively treat MS.

Reversing myelin loss after preterm hypoxia

White matter injury is common in premature infants, particularly those born before 32 weeks, and causes serious motor and neurological deficits. Vittorio Gallo from Children's National Medical Center studies preterm hypoxia by inducing myelin-damaging oxygen deprivation in early postnatal rodents. The manipulation causes a loss of oligodendrocytes, abnormal myelination, and poor performance on behavioral tests.

A genomic screen revealed that expression of the gene for epidermal growth factor (EGF), previously implicated in oligodendrocyte development, transiently increased by 8- to 10-fold shortly after hypoxia. In mice, overexpression of the EGF receptor in the oligodendrocyte lineage reversed some of the effects of hypoxia, increasing oligodendrocyte cell numbers and myelin thickness, and reversed performance deficits on two behavioral tests.

Delivering noninvasive intranasal injections of EGF within 5 days of hypoxia induction also attenuated the effects of hypoxia in mice. The treatment reached the white matter and activated EGF receptors, promoting oligodendrogenesis, reducing oligodendrocyte loss, and restoring levels of myelin protein expression. Myelin thinning was completely reversed and remained so 60 days later. EGF nasal treatment also reversed action potential firing deficits, myelin deficits observed by diffusion tensor imaging, and behavioral test results when administered just after hypoxia (but not when administered a week later). The team is working to establish the molecular mechanism for this amelioration and has so far identified a promising link to notch signaling, which is a known inhibitor of oligodendrocyte differentiation.

Speakers:
Luke L. Lairson, Scripps Research Institute
Jonah R. Chan, University of California, San Francisco
Wendy B. Macklin, University of Colorado School of Medicine
Charles ffrench-Constant, Centre for Regenerative Medicine, University of Edinburgh, UK
 
Hot Topic Talk Presenters:
Pedro Brugarolas, University of Chicago
Erin M. Gibson, Stanford University

Highlights

• A screen for molecules that promote OPC differentiation identified muscarinic receptor modulating agents and scaffold molecules, which are being pursued for preclinical development.
• A high-throughput assay for myelination should be used to identify promising FDA-approved compounds that could be taken forward quickly as MS therapeutics.
• Zebrafish embryos are powerful animal models for in vivo drug screening.
• The process of myelination appears to have both innate and adaptive elements.
• Fluorinated 4-AP is being developed as a tracer in PET imaging to track demyelination.
• Optogenetically induced neuronal activity spurs OPC differentiation in the motor cortex.

Identification of small molecule modulators of remyelination

Luke L. Lairson of Scripps Research Institute is working to identify compounds that promote OPC differentiation, based on results pointing to it as a driver of MS progression. A high-throughput screen his team developed uses a high content imaging-based phenotypic assay to identify promising small molecules. Using the assay to track in vitro differentiation of rat optic nerve-derived OPCs over 6 days, with thyroid hormone as a positive control, the researchers screened 100 000 drug-like molecules and 6000 molecules with known biological activity, identifying multiple known and novel modulators of OPC differentiation. An interesting finding identified a class of FDA-approved neurotransmitter modulating agents that could be fast-tracked for clinical use. The most efficacious of this group, benzatropine, is an antimuscarinic agent used to treat Parkinson's disease.

The researchers first treated OPCs with benzatropine along with agonists and antagonists of the M1 and M3 muscarinic receptors, showing that the compound increases OPC differentiation by signaling through these receptors. Next, they used an experimental autoimmune encephalomyelitis (EAE) mouse model of MS to show that the compound has clinical benefit in vivo. Histology, electron microscopy, and a second in vivo mouse model experiment (with demyelination chemically induced using cuprizone) suggested that benzatropine works by enhancing myelination. However, benzatropine's toxicity may limit its clinical use. They are also evaluating in vivo three agents with 100-fold better toxicity profiles.

The original screen also identified seven scaffold molecules that promote OPC differentiation. Three are being targeted for preclinical development, and the team is working to optimize the drugs' pharmacokinetic properties for in vivo evaluation.

