Scholarly article on topic 'Regulation of remyelination in multiple sclerosis'

Regulation of remyelination in multiple sclerosis Academic research paper on "Biological sciences"

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{Remyelination / Oligodendrocytes / Hyaluronan / Wnt / Notch1 / "Retinoid X receptor"}

Abstract of research paper on Biological sciences, author of scientific article — Khalid A. Hanafy, Jacob A. Sloane

Abstract Multiple sclerosis is a common demyelinating disease that worsens over the course of disease, a significant problem in clinical management. Disability in MS is significantly promoted by poor repair and remyelination of lesions. Both oligodendrocyte recruitment and maturation defects are seen as major causes of poor remyelination in MS. The mechanisms behind impaired remyelination in animal models include involvement of the Notch1, wnt, and hyaluronan/TLR2 pathways. RXR/PPAR signaling has also more recently been identified as an important regulator of remyelination. The local inflammatory milieu also appears to play critical and conflicting roles in promotion and inhibition of remyelination in MS. Understanding the forces regulating remyelination in MS represents an exciting and important initial step towards developing therapeutics targeting chronic disability in MS.

Academic research paper on topic "Regulation of remyelination in multiple sclerosis"

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Review

Regulation of remyelination in multiple sclerosis

Khalid A. Hanafy, Jacob A. Sloane *

Department of Neurology, Beth Israel Deaconess Medical Center, Center for Life Sciences, CLS615, 3 Blackfan Circle, Boston, MA 02215, United States

ARTICLE INFO

Article history: Received 3 March 2011 Revised 19 March 2011 Accepted 21 March 2011 Available online 30 March 2011

Edited by Richard Williams, Alexander Flügel and Wilhelm Just

Keywords:

Remyelination

Oligodendrocytes

Hyaluronan

Notch1

Retinoid X receptor

ABSTRACT

Multiple sclerosis is a common demyelinating disease that worsens over the course of disease, a significant problem in clinical management. Disability in MS is significantly promoted by poor repair and remyelination of lesions. Both oligodendrocyte recruitment and maturation defects are seen as major causes of poor remyelination in MS. The mechanisms behind impaired remyelination in animal models include involvement of the Notch1, wnt, and hyaluronan/TLR2 pathways. RXR/PPAR signaling has also more recently been identified as an important regulator of remyelination. The local inflammatory milieu also appears to play critical and conflicting roles in promotion and inhibition of remyelination in MS. Understanding the forces regulating remyelination in MS represents an exciting and important initial step towards developing therapeutics targeting chronic disability in MS.

© 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Demyelination is the pathological removal of myelin sheaths that surround axons and enhance axonal function. Multiple sclerosis is characterized by repeated focal demyelination of the brain, which accounts for a large degree of disability associated with this disease. Demyelinated lesions are often poorly repaired and this likely worsens the duration and chronicity of disability in the short term. The extent and prevalence of disability also increases in most patients with longstanding MS.

Two components of MS pathology are central to disability in MS: axonal injury and impaired remyelination within lesions. Remyelination is the process of creating new myelin sheaths on axons that have been demyelinated. Remyelination restores axonal

Abbreviations: 15d-PGJ(2), 15-deoxy-a12,14-prostaglandin J2; CNP, 2',3'-cyclic nucleotide 3'-phosphodiesterase; CNS, central nervous system; cKO, conditional knockout; dpl, days post lesion; DRG, dorsal root ganglion; EAE, experimental allergic encephalomyelitis; GalC, galactocerebroside; HDAC, histone deacetylase; HA, hyaluronan; HMW, high molecular weight; kDA, kilodalton; LINGO1, Leucine rich repeat and Ig domain containing 1; LMW, low molecular weight; LPS, lipopolysaccharide; MAG, myelin associated glycoprotein; MBP, myelin basic protein; MS, multiple sclerosis; Nfasc, neurofascin; Ngr1, Nogo-66 receptor 1; NICD, Notch1 intracellular domain; OPC, oligodendrocyte progenitor cell; PLP, proteolipid protein; PPAR, peroxisome proliferator activated receptor; RXR, retinoid X receptor; siRNA, small inhibitory RNA; TLR, Toll-like receptor * Corresponding author. Fax: +1 617 735 3252.

E-mail address: jsloane@bidmc.harvard.edu (J.A. Sloane).

function by enhancing saltitory conduction and axonal conduction velocity. Remyelination may also restore trophic support to axons and prevent further axonal damage and loss.

Animal models often show robust remyelination is attainable but remyelination appears to be limited in MS. In this article, we review what is known about remyelination in MS and describe potential mechanisms behind why remyelination fails in this disease. Lastly, we discuss the interplay between the local inflammatory milieu and remyelination in MS.

