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Central nervous system-directed effects of FTY720 (fingolimod)
Journal of the Neurological Sciences 274 (2008) 13–17
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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j n s
Central nervous system-directed effects of FTY720 (fingolimod) Veronique E. Miron a,⁎, Anna Schubart b, Jack P. Antel a a b
Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4 Novartis Institutes of Biomedical Research, Basel, Switzerland
A R T I C L E
I N F O
Article history: Received 10 April 2008 Accepted 27 June 2008 Available online 3 August 2008 Keywords: Sphingosine-1-phosphate FTY720 (fingolimod) Multiple sclerosis Central nervous system Immunotherapy
A B S T R A C T FTY720, also known as fingolimod, is an orally administered sphingosine-1-phosphate (S1P) analogue that is under investigation as a therapy for both relapsing–remitting (RR) and progressive forms of multiple sclerosis (MS). The demonstrated beneficial effect of FTY720 on disease activity in RR–MS patients and in the animal model experimental autoimmune encephalomyelitis (EAE) is largely attributed to effects on the systemic immune system. In addition, unlike other current systemic immuno-modulators used in MS, the lipophilic nature of FTY720 allows it to cross the blood-brain barrier (BBB). Since S1P receptors are expressed on all cell types, FTY720 has the potential to exert effects directly on the BBB and on resident cells of the CNS. The latter include cells implicated in regulating immune reactivity within the CNS (astrocytes, microglia), those that are targeted by the disease process (oligodendrocytes, neurons), and those involved in repair (oligodendrocyte progenitor cells). In vitro studies document the dose-dependent effects of FTY720 on neural cell survival, differentiation, and cytoskeletal dynamics. Animal model studies, specifically EAE, indicate an overall neuroprotective effect of FTY720 mediated at least in part by its actions within the CNS. Ongoing studies will need to define the direct and indirect (via immune-modulation) effects of FTY720 on the CNS across the broad clinical spectrum of MS. © 2008 Elsevier B.V. All rights reserved.
1. Introduction FTY720 (fingolimod) is a sphingosine-1-phosphate (S1P) analogue shown to reduce the frequency of clinical relapses and magnetic resonance (MR)-defined disease activity in multiple sclerosis (MS) patients [1]. These effects have mainly been attributed to the ability of FTY720 to restrict immune cell entry into the CNS via sequestration of T- and B-lymphocytes in secondary lymph nodes [2]. However, unlike other immuno-modulatory agents currently approved as therapies for relapsing forms of MS, FTY720 readily accesses the CNS by virtue of its lipophilicity [3]. S1P receptors are expressed on all cell types found within the CNS under physiologic and pathologic conditions [4]. This combination of drug entry into the brain parenchyma and presence of its receptors within this compartment highlights the importance of evaluating its effects on the CNS both via a direct neurobiological mechanism and an indirect mechanism consequent to its immunomodulatory effects. Both the direct and indirect mechanisms could have significant impacts on the extent of tissue injury and repair that occur in MS. We will discuss ongoing in vitro and experimental modelbased studies targeted at showing how the direct neurobiological and indirect immuno-modulatory actions of FTY720 can impact tissue injury and repair. Such studies should provide guidance on how to use and monitor this agent across the broad clinical spectrum of MS. ⁎ Corresponding author. E-mail address: [email protected] (V.E. Miron). 0022-510X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2008.06.031
2. Pharmacology of S1P receptors, their endogenous ligand, and FTY720 There are 5 known S1P G protein coupled receptor isoforms that belong to the endothelial differentiation gene-related (EDG) receptor family. The endogenous ligand for the S1P receptors, S1P, is a bioactive lysosphingolipid present in the circulation in micromolar concentrations [5] and in the CNS. S1P is produced by cells of the innate immune system [6] and by endothelial cells, neurons, and astrocytes in the CNS [7–10]. S1P can act in an autocrine or paracrine manner on immune and neural cells. There are suggestions that extracellular S1P levels in the CNS are increased under conditions of ischemia, spinal cord injury, and inflammation [11–14]. Raised levels of S1P in the MS-affected CNS could modify the effects of FTY720 on neural and immune cell responses. EDG receptors are upregulated in the periphery of MS lesions [15]. FTY720 was developed by chemical modulation of the fungalderived metabolite, myriocin. FTY720 is rapidly phosphorylated to its biologically active metabolite in vivo [16–18] and can then act on S1P1, 3, 4, and 5 [16]. Whereas S1P exposure causes receptor recycling, the binding of FTY720 to these receptors results in subsequent endocytosis, degradation, and downregulation at the mRNA level [19–21]. This suggests that FTY720 can potentially sequester receptors available to bind S1P. Also, S1P and FTY720 differ in their potencies and affinities to S1P receptor isoforms, and may therefore promote distinct responses in target cells [22].