High-throughput screening of therapeutic agents for myelin repair using micropillar arrays

Jonah R. Chan from the University of California, San Francisco, described the development of a high-throughput assay for myelination that can be used to screen FDA-approved drugs for efficacy in MS.

His group had previously designed a coculture system for studying myelination in a dish, but automating it seemed impossible, in part because the system cannot be used to track individual cells—a requirement for quickly quantifying the results. However, the researchers found that OPCs in culture could form myelin when plated onto fixed neurons, suggesting that myelination may not require an axonal signal but rather be triggered by the unique structure of the axon. They therefore designed silica-based micropillars on which myelin formation from individual oligodendrocytes could be detected and then quantified in a multiwall plate.

The team tested 1000 FDA-approved drugs in two weeks, identifying a group of anti-muscarinic compounds. The most promising is a first-generation antihistamine, clemastine, with an excellent safety profile. Validation studies in coculture showed that clemastine and another anti-muscarinic drug, benzatropine, promote differentiation of OPCs. Myelin formation could also be observed and quantified.

Next, Chan's team tested the compounds in vivo. When fed clemastine or benzatropine, adult mice whose white matter had been lesioned with the chemical lysolecithin exhibited increased myelin production and repair. Benzatropine reduced disease severity in an EAE model. Clemastine's action was more gradual, but it also appeared to reverse axonal loss, probably by preserving axons that were still myelinated. A small clinical trial of clemastine in MS is now underway. Chan is optimistic that many more potent therapies may be lurking in libraries of FDA-approved drugs and could be tested and quickly commercialized.

In vivo target discovery using a zebrafish model

Zebrafish provide an excellent model for in vivo studies of myelination. The zebrafish genome is fully annotated and many transgenic models already exist for studying oligodendrocytes. Zebrafish fertilization and development occur ex utero, so the embryos are easy to treat with drugs. The embryos are also transparent, making for easy imaging. Zebrafish development is rapid and comparable to mammalian development, and breeding can be achieved at a fraction of the cost of breeding mice.

Zebrafish embryos are also amenable to many analytical tools, including quantitative PCR, in situ hybridization, genetic manipulation, electron microscopy, and limited immunocytochemistry. Wendy B. Macklin of the University of Colorado School of Medicine uses zebrafish to study the role of retinoic acid signaling in myelination. She has found that the compound reduces gene expression for myelin formation in early development but not after oligodendrocyte differentiation.

Researchers can look for small molecules that enhance oligodendrocyte differentiation by analyzing myelination properties of approved drugs or by screening broader small molecule libraries of ~100 000 compounds. Macklin's lab uses fluorescence intensity as a readout, selectively imaging oligodendrocyte cell bodies, cell membranes, or cells at later developmental stages. The researchers can also modulate gene expression in oligodendrocytes to test the roles of specific genes. Studies in living animals allow researchers to detect toxic, behavioral, and other side effects of experimental drugs.

In a transgenic zebrafish in which myelin protein zero (mpz) is tagged with a green fluorescent protein, the internode structure of myelinated axons is visible by 6 days post-fertilization. (Image courtesy of Wendy B. Macklin)

An ideal drug screen would test compounds in the context of oligodendrocyte injury, not during normal development only. A zebrafish line developed in 2008 used transgenic technology to allow the spatial and temporal control of oligodendrocyte ablation, but so far it may not be sufficiently cell-specific for wide use. Macklin's lab is working toward automating such screens—another crucial goal for testing large compound libraries.

Identifying targets for remyelination: from cultures to animal models

Charles ffrench-Constant of the University of Edinburgh identified the need for more research on myelination and remyelination mechanisms, alongside efforts to develop screens for novel therapeutic targets.

One project in his lab examined the link between innate immunity and remyelination, finding that M2 macrophages and microglia promote remyelination after lysolecithin lesion. Researchers pinpointed activin A as a microglia-derived protein that has dose-dependent effects on regeneration, promoting proliferation at 1 ng/ml, differentiation at 10 ng/ml, and survival at 100 ng/ml. Other aspects of activin signaling were similarly dose-dependent, suggesting a complex regulation of myelination by the molecule.