2. Does remyelination occur in MS?

Many studies have demonstrated that remyelination can occur within MS lesions [1-7]. In 1965, investigators first documented incomplete remyelination within the margins of MS lesions [5]. Later, the term ''shadow plaque'' was coined to define homoge-nously remyelinated lesions observed in MS [8,9]. Shadow plaques are now commonly identified in MS, characterized by their well-demarcated borders and incomplete myelination compared to normal white matter. However, a large number of lesions exhibit no remyelination. Furthermore, extensive remyelination is observed only rarely [1-4,7], indicating remyelination is insufficient in most MS lesions.

In rather dichotomous fashion, remyelination occurs in some MS patients and not in others. In some MS patients, remyelination occurs in most lesions and up to 96% total lesion area. In others,

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remyelination is restricted to the margins of a few lesions and is largely absent [3,10]. Thus, remyelination is possible in some cases of MS and is blocked in a significant proportion of others.

Based on study of MS pathology, possible explanations for differences in remyelination potential include disease heterogeneity or differences in disease chronicity. Lucchinetti et al. identified four different subtypes of MS lesions that occurred in mutually exclusive fashion [11]. In type III and IV lesions, oligodendrocytes were relatively depleted and no shadow plaques were observed. In contrast, inflammation was more prominent in type I and II lesions and was associated with less oligodendrocyte destruction. A clear association between oligodendrocyte loss and lack of shadow plaques was observed, but more work is needed to strengthen the correlation.

However, it is clear that differences in remyelination potential are seen when comparing MS cases early and late in the course of disease. More remyelination in early stages of MS is observed by many groups [1,5,6], although not all reports agree [3]. Remye-lination may even begin within a month or two after active demy-elination [6]. In contrast, remyelination in late stage MS appears sparse and restricted to borders of inactive lesions [1,3,5].

Remyelinated areas frequently contained lipid-laden macrophages or microglia, which are in contact with thinly remyelinated fibers [6,9], suggestive of active cycling between demyelination and remyelination. In addition, recurrent demyelination within shadow plaques has been documented, and recurrent bouts of demyelination could lead to permanent demyelination [4,12].

Oligodendrocytes potentially can survive a bout of demyelination and may contribute to subsequent remyelination attempts [4,6,13-15]. However, oligodendrocyte depletion over time after repeated bouts of demyelination may make remyelination impossible. Consistent with this hypothesis, MS lesion cores late in the course of disease often contain only a few OPCs [13,14,16-18] but exhibit remyelination at the lesion edge, suggesting forces regulating remyelination are insufficient to reach the lesion core. Thus, at least in late stage MS, failure of oligodendrocyte recruitment is a significant reason for poor remyelination of lesions. It is less clear what prevents remyelination in early MS lesions, but could relate to characteristics of local inflammatory processes or intrinsic dysfunction of oligodendrocytes.

3. Does oligodendrocyte dysfunction prevent remyelination in MS?

For remyelination to occur, oligodendrocyte progenitor cells (OPCs) must survive, proliferate, and migrate to lesions. Once within MS lesions, OPCs must differentiate to pre-oligodendrocytes, premyelinating oligodendrocytes, and then mature myelinating oligodendrocytes to then regenerate myelin sheaths. In theory, remyelination can be blocked at any point in the remyelination process: oligodendrocyte survival, proliferation, migration, maturation, and/or myelin sheath formation. In late stage MS, remyeli-nation appears limited by oligodendrocyte density, which could be a product of impaired survival, proliferation, and/or migration of oligodendrocytes.

Alteration in oligodendrocyte maturation can be studied in MS by measuring densities of OPCs, pre-oligodendrocytes, premyeli-nating oligodendrocytes, and mature myelinating oligodendro-cytes within the MS brain. While low oligodendrocyte densities can be observed in early MS lesions, multiple studies clearly indicate that numerous OPCs and premyelinating oligodendrocytes are commonly found within MS lesions. Oligodendrocyte progenitor cells are numerous within MS lesions, at about half the density of normal appearing white matter [18,19]. In fact, periplaque white matter and shadow plaques showed a higher density of Olig2+ cells compared to controls. O4+ GalC- pre-oligodendrocytes are also plentiful in most lesions in chronic MS [17].

Interestingly, there was a significant positive correlation between the densities of pre-oligodendrocytes and macrophages in MS cord lesions [15,20], although this is controversial [14]. This finding suggests a possible benefit of chronic inflammation to maintaining elevated oligodendrocyte densities within MS lesions. Alternatively, this finding could reflect the notion that more recent lesions contain more oligodendrocytes and that oligodendrocyte density lessens as lesions age.

Oligodendrocyte differentiation in MS lesions appears to be blocked late in the oligodendrocyte lineage since very few GalC+ mature oligodendrocytes were found within MS lesions [17]. These cells were rounded and possessed few cellular processes consistent with an immature or aberrant state [17,20]. The density of PLP+ premyelinating oligodendrocytes is also significantly reduced in MS lesions compared to normal appearing white matter [21]. In about 7% of all MS lesions, the density of premyelinating oligodendrocytes approximated the density found in the developing brain.