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3. Effects on FTY720 on the blood-brain barrier (BBB) The BBB is composed of endothelial cells and serves as a tightly regulated semi-permeable barrier between the blood and underlying tissue. In MS, the disruption of the BBB likely contributes to entry of activated blood-derived lymphocytes, and resulting injury to the brain parenchyma [23]. Extensive studies have been conducted on the effects of S1P and FTY720 on an array of endothelial cells. Humanderived endothelial cells express high levels of S1P1 and S1P3 [24], and can also produce S1P themselves. Exposure of human pulmonary endothelial cells to S1P reduces permeability via S1P1- and Rac1 GTPase-mediated actin cytoskeleton rearrangement [25]. S1P3 activation, however, has been associated with Rho signaling and barrier disruption [25]. S1P treatment of endothelial cells recruits S1P receptors, Rac1-associated regulatory proteins, and actin cytoskeleton modulators to lipid raft-like domains [8]. The recruitment of both S1P1 and S1P3 to these domains may reflect negative regulation between receptors to balance barrier properties [8]. In addition, human umbilical vein endothelial cells respond to FTY720 by increased calcium mobilization and cell migration [26]. Human brain endothelial cells are one of the cell types in the CNS that are able to phosphorylate FTY720 to its activated form. FTY720 promotes adherens junction assembly and decreased permeability of the BBB via S1P1 [27,28]. The comparable effects of S1P and FTY720 treatment of mice on BBB permeability has led to suggestions that the S1P receptors are not internalized by FTY720 [27], and that these cells could continuously respond to chronic FTY720 treatment and contribute to clinical outcome. These studies suggest that restricting activated lymphocyte entry into the CNS may contribute to the therapeutic effect of FTY720 in MS patients. 4. Effects on FTY720 within the CNS 4.1. In vivo studies Orally administered FTY720 readily accesses the CNS [3] where neural cells have endogenous sphingosine kinase activity to contribute to generating the active form of the drug [17,29]. Levels of both the parent drug and active metabolite are found in higher concentrations in the brain parenchyma versus the blood in treated animals. While blood levels reach a plateau by 7 days of treatment, brain concentrations of FTY720 further increase between 7 and 13 days of treatment. After this, the levels appear to stay constant (until 23 days of treatment) [30]. FTY720 in the brain co-localizes with myelin, suggesting that a majority of this lipophilic molecule is integrated into the myelin membranes [30]. Levels in the CSF are in the subnanomolar range, which is, however, sufficient to act on CNS cells in vitro [31]. In mice, S1P5 is primarily expressed in CNS white matter [32,33]. S1P5 receptor knockouts have no myelin pathology likely due to redundancy in receptor-associated signaling pathways [33]. In contrast, there is a defect in the CNS development in mice deficient in S1P1 receptors [34]. These receptors are ubiquitously expressed and play a major role in vascular development, which might be causative for the effects on neurogenesis. No pathological changes were observed in the CNS of mice in which S1P1 was specifically deleted in neural cells (Nes-Cre; Boisclair, personal communications). 4.2. In vitro studies Such studies permit analysis of the direct effects of FTY720 on neural cells, however contributions of the CNS microenvironment are not considered (including actions of the endogenous ligand S1P). The optimal concentration of FTY720 as an immuno-modulatory therapeutic agent in MS remains under clinical trial study. The concentrations of FTY720 used in in vitro studies (10 nM–10 μM)