The lab is also investigating mechanisms of myelin sheath formation. Myelin-producing oligodendrocytes undergo a dramatic shape change, suggesting a role for polarity proteins in myelination. The researchers found that a basal polarity protein called Scribble is upregulated 5–14 days after a lesion, when remyelination occurs. Scribble is expressed in both OPCs and oligodendrocytes at the site of axo-glial adhesion. Its absence in vivo caused abnormal myelination and reduced the efficiency of remyelination.

To examine the cell-intrinsic elements of myelin sheath formation, the group cocultured oligodendrocytes with synthetic nanofibers 4 microns in diameter. The cells formed myelin sheathes with internode length—a critical factor in nerve conductance—identical to that found in oligodendrocytes in vivo. Increasing the diameter of the synthetic fiber increased the internode length of the myelin sheath, as did applying the medium from a culture in which neurons had been stimulated. These findings point to both innate and adaptive regulation of myelination. The lab is exploring both processes, with a focus on mechanisms regulating internode length.

Hot topic talk: a novel tracer for demyelination and remyelination

Pedro Brugarolas from the University of Chicago gave a hot topic talk on his work developing a radioactive tracer for positron emission tomography (PET) that labels demyelination with high sensitivity and specificity. In the paranode regions of myelinated axons, potassium channels cluster underneath the myelin sheath, but upon demyelination, the channels become exposed and dysfunctional, disrupting the neuron's ability to propagate action potentials. A drug called 4-aminopyridine (4-AP) that is approved to treat MS blocks the channels and restores action potentials.

Because 4-AP directly targets potassium channels on demyelinated axons, Brugarolas and colleagues radiolabeled the drug to see if it could be used to image demyelination. In a demyelinated mouse model and a mouse model with lysolecithin-induced focal lesions, radiolabeled 4-AP localized to demyelinated areas and correlated inversely with myelin content. After synthesizing several fluorinated derivatives of 4-AP, which are compatible with PET imaging, the researchers identified one, 3-F-4-AP, with pharmacological properties similar to the original compound. PET studies of the compound in animals are planned.

Hot topic talk: neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain

A second hot topic talk by Erin M. Gibson from Stanford University focused on the role of neuronal activity in myelination. Studies suggest that neuronal activity promotes oligodendrogenesis in the mammalian brain, but in vivo evidence of the effect has been lacking.

The researchers used a mouse model with light-sensitive channel thy-1 channelrhodopsin expressed in cortical layer 5 pyramidal neurons. A light source inserted into nearby tissue (in this case, premotor cortex) stimulates the apical dendrites of these neurons to depolarize. Thirty-second light pulses, which caused the animals to walk in circles, were administered in two time sequences, and the animals were sacrificed three hours, four hours, or four weeks later.

Using the thymidine analogue EdU to mark proliferating cells, the researchers found an increase in new OPCs, and four weeks later an increase in immature and mature oligodendrocytes, in response to the neural activity induced by light pulses. Compared to control animals, the stimulated animals had thicker myelin sheaths and faster forepaw gait. Optogenetically stimulated animals treated with HDAC inhibitors, which block OPC differentiation, did not show myelin thickening or gait enhancements, suggesting that the newly proliferating OPCs were responsible for these changes.

Speakers:
Douglas L. Arnold, McGill University and Montreal Neurological Institute, Canada
Bruno Stankoff, University Pierre & Marie Curie, France
Steven A. Goldman, University of Rochester Medical Center; University of Copenhagen, Denmark
David H. Rowitch, University of California, San Francisco
Andrew Eisen, Acorda Therapeutics
Sha Mi, Biogen Idec
 
Moderator:
Daniel Pelletier, Yale University School of Medicine

Highlights

• Glial progenitor cell transplantation could restore myelin in people with progressive MS.
• Neural stem cell transplantation appeared to be safe in a phase I trial for Pelizaeus-Merzbacher Disease, a rare genetic demyelinating syndrome.
• A monoclonal antibody called rHIgM22 that promoted remyelination in vitro and in animal models of MS is undergoing testing in a phase I clinical trial.
• Anti-LINGO-1, a therapeutic antibody in phase II trials, shows promise as a myelin repair therapy.