As mentioned above, the regional distribution of oligodendro-cytes within the MS lesion and its margins has been studied as well. While all demyelinated lesions often have reduced oligoden-drocyte densities roughly 0-50% that of normal white matter, remyelinated regions have oligodendrocyte densities approaching or exceeding normal white matter [7,13,14]. This can vary when examining MS lesions early and late in the course of disease. Interestingly, some investigators have observed significantly increased oligodendrocyte densities at the lesion edge, although this has never been adequately quantified [1-4]. Oligodendrocytes within lesion edges or in remyelinating areas do not appear to be in a pro-liferative state [17,18,22]. Thus, given an abnormal low density of oligodendrocytes within lesion cores, the increase in oligodendro-cyte density within lesion margins may indicate migration to lesion cores is impaired. This may be especially true for late stage MS lesions that show remyelination restricted to margins, a possibility our group is actively investigating.

Beyond the oligodendrocyte recruitment and maturation, mye-lination also requires contact between axons and oligodendrocytes and creation of multiple wraps of oligodendrocyte processes around the axon, culminating in the myelin sheath. Clearly, oligo-dendrocytes present in MS lesions express PLP, and variants of MBP [21,23], which are important myelin sheath proteins. Thus, these premyelinating oligodendrocytes contact axons and appear primed to myelinate the sheath. This suggests a block in further maturation beyond the premyelinating state, or in the ability of the oligodendrocyte to myelinate the axon. Axons may also lose receptivity to myelination in MS lesions [24].

Overall, there are a number of possible issues that limit remye-lination within MS lesions. These include most prominently problems with oligodendrocyte maturation and the formation of myelin sheaths. Other significant factors include impaired oligodendrocyte migration and/or proliferation in lesions. Several labs have begun working of deciphering the molecular pathology that limits remyelination in hopes of some day enhancing remyelina-tion in MS patients. Initial data indicate a number of possible culprit molecular pathways or signals play a role in blocking remyelination, including Notch1 signaling, wnt/K-catenin, hyalu-ronan, L1NGO1, and RXR among others.

4. Is remyelination normal?

Although remyelination does occur in MS, myelin sheaths formed in remyelination are structurally abnormal [1,5,9,10]. In the first observations of MS lesions by EM, thinly myelinated axons were observed within chronic MS lesions [5]. Further studies found thinly myelinated fibers primarily at the margins of chronic lesions [1]. The internodal length of these remyelinating sheaths was also found to be shorter than normal [1,6]. Nodal length also is widened

[3]. The shortened and thin myelin sheaths suggest that remyeli-nated axons show altered saltitory conduction and reduced conduction velocity compared to normal myelinated axons, which can often be observed in MS patients by physical exam or evoked potentials.

In spite of abnormal myelin sheath measurements, all remyeli-nating internodes within MS lesions appear to have normal paran-odal architecture [1,9]. In addition, short myelin sheaths within shadow plaques show proper localization of paranodal Caspr, nodal Nav channels, and juxtaparanodal Kv channels, while demyeli-nated axons had more diffuse localization of these proteins [25,26]. Furthermore, the length of Caspr, Kv channel, and nodal sodium channel aggregates in shadow plaques is identical to normal MS white matter. In addition, Nfasc186 and MOG appeared to localize appropriately in myelin sheath domains in shadow plaques [27]. Only Nfasc155 showed slightly diffuse paranodal mislocalization in remyelinating areas [27], suggesting Nfasc155 localization to paranodes is a late event in remyelination.

Whether abnormal myelin sheath ultrastructure represents a problem specific to myelination itself or a more general problem with oligodendrocyte function is unclear at present. For example, the cultured oligodendrocyte generates multiple branching processes in culture that can be short/long and branched/unbranched depending on various conditions. A short oligodendrocyte process would likely result in a thin myelin sheath since the process would be unable to wrap as many times as a longer process. This is consistent with findings of oligodendrocyte process number and number of associated internodes in MS lesions [21]. Nevertheless, it is equally possible regulatory signals and sheath components that regulate the formation of the very specialized sheath structure could restrict the sheath thickness and lengths of internodes and nodes in MS lesions. This is a poorly understood but important aspect of myelin biology that should develop more rapidly with better scientific tools.

5. What pathways regulate remyelination?

5.1. Notchl pathway

Expressed by oligodendrocytes, Notchl receptor is a well-known regulator of OPC maturation [28-30]. Notchl responds to membrane-bound ligands, Jaggedl and Delta, and inhibits OPC maturation [28,31]. Since Jaggedl can be expressed by axons, neurons, and astrocytes throughout the brain [32], increases in Jaggedl may stimulate Notchl on oligodendrocytes and block remyelination in MS.

