2.1.1. S1PR-1

During CNS inflammation, both S1PR-1 and S1PR-3 are up-regulated by astrocytes and are associated with astrocyte activation and increased production of glial acidic fibrillary protein (GFAP) [3]. The up-regulation of S1PR-1 is seen on activated GFAP-expressing astrocytes in or near active MS lesions [43–45]. During active inflammation there is also S1PR-1-dependent up-regulation of microglia (MGL) that is enhanced in the presence of S1P to further increase inflammation [46]. By contrast, in EAE produced in astrocyte S1PR-1 knockdown mice, there is an inhibition of astrocyte activation [38]. Furthermore, selective S1PR-1 blockade enhances maturation of OLGs [40]. Thus S1PR-1 blockade is a potentially important pharmacological target to reduce astrogliosis and promote remyelination in MS. Although S1PR3 up-regulation occurs in activated astrocytes, the role of S1PR-3 in producing significant cardiovascular e ffects, via its regulation of the KACh potassium channel [47–50], has diminished interest in it as a target for modulation in MS.

### 2.1.2. S1PR-2

S1PR-2 is expressed on oligodendroglial progenitor cells (OPC) and macrophages (MPG) [51,52]. In mice with EAE, S1PR-2 activation promotes breakdown of BBB, which is prevented by the use of selective endothelial cell S1PR-2 blocking agents [53], and in endothelial S1PR-2 knockout EAE mice. In these latter models there is also an inhibition of fibrinogen extravasation into the CNS, as well as the inhibition of MPG and MGL recruitment into CNS lesions [41]. To eliminate the potentially confounding e ffects of systemic inflammation in EAE, models employing in situ injection of lyolecithin-induced demyelination, have produced the same results: S1PR-2 knockout or pharmacologic S1PR-2 inhibition reduced MCP and MGL infiltration of the lesions [41]. S1PR-2 is also a negative regulator of oligodendroglial progenitor cell (OPC) maturation into OLGs. Following demyelination, the inhibition of S1PR-2 promoted maturation of OPCs, increased re-myelination and increased the number of mature myelinated axons [41]. In addition to being a receptor for S1P, S1PR-2 has a high affinity for NOGO A, which also inhibits remyelination [54], providing a second S1PR-2 pathway for the inhibition of re-myelination [55–57]. Consistent with these observations, the pharmacological inhibition of S1PR-2, or use of S1PR-2 knockout mice, results in improved EAE scores, reduced BBB leakage, less fibrinogen extravasation, reduced macrophage (MPG) and MGL recruitment, as well as increased OLG accumulation and re-myelination [53]. Although not a target of any current clinically employed S1PR modulators, S1PR-2 activation, because of its e ffects on BBB integrity, macrophage/MGL activation, and myelination, could be an important potential therapeutic target of future drug development.

### 2.1.3. S1P Neurotropism and S1PR-1/S1PR-2 Synergy

Sphingosine-1-P, by stimulating both S1PR-1 and S1PR-2, may induce gene expression encoding for the production of protective astrocyte neurotrophic factors, not achieved by S1PR-1 modulation alone [58]. Bi-receptor S1PR-1 and S1PR-2 stimulation [59] appears to be necessary to up-regulate neurotrophic mRNA expression, which only takes place in astrocytes [60,61]. To date there are no pharmacological agents of which we are aware that operate jointly or exclusively at both S1PR-1 and S1PR-2 under investigation as potential medications for MS, ye<sup>t</sup> the importance of regulating astrocyte

proliferation and astrogliosis would be reasons enough to examine agents that can synergistically modulate both receptor subsets.

### 2.1.4. S1PR-5

The S1PR-5 is mainly expressed on OLGs in white matter tracts, and on brain endothelial cells [51,62]. S1P5 receptor mRNA has been found primarily in the white matter of the CNS, but has not been detected in neurons, microglia, or astrocytes [63,64]. S1PR-5 is expressed throughout the development timeline of OLGs, from immature stages to the mature myelin-forming cell [62,65,66]. In mature OLGs, S1P5 receptors co-localize with myelin basic protein exclusively on myelinated axons and not on OLG cell bodies [64].

Although S1P5 appears to mediate S1P-induced survival of mature OLGs, it may also induce cell process retraction on immature OLGs [51,62], thus regulating a dual-signaling developmental pathway. It appears that activation of S1PR-5 may be important in the maturation and survival of OLGs by playing a role in their modulation, formation, and myelin repair [62].

As has been shown for S1PR-2, S1PR-5 influences BBB integrity and is highly expressed on human brain endothelial cells. Studies have demonstrated that S1PR-5 plays a key role in BBB maintenance and modulation of endothelial cell inflammatory state. S1PR-5 knockdown endothelial cells have reduced barrier integrity [67]. Activation of S1PR-5 reduces the expression of inflammatory cell adhesion molecules on their surface, and enhances the capacity of brain endothelial cells to prevent monocyte penetration. It has been suggested [67] that the role of S1PR-5 may include induction of specific BBB properties such as low paracellular permeability and increased expression of key brain endothelial proteins such as tight junction, ATP binding cassette and glucose transporter molecules. These results point out the potential importance of investigating pharmacological agents which have high a ffinity and selectivity to promote S1PR-5 preservation in maintaining BBB integrity and protecting OLG maturation and myelination.

#### *2.2. Direct Pharmacological Actions of the S1PR Modulators in the CNS*

Established and approved S1P agonists include fingolimod (FGM), siponimod (SPM), and ozanimod (OZM). Ponesimod (PNM) has recently been submitted for regulatory approval based upon Phase III clinical study outcomes. Because it has been available the longest, most data on modulation of S1P receptors by these agents come from studies utilizing FGM. At low doses, FGM has been shown to cause process extension of human OPCs via interaction with S1PR-1, but at high doses, causes process retraction via S1PR-3 and S1PR-5 [42]. Furthermore, short-term FGM treatment causes retraction and prevention of migration of OPCs via an S1PR-3 and S1PR-5-mediated pathway, whereas long-term exposure led to increased cell survival via an S1PR-1-driven pathway [42,51]. Fingolimod also enhances BBB integrity, probably by modulation of S1PR-5, reducing transendothelial migration of monocytes in vitro, the latter being an initial step in the formation of new MS lesions [67].
