6.1.1 Aiding Migration of Oligodendrocytes
At least 30 % of MS lesions lack OPCs, pointing towards insufficient OPC recruitment [
3,
90]. One research goal has therefore been to identify targets that will aid the migration of OPCs to the demyelinated area. PDGFα is the archetypal chemoattractant for OPCs, although it is difficult to separate this effect from its effect on OPC proliferation. OPCs express the PDGFα receptor, and repopulate the lesion site during the early remyelination process in successful remyelination in a mouse model [
91]. In a rat model with toxin-induced demyelination, addition of PDGF increased both OPC number (mitogenic) and presence in lesions though with no increases in remyelination [
92]. However, using the same model but with a different delivery system of PDGF resulted in an increased number of oligodendrocytes and enhanced remyelination [
93]. Thus, improving the number of OPCs in the correct place may help, but in animal models where remyelination is normally robust and does not appear to fail due to insufficient OPC recruitment, this may be difficult to ascertain. A link may exist between PDGF and the human monoclonal IgM (rHIgM22), which is currently in clinical trial as described above. Evidence from in vitro studies suggests that activation of the PDGF-alpha receptor by rHIgM22 is necessary for stimulation of OPC proliferation and promotion of remyelination [
89].
The semaphorins have also been identified as potential targets to promote OPC recruitment. These are a class of secreted and transmembrane proteins that were primarily described as axonal guidance molecules in the developing nervous system. Class 3 semaphorins have also been identified as chemotactic factors for oligodendroglial cells during development. Semaphorin 3A (Sema3A) and 3F (Sema3F) aid the recruitment of glial precursor cells from the brain to the developing optic nerve by their repulsive and attractive properties, respectively [
94,
95]. These molecules are down-regulated in normal brain white matter but are re-expressed after injury, including demyelination in rodents and humans [
96]. Moreover, expression of the semaphorin 3 receptors, neuropilins and plexins, is increased on OPCs after demyelination in mice, and upon Sema3A/3F overexpression around lesions, OPC recruitment to lesions was reduced or increased, respectively [
97]. In human MS post mortem tissue, those lesions expressing the chemorepellent Sema3A were more likely to contain few OPCs and not to show remyelination [
52]. Furthermore, in a model of focal toxin-induced demyelination in the mouse corpus callosum, addition of recombinant Sema3A to the lesion decreased OPC recruitment and remyelination, and rSema3F addition or use of a Sema3A knockdown mouse increased OPC recruitment and remyelination. These results give us two new therapeutic targets for promotion of remyelination.
Other chemokines that are able to influence immune cell migration have also been detected around demyelinating lesions. Omari et al. [
98,
99] found that immature and more mature OPCs expressed chemokine receptors CXCR1-3 in vitro. The respective ligands CXCL8, 1 and 10 were absent from normal CNS tissue, but were detected in reactive astrocytes bordering MS lesions, providing possible cues for OPC recruitment. Overexpression of CXCL1 led to a milder disease course, decreased neurodegeneration and more prominent remyelination in an immune-mediated mouse model of MS [
100], supporting a potential pro-migratory, pro-myelinating and neuroprotective function of CXCL1.
Thus, a new group of pro-migratory or anti-repellent molecules have been generated to be investigated further for therapies.
Around 70 % of demyelinated MS lesions contain immature oligodendroglial cells that appear to be in an arrested state, unable to fully differentiate [
3,
90]. As well as the LINGO-1 molecule, discussed above, there are now more than ten targets in this group. These have been identified either due to their importance in developmental myelination or from expression screens of demyelinated tissue. Two pathways known to be important in developmental myelination are the Notch-1 and Wnt pathways. The Notch-1 receptor ligand Jagged-1 is expressed by reactive astrocytes around injury sites and was first identified as a potential negative regulator of remyelination in a microarray screen [
101]. During development, Notch–Jagged signalling inhibits OPC differentiation; with Notch-1-null oligodendroglial cells exhibiting accelerated oligodendrocyte differentiation and myelination [
102,
103]. Notch-1 also appears to be a key regulator of remyelination as Notch-1 and its effector Hes5 are detected in OPCs in demyelinating lesions in rodent models in vivo, with Notch signalling hindering OPC maturation and myelin formation in vitro [
104]. Moreover, transforming growth factor (TGF)-ß, a cytokine upregulated in MS, was able to trigger Jagged-1 expression in reactive human astrocytes, which are known to border MS plaques lacking remyelination, while Notch-1 and the downstream effector gene Hes5 localised to immature oligodendroglial cells [
101,
105].