John et al. found that TGF-ßl, an upregulated cytokine in MS, induced expression of Jaggedl in reactive astrocytes [3l]. Jaggedl was also expressed by reactive astrocytes in demyelinated but not remyelinated MS lesions. The borders of acute MS lesion borders demonstrated staining for Jaggedl as well as Notchl and the inhibitory basic helix-loop-helix protein, Hes5, suggesting the presence of active Jaggedl/Notchl signaling.

However, initial animal models examining Notchl function in remyelination contradicted in vitro blockade of OPC maturation by Notchl signaling. First, alterations in Jaggedl and Notchl expression in animal models did not match what was found for MS lesions. Lesioning of the trigeminal tract by ethidium bromide increased rather than decreased Jaggedl expression within remyelinating lesions [32]. Similarly in EAE, with remyelination, Notchl expression on oligodendrocytes was only observed in remyelinat-ing lesions [33]. Secondly, conditional deletion of Notchl in PLP+ oligodendrocytes had no effect on remyelination after cuprizone mediated demyelination, based on G ratio and percent remyelina-tion [32]. Oligodendrocyte recruitment within lesions was also

unaffected. Thus, these findings did not support a significant role of Notchl in remyelination in animal models.

However, PLP+ oligodendrocytes may be too far along in maturation to respond to Jaggedl/Notchl signaling in the above in vivo experiments. In support of this possibility, experiments using CNP-cre mice instead showed conditional deletion of Notchl in CNP+ oligodendrocytes promoted precocious oligodendrocyte maturation [34]. In addition, using Olig2-driven cre mice, Zhang et al. showed that conditional deletion of Notchl promoted an earlier pattern of oligodendrocyte maturation in development [34]. After lysolecithin injection, remyelination was more extensive in conditional Notchl deleted mice. More remyelinated sheaths were observed in these mice as well. In addition, in vitro myelination experiments utilizing OPCs and DRGs found that Notchl siRNA enhanced myelination [34]. Therefore, Notchl signaling may block oligodendrocyte maturation in earlier stages differentiation.

Recently, activation of Notchl, characterized by cleavage and generation of an intracellular fragment (NICD), was observed within oligodendrocytes within MS lesions [29]. Nuclear translocation of NICD is required for complete Notchl signaling. However, NICD forms aggregates outside nucleus and its nuclear translocation does not occur, suggesting that Notchl signaling was not completely activated.

This finding argued that canonical Notchl signaling, via ligand delta, Jaggedl, or Lag2 [35], was not operative in MS. In contrast, contactin, a non-canonical Notchl ligand [30], was upregulated in MS lesions [29]. Contactin/Notchl stimulation actually induced MAG upregulation in vitro while Jaggedl stimulation had no effect, in contrast to previous findings [3l]. Because contactin is normally expressed at the axonal paranode, unmyelinated axons receptive to myelination may upregulate contactin to signal oligodendro-cytes to mature and begin myelination.

The blockade in NICD nuclear translocation is dependent on expression of T1P30, an inhibitor of Importin К [29]. Both Lamin Bl and T1P30 were increased in MS lesions. In remyelinated shadow plaques, T1P30-cells with nuclear N1CD+ increased in shadow plaques compared to chronic inactive lesions. However, more T1P30+ cytoplasmic N1CD+ cells were observed in chronic inactive plaques, suggesting T1P30 blocked remyelination in these lesions. Consistent with this hypothesis, expression of T1P30 blocked contactin dependent expression of MAG and cellular differentiation in vitro. 1n addition, T1P30 blocked nuclear translocation of N1CD.

Overall, the role of Notchl in remyelination in MS is becoming more, not less, complicated over time. 1n animal models of MS, Notchl appears to block remyelination, although it is unproven whether Jaggedl is the responsible Notchl ligand [34]. Studies utilizing MS tissue show that Notchl signaling is blocked by T1P30 upregulation [29]. Because Notchl signaling is interrupted within chronic lesions lacking remyelination, Notchl signaling appears to promote remyelination. While in vitro experiments support this claim, animal experiments still are needed to fully prove the bidirectional function of Notchl in remyelination.

5.2. LINGO1 pathway

Leucine rich repeat and 1g domain containing l (or L1NGOl) was first shown to be expressed exclusively in brain [36], and specifically by neurons and oligodendrocytes in normal brain and activated microglia and astrocytes in MS [36-39]. 1n CNS development, L1NGOl expression is regulated by NGF/TrkA signaling [40].

Since L1NGOl expression was found to increase in spinal cord injury [36], L1NGOl was originally tested for effects on CNS repair. Myelin debris inhibits neurite outgrowth and dominant-negative L1NGOl blocks these effects [36]. L1NGOl appears to act through at least NgRl since L1NGOl binds strongly to cells expressing

human NgR1. Binding is saturable, consistent with receptor ligand binding. Pulldown experiments also show that LINGO1 directly interacts with both p75 and NgR1 and overexpression of each receptor enhances LINGO1 effects in vitro.