will need to be considered in context of concentrations of FTY720 in the CNS of patients. 4.2.1. Astrocytes Astrocytes are glial cells that have supportive, metabolic, and homeostatic functions in the CNS. Upon injury, astrocytes adopt a hypertrophic phenotype, invade the lesion, and proliferate to create the glial scar. This is thought to create a barrier between damaged and healthy cells, but is also hypothesized to impair repair processes in MS lesions [35]. S1P treatment of astrocytes in vitro stimulates their proliferation and activation of the extracellular signal regulated kinases (ERK) 1/2, implicated in cell survival signaling [36]. S1P treatment of astrocytes and other glial cells also stimulates their production of growth factors [11,37] that can affect the proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) and support survival of neural cells. FTY720 can also induce ERK1/2 activation in cultured astrocytes and promotes their migration via S1P1 activation [31,38]. Others have suggested that S1P receptor activation on astrocytes may impact their role in BBB regulation [30,39]. These findings have implications for glial scar formation and repair processes in FTY720treated MS patients. 4.2.2. Microglia As the resident macrophages of the CNS, microglia have the capacity to phagocytose dying cells and debris. In addition, they have a major role in regulating the innate and adaptive immune responses in the CNS. Microglial reactivity in the MS brain has the potential to contribute both to tissue injury (e.g. by cytokine profiles) and repair (e.g. via growth factor production) [40,41]. In vitro studies using rat CNS-derived microglia have demonstrated that their S1P receptor levels are modulated in response to activation, and S1P treatment alters cytokine production [42]. This likely reflects a mechanism whereby microglia modulate their responses based on environmental signals (i.e. increased S1P levels following trauma/inflammation), but also highlights the importance of S1P signaling in this regulation. FTY720 is able to inhibit macrophage infiltration into inflammatory lesions [43]; whether this impacts microglial activation, migration, and phagocytic properties in MS brain remains to be determined. 4.2.3. Mature oligodendrocytes (OLGs) The mature OLGs in the adult human CNS are post-mitotic cells that are involved in maintenance and regular turnover of the myelin membrane. Initial studies studying S1P receptor activity in mature rat OLGs were conducted by differentiating OPCs in vitro. These demonstrate that S1P treatment promotes their survival in deathpromoting environments via S1P5 activation of Akt, but does not have any notable effects on cytoskeletal dynamics [33,44]. Similar conclusions were reached with exposure of these cells to FTY720 [44,45]. Adult human CNS-derived OLGs respond to FTY720 by modulating their process outgrowth in a dose- and treatment duration-dependent manner [46]. These mature OLGs express high levels of S1P5 and relatively lower levels of S1P3 and S1P5 [46], contrasting with OPCs that predominantly express the receptors in relative abundance of S1P1, S1P5 and S1P3 [47]. 4.2.4. Oligodendrocyte progenitor cells (OPCs) Remyelination has been documented to occur in MS lesions [48,49] and in animal models of immune-(EAE) [50] and toxin-induced demyelination [51]. Remyelination requires OPC proliferation and migration into demyelinated lesion sites, and subsequent differentiation into mature myelinating phenotypes [52]. 4.2.4.1. Survival. Overnight FTY720 treatment (1 μM) of rodentderived OPCs rescues these cells from death induced by growth
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factor withdrawal, treatment with cytokines, and exposure to activated microglia conditioned media via ERK1/2 and Akt signaling [44]. In addition, synergistic effects on survival are observed when rat OPCs are co-treated with the growth factor neurotrophic factor 3 (NT-3). However, this rescue effect with FTY720 (1 μM) is abrogated with more prolonged growth factor deprivation (2 days) [45]. Human CNS-derived OPCs are rescued from prolonged (2 day)growth factor withdrawal-induced apoptosis via S1P1-dependent ERK1/2 activation by FTY720 [47]. This is in contrast with the S1P5dependent rescue of rodent OLG from death-promoting environments with S1P treatment [33], and may represent a species-specific survival response, differences between S1P and FTY720 receptor binding, or a developmental switch in S1P-receptor mediated cell survival. 