Tracking remyelination using magnetic resonance techniques

Clinical markers are insufficient to determine the efficacy of an experimental remyelination therapy in an MS clinical trial, so biologically specific imaging is needed. Douglas L. Arnold of McGill University and Montreal Neurological Institute described an approach that uses magnetic resonance imaging (MRI).

Two commonly used MRI techniques, T2 relaxometry and diffusion tensor imaging (DTI), have several limiting disadvantages. Both have low signal-to-noise ratio and reflect measurements that are not specific to myelin; in addition, T2 relaxometry is not built into commercial scanners and therefore cannot be uniformly implemented across clinical trial sites.

A third approach called magnetic transfer ratio imaging (MTR), however, provides a practical way to clinically assess remyelination efficiency in vivo. Arnold explained that it measures the exchange of magnetization between hydrogen nuclei in water and in membrane macromolecules, which are highly enriched in myelin. This method's specificity should be similar to that of Luxol fast blue staining, a classical marker of myelin.

A comparison to a baseline MTR image shows lesions newly forming and resolving. (Image courtesy of Douglas L. Arnold)

MTR is not without drawbacks: many commercial MRI scanners have MTR capability, but it is implemented differently in different machines, complicating its use in multicenter trials. Although signal specificity is also variable, the signal can be normalized across different machines, and MTR is considerably more specific than other techniques. MTR has a high signal-to-noise ratio and is already in use in clinical trials.

Arnold and his colleagues have developed a technique called delta MTR for defining acute demyelinating lesions. Images taken at different time points are aligned to a reference scan to create a magnetization ratio that more accurately reflects demyelination and remyelination over time than does tracking gadolinium enhancement, the MRI contrast agent commonly used for clinical trials of MS.

Tracking remyelination using positron emission tomography imaging

PET imaging is specific, sensitive, and quantifiable and would be a powerful clinical and research tool in MS if it could track myelin. Bruno Stankoff of the University of Pierre & Marie Curie described his group's work to develop a PET tracer to assess myelin content in white matter.

In 2006 Stankoff's group identified a compound called 1,4-bis(p-aminostyryl)-2-methoxy benzene (BMB) that could stain myelin in the normal brain; other related compounds with potential use in MS clinical imaging have since been identified. Experiments in a demyelinated mouse model showed that these compounds can be used to accurately monitor demyelination and remyelination over time.

The team also investigated the myelin-staining properties of thioflavin derivatives, which are easier to work with clinically. Based on promising experiments in mice and baboons, they tested one molecule, radiolabeled Pittsburgh Compound B (PIB), in humans. The compound produced a well-defined myelin stain in the white matter of young, healthy subjects and could also identify MS lesions.

The researchers then developed a noninvasive quantification method for PIB imaging. In a comparison of three data sets—quantified maps of myelin mRNA, myelin water fraction data, and PIB distribution in healthy adults—from the same brain regions, PBI PET imaging provided more specific pictures of myelin protein distribution than did MRI imaging of myelin water fraction. In a clinical trial of 20 patients with relapsing-remitting MS and 10 healthy volunteers, researchers could estimate myelin dynamics within lesions and correlate their estimates with clinical activity. These studies suggest that PIB PET imaging can be used to investigate the timeframe of remyelination in MS.

Human glial progenitor cell-based treatment and modeling of neurological diseases

Glial progenitor cells are highly abundant in the adult human brain. Steven A. Goldman of the University of Rochester Medical Center and the University of Copenhagen explained how transplantation of glial cells could be used to treat myelin diseases.

Goldman's lab found that a purified population of glial progenitors obtained from 18- to 22-week-old human fetal tissue could myelinate and migrate in animal models. Transplanting these cells into several CNS sites in the shiverer mouse—which lacks myelin, is therefore ataxic, and dies at 20 weeks old—rescued the motor deficit almost completely in 30% of animals, producing extensive donor-derived myelination and restoring a normal lifespan.