Not only does LINGO1 appear to have a repressive role in neu-rite outgrowth, but LINGO1 also restricts oligodendrocyte maturation and myelination as well [37,38,40]. LINGO1 blocks oligodendrocyte differentiation, MAG and MBP expression, and myelin formation in vitro in a dose dependent manner [37,40]. In contrast, siRNA directed at LINGO1 enhances oligodendrocyte process length and myelin sheet formation [37]. Repression of LINGO1 function by blocking antibodies or dominant negative-LINGO1 transfection enhances myelin sheet formation, induction of MBP expression, and myelination in vitro [37,38,41]. These effects are specific to CNS myelination as LINGO1 has no effects on Schwann cell myelination in vitro [40]. The repressive effect of LINGO1 appears to work through RhoA activation. LINGO1 antagonists block RhoA activation in oligodendrocytes in vitro [37]. Dominant negative LINGO1 also enhances fyn phosphorylation [37], which regulates RhoA function [42].

In vivo expression of LINGO1 also affects myelination as transgenic mice expressing axonal LINGO1 show reduced cord myelination [40]. Conversely, LINGO1 KO mice show increased myelination in development and oligodendrocyte cultures contain an increased percentage of mature oligodendrocytes [37]. Because neurons and oligodendrocytes cooperate in the formation of myelin in vivo, it is unclear what cell(s) are responsible for effects of LINGO1 on myelination. However, LINGO1 most likely regulates oligodendrocyte function based on in vitro studies utilizing purified oligodendrocytes.

Inhibition of LINGO1 function enhances remyelination in several models of de/remyelination. EAE in LINGO1 null mice was less severe, probably due to non-hematologic effects as T cell responses were no different between genotypes in terms of cytokine production [38]. In contrast, more remyelination was seen in LINGO1 null mice, and possibly accounted for lower clinical EAE scores. It is important to note that peak EAE scores were also significantly lower in LINGO1 null mice, indicating that loss of LINGO1 expression lessens the severity of active EAE as well as improving remyelina-tion in chronic stages. In keeping with this broad effect of LINGO1, anti-LINGO1 antibodies blocked severity of EAE at all stages of disease [38]. Histological evidence of remyelination and fractional anisotropy, an MRI measure of myelin content, was increased with LINGO1 antibody treatment. LINGO1 antibody treatment started after onset of EAE was also beneficial, suggesting LINGO1 can negatively impact chronic EAE in isolation from acute.

This work with LINGO1 was extended to other de/remyelination models. LINGO1 blocking antibodies enhanced remyelination of cerebellar slice cultures after chemical demyelination [41]. Cocul-tures of oligodendrocytes and dorsal root ganglion cells produced more myelinated segments after LINGO1 antibody treatment as well. LINGO1 blocking antibodies also accelerated myelination of neonatal cerebellar slices in vitro. Remyelination after lysophos-phatidylcholine injection to the dorsal columns is enhanced by LINGO1 blocking antibody. Both ultrastructural evidence of remye-lination as well as motor evoked potentials support remyelination was enhanced by LINGO1 blocking antibodies. In the cuprizone model, LINGO1 blocking antibodies enhanced remyelination as well as increased mature CC1+ oligodendrocyte numbers. Thus, many reports provide substantial support for the hypothesis that LINGO1 expression in MS blocks remyelination.

5.3. Hyaluronan pathway

While other glycosaminoglycans accumulate at lesion borders, hyaluronan (HA) appears to accumulate within lesion cores

[43-45]. Elevations in hyaluronan are also seen in EAE lesions [45]. At peak EAE (14 days post inoculation), HA molecular weight broadly ranged from 6150 to 1000 kDa in size. This changed to primarily 1000 kDa in size at 30 dpi. T cells and microglia produce 200-400 kDa whereas astrocytes produce 900-1000 kDa HA. To test the hypothesis that HA blocks remyelination in MS lesions, HA was coinjected with a demyelinating agent, lysolecithin. While high molecular weight (HMW) HA (900 kDa) blocked remyelina-tion based on MBP staining, LMW HA (300 kDa) had no effect. Neighboring oligodendrocytes were identified as pre-oligodendro-cytes with no staining for mature oligodendrocytes. Cultured oligo-dendrocytes also did not mature to MBP+ oligodendrocytes in the presence of HMW HA. In this paper only, LMW HA had no effect as well on maturation state.