4.2.4.2. Proliferation. Although S1P treatment of neural progenitor cells induces their proliferation [53], FTY720 does not have any effect on mitotic properties of OPCs [44,45,47]. However, FTY720 can synergistically increase platelet-derived growth factor (PDGF)-dependent cell cycle progression of rat OPCs [45]. PDGF can also modulate S1P transcript levels in OPCs [45], thereby potentially modulating responses to FTY720 and S1P. 4.2.4.3. Migration. Treatment with either S1P or FTY720 can hamper OPC spontaneous migration via S1P5 activation [54]. Nevertheless, FTY720 does not have any effect on directed migration towards PDGF [45]. Whether OPC migration in response to demyelination in MS is predominantly spontaneous or directed is unknown. 4.2.4.4. Process dynamics. S1P induces transient process retraction in rodent pre-OLGs in vitro via S1P5-dependent activation of Rho GTPase and the microtubule-associated collapsing response mediator protein 2 (CRMP2) [33]. This contrasts with the aforementioned S1P5driven effects on mature OLGs, suggesting that with rodent oligodendroglial maturation, there is either a switch in the G proteinassociated pathways downstream of S1P5, or changes in G protein coupling efficiency due to alternative splicing of receptor transcripts [55]. The former hypothesis is supported by S1P5 having the potential to be associated with both Gαi and G12 [55]. Whereas a high dose of FTY720 does not modulate rat OPC cytoskeleton, simultaneous exposure to FTY720 and NT-3 for less than 24 h causes an increase in branching of processes [44]. Conversely, FTY720 treatment alone of human OPCs for 1 day induces an initial process retraction, reversed by uncoupling S1P3/5 from their G protein, and associated with RhoA GTPase cytoskeletal signaling (myosin light chain II and cofilin) [47]. Although some degree of process extension is required for remyelination, process retraction is critical in the regular course of terminal oligodendroglial differentiation [56]. Prolonged treatment with higher doses induces process extension associated with Rac1-linked cytoskeletal signaling cascades (ERK1/2), mimicked with an agonist of S1P1 [47]. This may highlight a fundamental difference between human and rodent S1P receptor signaling in OPCs, although potential technical contributions are also considered (sample age, culture conditions, etc.). 4.2.4.5. Differentiation. Low nanomolar doses of FTY720 promote rat OPC differentiation, whereas an S1P1 agonist does not enhance maturation [45]. However, higher doses of FTY720 inhibit the differentiation of these cells into mature OLGs [44,45,47]. In human OPCs, both low and high doses of FTY720 inhibit differentiation, which is rescued by blocking S1P3/5 signaling with suramin cotreatment. Again in contrast to the rodent counterparts, human OPCs demonstrate enhanced differentiation when treated with an S1P1 agonist [47]. The inhibitory effect of FTY720 on progenitor differentiation is reversed by co-treatment of rat OPCs with NT-3
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[44], or by co-treating human OPCs with the S1P3/5 pathway inhibitor, suramin [47]. These studies may reveal dose-dependent affinities to different S1P receptors with opposing effects on differentiation, and may also indicate differences between human and rodent OPC signaling in response to FTY720 treatment. These studies indicate the importance of S1P receptor signaling in oligodendroglial functions, and highlight the potential effects of FTY720 on myelin maintenance and remyelination in MS. 4.2.5. Neurons The endogenous ligand S1P is shown to have multiple effects on neurons in vitro. S1P is critical in potentiating growth factorregulated neuronal differentiation, survival, and process extension [57,58]. Prolonged S1P treatment of hippocampal neurons results in apoptosis [59]. S1P binding to S1P1 has a synergistic effect on NGFinduced neurite extension via Rac1 GTPase [58]. However, S1P can also cause neurite retraction in primary neuronal cultures and neuronal cell lines via S1P3/5- and Rho GTPase-signaling cascades [60,61]. Neurotransmitter release and modulation of neuronal excitability can be regulated by S1P receptor activity [62–64]. Interestingly, desensitization to chronic S1P exposure in these cells suggests that neurons would only respond to changes in S1P levels, for instance under inflammatory conditions. Although FTY720 is distributed to the brain, it does not seem to be primarily located in neurons suggesting that impacts on neuronal function may be limited. Accordingly, in vitro studies indicate that only supra-physiological micromolar concentrations of FTY720 can lead to apoptosis in rat cerebellar neurons [65]. 