However, myelination occurred slowly, over nine months, with each axon taking 1.5 days to myelinate. The researchers sped the process considerably by further purifying the original progenitor pool. A genomic cluster analysis identified the PGFα receptor as an oligodendrocyte lineage-specific marker on glial progenitors and identified an epitope on the protein that could be used to segregate the cells. Transplanting these cells alone completely remyelinated shiverer mice within 3 months.

The researchers also tested the cells' therapeutic potential using adult animal models. Transplantation into adult shiverer mice resulted in phenotypic rescue; it also ameliorated demyelination in a cuprizone mouse model of toxic demyelination and in rats treated with lysolecithin. A pilot clinical trial of this therapy in 30 patents with secondary progressive MS is pending FDA approval. Meanwhile, because fetal tissue is limited, the group has also developed a protocol for generating OPCs from embryonic stem cells or from induced pluripotent stem (iPS) cells. Initial results suggest iPS cells may be more efficacious than fetal tissue.

Myelin development, disease, and cell-based therapy

Pharmacological approaches can be used to stimulate progenitors to overcome signaling blockades of OPC differentiation and myelination, but when oligodendrocytes have cell-intrinsic deficits, cell-based approaches may be more appropriate. David H. Rowitch of the University of California, San Francisco, described his group's work to develop an experimental therapy for Pelizaeus-Merzbacher Disease (PMD), a hypomyelinating syndrome that is recessive, X-linked, and oligodendrocyte-specific, and in its most severe form is fatal by about age 5.

MRI shows the absence of myelin tracts in the brain of a 2-year-old boy with PMD compared to the brain of a healthy 2-year-old boy. (Image courtesy of David H. Rowitch)

In a safety study conducted between 2010 and 2012 in four boys with PMD, Rowitch and his colleagues worked with Stem Cells Inc. to transplant 75 million neural stem cells into the subcortical white matter and then followed the subjects' clinical parameters and MRI data. The cells were found to be safe 2–4 years after transplant. In two patients, diffusion tensor imaging parameters reflected durable changes suggestive of axonal stabilization or myelin production. However, this small study, without a control group or histological proof of biological activity, was not intended to determine efficacy. A phase II study is planned.

A Stanford investigator is working to create iPS cells from one patient's tissue and differentiate the cells into oligodendrocytes. One goal is to speed OPC differentiation in culture. In addition to testing the biological properties of iPS-derived oligodendrocytes, researchers can use these cells to explore gene correction strategies with new gene editing technology called the CRISPR/Cas system. Rowitch suggested that the successful treatment of PMD with neural progenitor transplantation may be considered a proof-of-concept for similar pro-myelination approaches in MS.

Clinical investigation of rHIgM22 as a potential remyelinating agent

Andrew Eisen of Acorda Therapeutics described the development of recombinant human IgM22 (rHIgM22), a monoclonal antibody in phase I clinical trials for treating demyelination in MS. The molecule was first identified in Mayo Clinic experiments in mice inoculated with spinal cord homogenate. Of the germline antibodies produced, rHIgM22, which binds to a proteolipid found in lipid rafts on oligodendrocytes, promoted remyelination in vitro and in mice infected with Theiler's encephalomyelitis virus (TMEV), an MS animal model. TMEV mice also had reduced lesion volume after treatment with the antibody and showed other signs of improved neuronal health.

Human recombinant IgM22 stimulates remyelination in mice after Theiler's encephalomyelitis viral infection, which causes demyelination. (Image courtesy of Andrew Eisen)

Based on these findings, Mayo Clinic researchers worked with Acorda Therapeutics to launch a phase I multicenter, double-blind, placebo-controlled, dose-escalating study of safety and tolerability of rHIgM22 in MS. The trial also interrogated biological effects of the drug. Of the 72 patients, none have experienced any serious safety issues or adverse events. Patient assessment includes clinical measures, MR imaging, and spectroscopy, as well as measures of biological effects, such as heavy water loading to look at myelin dynamics. Results from the clinical trial are expected in early 2015.

The investigators also completed a lipidomics study, collecting serum samples from patients in acute relapse or at defined intervals after acute relapse. The aim was to search for lipid biomarkers that can determine the extent of demyelination and remyelination. The lipid changes identified, if validated, may serve as biomarkers for the natural history of MS following a relapse and for treatment-induced improvement.