Our group hypothesized that hyaluronan could stimulate Tolllike receptors, TLR2 and TLR4, which are known HA receptors [46-49]. In addition, TLRs block the differentiation of a variety of cell types [50]. We found that oligodendrocytes strongly expressed TLR2 [50], whereas astrocytes and microglia expressed multiple TLRs including TLR2 and TLR4 [51-54]. Known agonists of TLR2 but not of TLR4 blocked oligodendrocyte maturation, indicating some TLR2 specificity to oligodendrocyte maturation. In dose dependent manner, hyaluronan also blocked oligodendrocyte maturation in vitro. Neutralizing antibodies to TLR2 but not TLR4 or CD44, another hyaluronan receptor, blocked effects of hyaluronan on oligodendrocyte maturation. We repeated lysolecithin induced demyelination experiments with coinjections of hyaluronan and found that TLR2 null mice exhibited more rapid and efficient remyelination in vivo.

Because LMW HA but not HMW HA stimulates TLR2 and TLR4 [46-49], we hypothesized that HMW HA may be processed by hyaluronidases to smaller hyaluronan fragments that then stimulate TLR2. Hyaluronidases are expressed by oligodendrocytes and hyaluronidase activity was required for blockade of oligodendrocyte maturation by HMW HA [50], although other groups initially found otherwise [45]. Thus, remyelination blockade in MS may require hyaluronan synthesis, partial hyaluronan degradation, and stimulation of TLR2 on oligodendrocytes. Many of these steps remain to be tested, and we are currently pursuing these goals.

5.4. Wnt/fi-catenin pathway

Another novel pathway involved in both myelination and remyelination is the canonical wnt signaling pathway [55-57]. The wnt/E-catenin pathway is an important negative regulator of dorsal oligodendrocyte maturation in development [55]. Treatment of cord explants or primary mixed glial cultures with wnt conditioned media suppresses O4+ oligodendrocyte development. In addition, pharmacological inhibition of GSK3K, which is downstream of wnt signaling, blocked O4+ oligodendrocyte development without effects on OPC numbers.

The role of the wnt pathway in remyelination was uncovered by screening 1040 in situs of transcription factors in a mouse lysolecithin injection model at 5, 10 and 14 days post lesion [56]. From this initial screening, Fancy et al. found altered gene expression in positive and negative regulators of the wnt pathway. Three murine multiple sclerosis models were used to confirm wnt's role in remyelination: lysolecithin cord injections, cuprizone intoxication, and ethidium bromide injection into the cerebellar peduncles. In the lysolecithin model, TCF4, a major transcription factor involved in wnt signaling, is co-expressed in Olig2-postive cells in lesioned adult spinal cord, but not the unlesioned adult spinal cord. TCF4 was expressed by oligodendrocyte progenitor cells, but was not observed in cells containing cytoplasmic Olig1 and PLP, both markers of the mature oligodendrocyte. Confirming relevance of TCF4 to

oligodendrocyte maturation in development at least, a transgenic mouse expressing dominant negative TCF4 exhibited normal OPC development but grossly impaired oligodendrocyte maturation [57].

Mice expressing a K-catenin-TCF4- K-galactosidase reporter were then used to demonstrate wnt pathway activity during development and remyelination in the adult after injury [56]. In these reporter mice they found galactosidase activity in Olig2+ cells in the spinal cord on postnatal day 1 and in portions of the corpus callosum on postnatal day 5. In adult spinal cord, only following lysolecithin injection, was there significant upregulation of K-galactosidase activity. Thus, the wnt pathway is likely involved in developmental myelination as well as adult remyelination after injury.

Next, the authors generated mice expressing dominant active p-catenin in Olig2+ cells to determine whether K-catenin could suppress developmental myelination [56]. At postnatal days 9 (p9) and 15 (p15), these mice had significantly reduced PLP+ oligodendrocytes in spite of normal Olig2+ densities. These mice also exhibited significant ataxia, tremor as well as hypomyelination at p15 but not in adults, indicating a delay rather than a block in developmental myelination. 1n contrast, when K-catenin was conditionally knocked out of Olig1+ cells, precocious oligodendrocyte development in spinal cord was observed during embryogenesis [57].

1t remains to be seen whether the wnt pathway is involved in remyelination in MS. Wnt pathway and protein expression was observed in active MS lesions but not in chronic lesions or normal adult white matter [56]. As yet, it is unclear whether remyelination is inhibited in areas where wnt signaling is present.

Histone deacetylasese (HDACs) have also recently been identified as key regulators of oligodendrocyte maturation in development and remyelination [57-59]. Since HDAC1 negatively regulates wnt and notch signaling in the retina [60] and is required for oligodendrocyte lineage specification [61], there may be a connection between HDACs, wnt and/or notch signaling, and oligoden-drocyte maturation and remyelination. To study whether specifically HDAC1/2 regulate oligodendrocyte lineage fates and oli-godendrocyte maturation in development, Huang et al. generated HDAC 1/2 double cKO mice mediated by Olig1-Cre [57]. No oligodendrocytes or OPCs were observed in double cKO mice. In culture experiments, oligodendrocytes were observed but oligodendrocyte maturation was completely blocked without affecting motor neuron or astrocyte numbers. Although HDACs can regulate wnt and notch signaling [60], elevations in only K-catenin activity were seen in double cKO HDAC1/2 mice [57] indicating that HDAC1/2 regulate wnt signaling but have no effect on notch signaling.