5. Effects of FTY720 in an animal model of MS (EAE) Many studies have shown that FTY720 is highly effective in different EAE models, both prophylactically and therapeutically [65–69]. In both mouse and rat models, treatment with FTY720 is associated with a pronounced reduction in inflammatory infiltrates (T cells, B cells and macrophages), demyelination and axonal loss. A reduction of macrophage recruitment into the brain is also observed by MRI-based in vivo tracking using USPIOs [67]. This is paralleled by reduced levels of transcripts of for pro-inflammatory cytokines and chemokines [69,70] and increased levels of transcripts for myelin genes [70]. Despite the clear effects on astrocyte cultures in vitro, there is no indication for glial abnormalities in the EAE models. Even a treatment initiation of animals entering a chronic disease stage of EAE results in almost complete suppression of clinical symptoms as well as a normalization of electrophysiological responses and visual/somatosensory evoked potentials in EAE-afflicted animals [69]. Also, a partial reduction in clinical disease is observed after therapeutic administration with very low doses of FTY720 that are ineffective prophylactically [30], indicative of a neuroprotective effect. 6. Conclusion Together, the above outlined studies indicate the importance of S1P receptor signaling cascades in regulating neural cell functions. Fully understanding the net effect of FTY720 on the CNS is a daunting task given the complexity of S1P receptor-associated signaling and regulation, and the ubiquitous expression of these receptors on most cell types. The impact of FTY720 on neural cells in the MS brain will depend on the relative S1P receptor levels of a given cell type, modulation of these receptors in response to the drug and other environmental cues, and putative counteracting signals from the inflammatory environment. The efficacy of FTY720 in late stages of EAE and in the absence of adverse neurologic effects in animal models and in phase II clinical trials, suggest neuroprotective rather than
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Contents lists available at ScienceDirect
Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j n s
Central nervous system-directed effects of FTY720 (fingolimod) Veronique E. Miron a,⁎, Anna Schubart b, Jack P. Antel a a b
Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4 Novartis Institutes of Biomedical Research, Basel, Switzerland
A R T I C L E
I N F O
Article history: Received 10 April 2008 Accepted 27 June 2008 Available online 3 August 2008 Keywords: Sphingosine-1-phosphate FTY720 (fingolimod) Multiple sclerosis Central nervous system Immunotherapy
A B S T R A C T FTY720, also known as fingolimod, is an orally administered sphingosine-1-phosphate (S1P) analogue that is under investigation as a therapy for both relapsing–remitting (RR) and progressive forms of multiple sclerosis (MS). The demonstrated beneficial effect of FTY720 on disease activity in RR–MS patients and in the animal model experimental autoimmune encephalomyelitis (EAE) is largely attributed to effects on the systemic immune system. In addition, unlike other current systemic immuno-modulators used in MS, the lipophilic nature of FTY720 allows it to cross the blood-brain barrier (BBB). Since S1P receptors are expressed on all cell types, FTY720 has the potential to exert effects directly on the BBB and on resident cells of the CNS. The latter include cells implicated in regulating immune reactivity within the CNS (astrocytes, microglia), those that are targeted by the disease process (oligodendrocytes, neurons), and those involved in repair (oligodendrocyte progenitor cells). In vitro studies document the dose-dependent effects of FTY720 on neural cell survival, differentiation, and cytoskeletal dynamics. Animal model studies, specifically EAE, indicate an overall neuroprotective effect of FTY720 mediated at least in part by its actions within the CNS. Ongoing studies will need to define the direct and indirect (via immune-modulation) effects of FTY720 on the CNS across the broad clinical spectrum of MS. © 2008 Elsevier B.V. All rights reserved.