Anti-LINGO-1 to target myelin repair

Oligodendrocytes are abundant in MS lesions but seem unable to remyelinate axons, perhaps because of inhibitors in the environment. Sha Mi from Biogen Idec, who presented on behalf of her colleague Diego Cadavid, talked about their team's work to identify LINGO-1, a membrane-associated glycoprotein specifically expressed in neurons and oligodendrocytes in the brain, as a potent inhibitor of remyelination.

Inhibiting LINGO-1 promoted oligodendrocyte differentiation as well as remyelination, and LINGO-1 knockout mice developed normally but showed early-onset myelination. An anti-LINGO-1 antibody developed as an experimental therapeutic promoted oligodendrocyte differentiation and remyelination in several animal models of demyelination. In a rat EAE model, the antibody ameliorated a motor deficit, allowing the animal to bear weight on its hind paws. The group then showed in a double-blind study of lysolecithin-treated rats that MTR imaging could be successfully used to monitor remyelination. Imaging results also correlated with a histological assessment of myelin. Blocking LINGO-1 after crushing the rat optic nerve reduced axonal degeneration, suggesting that the compound targets both oligodendrocyte and axonal injury.

In two phase I studies, anti-LINGO-1 antibody was well tolerated by MS patients. It is now being investigated in two phase II trials, one (RENEW) in acute optic neuritis and one (SYNERGY) in relapsing forms of MS. The SYNERGY trial will investigate whether the antibody facilitates neurological repair when given along with interferon-β, a standard anti-inflammatory treatment for MS. This is the first MS trial to use improvement as a primary endpoint. Both phase II trials will help determine dosing for a future phase III trial.

Panel discussion: validating clinical imaging methods to measure remyelination

There is a huge gap between what is possible in clinical imaging and what researchers can image in vitro and in animal models. Panel moderator Daniel Pelletier of Yale University School of Medicine began the discussion by asking panelists to rank imaging modalities based on how well each detects changes in myelination alone in a multicenter clinical trial.

Stankoff noted that the first requirement for such a tool, before specificity and sensitivity, is availability, making MTR the top choice. Arnold concurred, adding that MTR also ranks higher in sensitivity and specificity than other MRI techniques, such as T2 relaxation. In new lesions, MTR can show demyelination by as much as 80%, but part of that effect is probably due to edema. Changes in chronic lesions and in normal-appearing white matter have also been successfully tracked with MTR. Nancy Richert of Biogen Idec, who joined the panel discussion, stressed that MTR is an evolving technique. Initially, researchers compared average change in MTR within an entire lesion, but more recent work has shown that the central portion of the lesion will never recover and can therefore be removed from the analysis.

The panelists agreed that more work is needed to evaluate these techniques—for example, by studying performance using interventions that are known to work. One concern for small biotech companies is the cost of clinical trials. The various imaging modalities require different numbers of subjects for statistical validity. One audience member asked whether the results of smaller studies could motivate larger ones, but Pelletier noted that different modalities provide different information and are thus not always additive. Arnold added that MTR can be used with as few as 30 patients, with certain limitations.

When asked about whether the field is capable of multicenter PET trials, Stankoff replied that the time for such studies has not yet arrived; first, tracers must be developed that are labeled with fluorine rather than carbon for easier use in a medical context. Other audience members asked about finding ways to specifically image newly formed myelin or the absence of myelin. Panelists noted that methods are in development for the latter, but the techniques would probably need to be used in conjunction with other approaches, because once myelin is gone the signal will disappear completely.

What extracellular cues block remyelination potential over time in MS?

Can therapeutic strategies increase not just myelin quantity but also myelin quality?

Which mechanisms are the most important for remyelination?

Which pathways do remyelinating agents identified in drug screens act on?

Will therapies for developmental demyelinating conditions work similarly to those used in chronic diseases such as MS?

To what extent will gliosis prevent remyelination?

Where do functional measures of myelination, such as evoked potentials, fit in to clinical trials and drug development?

How should newer imaging strategies or tools best be validated?