Similarly, the Wnt pathway negatively regulates developmental myelination. In transgenic mice overexpressing ß-catenin, a protein complex acting as an intracellular signal transducer in the Wnt pathway, there is no impairment of embryonic development of oligodendrocytes but later differentiation is affected, resulting in fewer oligodendrocytes expressing mature myelin markers (PLP) in the white matter [
106]. Consequently, in these mice, electron microscopic analysis at postnatal day 15 (P15) showed hypomyelination of axons, which reached normal wild-type levels by P50, suggesting that Wnt signalling delays rather than blocks oligodendrocyte maturation. Similarly, experimental toxin-induced demyelination in these transgenic mice also resulted in delayed oligodendrocyte differentiation during remyelination [
118]. Tcf4, an intranuclear binding partner of ß-catenin that is normally detected in developing mice but not in adult white matter, is re-expressed upon white matter demyelination in adult animals [
106,
107]. As Tcf4 locates to OPCs, which are recruited to the lesion site in experimental models, and as it is also highly expressed in MS lesions, Tcf4 may also play a role in regulating oligodendrocyte differentiation and myelin repair through active Wnt signalling.
A further molecule critical in development is sonic hedgehog (Shh), which is essential for oligodendrocyte specification. Studies in the adult brain have shown that Shh is still expressed in certain areas, for maintenance of stem cell niches, which show reduced cell proliferation in vivo if Shh signalling is blocked [
108,
109]. Viral application of Shh into the lateral ventricle of adult mice results in increased OPC proliferation, with subsequent differentiation, suggesting that overexpression of Shh modifies the stem cell niche to increase the production of precursor cells, which will ultimately differentiate along the oligodendroglial lineage [
110]. In experimental demyelinating lesions, Shh upregulation in oligodendroglial cells results in increased OPC proliferation and a Shh antagonist impairs myelin repair [
111].
To discover new pathways involved, expression screens have been used. Microarray analysis of demyelinated lesions from rat brain at different stages of remyelination identified the nuclear retinoid X receptor (RXR)-γ pathway as a positive regulator of endogenous remyelination [
112]. OPCs express this receptor, and treatment of OPCs with an RXR-γ antagonist in vitro leads to impaired OPC maturation, whereas incubation with 9-
cis-retinoic acid, a RXR agonist, stimulates differentiation, myelination and remyelination in culture. Furthermore, focal demyelinating lesions in RXR-γ knockout mice show accumulation of immature oligodendrocyte lineage cells, and treatment of rats with the RXR-γ agonist improves remyelination [
112]. Small molecule agonists of this receptor are currently being sought.
Another screen to identify molecules increased in inflammation that aid myelination has identified endothelin 2 as a stimulus for remyelination [
113]. Endothelin 2 enhances OPC differentiation, and promotes myelination and remyelination in ex vivo explant cultures. The endothelin receptor type B is expressed by rodent and human oligodendroglial cells [
114,
115] and by human oligodendroglial cells in MS brain lesions. Treatment of rats with the agonist of this receptor after a focal demyelinating lesion led to increased remyelination by enhancing differentiation of oligodendrocytes [
119].
Other molecules involved include the chemokine CXCL12 and its receptor CXCR4, which promote migration, proliferation and differentiation of neural precursors and, if inhibited, reduce remyelination [
116]. The bone morphogenic proteins (BMPs) 4, 6 and 7 are up-regulated in immune-mediated demyelinating lesions in mice [
117], and inhibition of BMP4 increases remyelination in areas of focal CNS demyelination in mice [
118]. However, this success may also be related to BMP-mediated astrogliosis. The extracellular matrix protein hyaluronan is expressed in human MS lesions and, in immune-mediated demyelinated lesions in mice, hyaluronan blocks OPC differentiation in vitro and in vivo, and may be mediated through the Toll-like receptor (TLR)-2, as this receptor is strongly expressed by oligodendrocytes and TLR2-agonists are able to inhibit OPC differentiation in vitro [
119]. Moreover, neutralising antibodies of TLR2 have been shown to prevent hyaluronan-mediated failure of OPC differentiation and TLR-null mice exhibit faster and more effective remyelination.
It is clear even from this selected list that remyelination is not regulated by one single molecule but instead through a combination of signalling pathways acting on OPCs and oligodendrocytes, but also no doubt on the other cell players, the microglia/macrophage, astrocytes and even the blood vessels. There is much to understand about this pathology and regenerative response, but at least the current targets have been identified, and can now be evaluated in further studies moving towards human clinical trials.