5.5. RXR pathway

Using a microarray discovery approach, retinoid X receptor gamma (RXRy) expression was shown to be significantly upregu-lated in focal demyelination of rat cerebellar peduncle at 14 and 28 days post lesion (dpl), compared with 5 days [62]. The expression of RXRy increased significantly from 5 through 28 dpl in both Olig2+ cells and CC1+ cells, indicating that oligodendrocytes were important RXRy expressing cells. Furthermore, RXRy had differential subcellular localization depending on oligodendrocyte matura-tional state, as RXRy was found cytosol of Nkx2.2+ OPCs and in the nuclei of CC1+ mature oligodendrocytes. Because of this differential subcellular localization, sequestering RXRy in cytosol may inhibit oligodendrocyte differentiation. Likewise, stimulation and nuclear localization of RXRy leads to oligodendrocyte maturation in vitro. Consistent with a role for RXRy in promoting oligodendrocyte maturation, in vitro transfection of RXRy siRNA promoted a significant decrease in MBP expression. In addition, RXR antagonists blocked oligodendrocyte maturation and myelination in vitro.

1n MS lesions, subcellular localization of RXRy correlated with remyelination status. More nuclear RXRy localization was observed in remyelinating lesions compared to chronic inactive lesions. 1n addition, nuclear RXRy was seen in active lesions and periplaque white matter compared to normal control white matter, supporting RXRy activation even in relatively unaffected areas of the MS brain. 1n vivo confirmation of RXR function in remyelina-tion was shown when treatment with 9-cis retinoic acid improved remyelination after toxin-induced demyelination of the cerebellar peduncles. Furthermore, a transient decrease in CC1+ mature oligodendrocytes was also observed in RXRy knockout mice after lyso-lecithin induced demyelination, suggesting of transient delay in oligodendrocyte maturation during lesional remyelination and repair.

RXR signaling occurs via three pathways: homodimerization, li-gand-associated (permissive) heterodimerization, and non-permissive heterodimerization. Since 9-cis retinoic acid enhanced remyelination in vivo [62], RXRy likely functions through heterodi-merization with other nuclear receptors, including other retinoic acid receptors, liver X receptors, peroxisome proliferator activator receptors (PPAR), vitamin D receptors, thyroid hormone receptors, as well as nuclear receptors, Nr2f1 and Nr4a2, most of which have known roles in MS or MS animal models [62-65].

5.6. Other factors

There are a number of relatively new factors that may influence remyelination in MS. NG2, a chondroitin proteoglycan expressed by oligodendrocytes, is upregulated in lysolecithin lesions in the spinal cord [66]. A substrate coated with NG2 blocks OPCs maturation to O1+ status but not OPC process outgrowth. As NG2 is partially degraded by MMP9, pretreatment by MMP9 reduced effects of NG2 on OPC maturation in vitro [66]. Although NG2 appears to inhibit OPC maturation, it is unclear whether NG2 blocks remye-lination at present.

Myelin transcription factor (Myt1) is a zinc-finger transcription factor that regulates PLP transcription [67]. Myt1 localizes to nuclei of immature oligodendrocyte progenitors and its expression is downregulated in mature oligodendrocytes [67-69]. Dominant negative Myt1 blocks OPC maturation and slightly inhibits proliferation, indicating Myt1 is primarily required for OPC maturation [69]. When Myt1 was examined in MS tissue, Myt1 localized to nuclei of presumed oligodendrocytes in periplaque white matter and cores of acute and chronic plaques [69]. More Myt1+ cells were observed in periplaque white matter than in normal appearing white matter. 1n a murine hepatitis virus infection animal model of demyelination/remyelination, Myt1 localized to nuclei of OPCs in lesions and increases in Myt1+ OPC density correlated with remyelination timing [69]. At least in Xenopus, Myt1 functions in cooperation with bHLH transcription factor X-NGNR-1 and is negatively regulated by the Notch/Delta signal transduction pathway [70]. Thus, Myt1 may be intimately associated with the function of Olig1/2 and/or Notch1 in terms of OPC maturation.

6. The role of innate immunity in remyelination

1nflammation has an important and poorly understood role in remyelination in MS. Clearly, the primary mechanism of demyelin-ation in MS is via immune related pathways. A number of beneficial treatments for MS are based on modulation or inhibition of immune mechanisms. However, non-immune processes could dominate the chronic later phases of MS. This hypothesis is supported by the relative paucity of infiltrating leukocytes late in disease and that current MS treatments appear to have little impact on progression.

Many researchers have documented a dual role of several proinflammatory cytokines in remyelination. Deletion of pro-inflammatory cytokines, including ILlfê and TNFa result in impaired remyelination indicating that components of the inflammatory cascade in MS may create a beneficial environment for remyelination [71-75]. As discussed above, inflammatory infiltrates are often associated with increased remyelination and a reduction in inflammation seen in chronic MS may reduce remyelination potential late in disease. Thus, inflammatory processes may also enhance remye-lination and repair of MS lesions.

Many of the pathways tied to remyelination have additional roles in modulating inflammatory processes in MS and animal models. Most prominently, Toll-like receptors, including TLR2, have a prominent role in EAE induction as well as chronic phases of EAE. Initial work found that TLR4 and TLR9 expression is essential to EAE induction while TLR2 expression has no role in EAE [76]. However, further work by our group and others found that TLR2 expression worsened chronic phases of EAE [77] (data not shown). While TLR2 was hypothesized to maintain chronic inflammation in EAE by one group [77], we hypothesize that TLR2 may directly block oligodendrocyte maturation and remyelination independently of inflammatory stimulation [50]. We are currently working on resolving this particular controversy.

RXRs and PPARs, well known for their role in MS, likely play a cooperative role in regulating inflammation and remyelination. In fact, PPAR activity requires heterodimerization of RXRs and PPARs [78,79]. Stimulation by either PPAR and RXR agonist blocks inflammatory activation of microglia in vitro [80,81]. However, PPARy stimulation with 15d-PGJ(2) acts cooperatively with 9-cis retinoic acid, an RXR agonist, to inhibit microglial activation, suggesting PPARy/RXRy heterodimerization occurs within microglia blocking inflammatory activation [81]. Although not proven, RXRy may also heterodimerize with PPARs in oligodendrocytes to permit remyeli-nation as suggested by Huang et al. [62].

Because RXRs are required for PPAR function, RXRs may be required for the many known effects of PPARs in MS. First, the PPAR agonist, 15d-PGJ(2), improved clinical incomes in EAE and decreased macrophage and CD4+ T cells infiltrating spinal cord in EAE mice [63]. In addition, PPARy and PPARS agonists promote oligodendrocyte process extension and cellular maturation [82-84]. Thus, PPARs may require heterodimerization with RXRs to promote oligodendrocyte maturation and remyelination while also limiting local inflammatory processes.

Finally, many regulators of remyelination are modulated by innate immune mechanisms. TGFfêl as well as lipopolysaccharide, a TLR4 agonist, promotes upregulation of Jaggedl in astrocytes and macrophages [31,85,86]. Increased Jaggedl expression leads to Notchl activation and further autoamplification of Notch signaling [86]. In addition, TLR stimulation can upregulate wnt protein expression and pathway function [87,88]. Thus, significant interplay likely exists between remyelinating and inflammatory stimuli in MS lesions.

There is also potential for feedback from remyelination regulators on innate immune activation. RXR stimulation appears to upregulate TLR4 expression but also inhibits microglial activation [81,89]. Wnt signaling reduces TNFa production, suggestive of anti-inflammatory effects of the wnt pathway [87]. WntD, a wnt homolog in Drosophila, acts as a feedback inhibitor of Toll and the NF-kappaB homologue Dorsal [90]. Hyaluronan and other TLR agonists have potential to enhance local inflammation at least in the short term [91,92]. However, chronic LPS stimulation (i.e. LPS tolerance) converts a pro-inflammatory signal to a protective function of macrophages [92]. Therefore, understanding the dynamics of acute and chronic stimulation of microglia, astrocytes and other innate immune sensing cells is of critical importance for complete understanding of remyelination processes in MS.

7. Conclusions

In MS pathology studies, two major factors appear to contribute to impaired remyelination in MS. In MS lesions late in the course of disease, oligodendrocyte recruitment is deficient and appears to be the primary reason for poor remyelination in late stage MS. In lesions containing more oligodendrocytes, impaired oligodendrocyte maturation is a major problem with efficient remyelination of lesions. While very little work has examined why few oligodendro-cytes are found within late stage lesions, several mechanism for blockade of oligodendrocyte maturation have been described, including Notch1, wnt/fê-catenin, and hyaluronan/TLR2 signaling. Whether all, some, or none of these possible mechanisms are involved in MS is yet to be established, and this difficulty is amply demonstrated by the recent conflicting data on Notch1 and, to some extent, hyaluronan function in remyelination. Remyelination and oligodendrocyte maturation also appears to be significantly influenced by the local inflammatory milieu both in ways that promote and inhibit remyelination. Myelin formed in remyelination attempts in MS is ultrastructurally abnormal and may also limit better clinical outcomes. Over the next several years, we anticipate that a number of these controversies will be resolved and lead to clinical approaches to enhance remyelination and clinical recovery in MS.

Acknowledgments

This work was funded by the National Multiple Sclerosis Society (RG4116A2; J.A.S.) and the American Academy of Neurology (K.A.H.). The authors declare no financial or commercial conflicts of interest.

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