1. Introduction FTY720 (fingolimod) is a sphingosine-1-phosphate (S1P) analogue shown to reduce the frequency of clinical relapses and magnetic resonance (MR)-defined disease activity in multiple sclerosis (MS) patients [1]. These effects have mainly been attributed to the ability of FTY720 to restrict immune cell entry into the CNS via sequestration of T- and B-lymphocytes in secondary lymph nodes [2]. However, unlike other immuno-modulatory agents currently approved as therapies for relapsing forms of MS, FTY720 readily accesses the CNS by virtue of its lipophilicity [3]. S1P receptors are expressed on all cell types found within the CNS under physiologic and pathologic conditions [4]. This combination of drug entry into the brain parenchyma and presence of its receptors within this compartment highlights the importance of evaluating its effects on the CNS both via a direct neurobiological mechanism and an indirect mechanism consequent to its immunomodulatory effects. Both the direct and indirect mechanisms could have significant impacts on the extent of tissue injury and repair that occur in MS. We will discuss ongoing in vitro and experimental modelbased studies targeted at showing how the direct neurobiological and indirect immuno-modulatory actions of FTY720 can impact tissue injury and repair. Such studies should provide guidance on how to use and monitor this agent across the broad clinical spectrum of MS. ⁎ Corresponding author. E-mail address: [email protected] (V.E. Miron). 0022-510X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2008.06.031
2. Pharmacology of S1P receptors, their endogenous ligand, and FTY720 There are 5 known S1P G protein coupled receptor isoforms that belong to the endothelial differentiation gene-related (EDG) receptor family. The endogenous ligand for the S1P receptors, S1P, is a bioactive lysosphingolipid present in the circulation in micromolar concentrations [5] and in the CNS. S1P is produced by cells of the innate immune system [6] and by endothelial cells, neurons, and astrocytes in the CNS [7–10]. S1P can act in an autocrine or paracrine manner on immune and neural cells. There are suggestions that extracellular S1P levels in the CNS are increased under conditions of ischemia, spinal cord injury, and inflammation [11–14]. Raised levels of S1P in the MS-affected CNS could modify the effects of FTY720 on neural and immune cell responses. EDG receptors are upregulated in the periphery of MS lesions [15]. FTY720 was developed by chemical modulation of the fungalderived metabolite, myriocin. FTY720 is rapidly phosphorylated to its biologically active metabolite in vivo [16–18] and can then act on S1P1, 3, 4, and 5 [16]. Whereas S1P exposure causes receptor recycling, the binding of FTY720 to these receptors results in subsequent endocytosis, degradation, and downregulation at the mRNA level [19–21]. This suggests that FTY720 can potentially sequester receptors available to bind S1P. Also, S1P and FTY720 differ in their potencies and affinities to S1P receptor isoforms, and may therefore promote distinct responses in target cells [22].
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3. Effects on FTY720 on the blood-brain barrier (BBB) The BBB is composed of endothelial cells and serves as a tightly regulated semi-permeable barrier between the blood and underlying tissue. In MS, the disruption of the BBB likely contributes to entry of activated blood-derived lymphocytes, and resulting injury to the brain parenchyma [23]. Extensive studies have been conducted on the effects of S1P and FTY720 on an array of endothelial cells. Humanderived endothelial cells express high levels of S1P1 and S1P3 [24], and can also produce S1P themselves. Exposure of human pulmonary endothelial cells to S1P reduces permeability via S1P1- and Rac1 GTPase-mediated actin cytoskeleton rearrangement [25]. S1P3 activation, however, has been associated with Rho signaling and barrier disruption [25]. S1P treatment of endothelial cells recruits S1P receptors, Rac1-associated regulatory proteins, and actin cytoskeleton modulators to lipid raft-like domains [8]. The recruitment of both S1P1 and S1P3 to these domains may reflect negative regulation between receptors to balance barrier properties [8]. In addition, human umbilical vein endothelial cells respond to FTY720 by increased calcium mobilization and cell migration [26]. Human brain endothelial cells are one of the cell types in the CNS that are able to phosphorylate FTY720 to its activated form. FTY720 promotes adherens junction assembly and decreased permeability of the BBB via S1P1 [27,28]. The comparable effects of S1P and FTY720 treatment of mice on BBB permeability has led to suggestions that the S1P receptors are not internalized by FTY720 [27], and that these cells could continuously respond to chronic FTY720 treatment and contribute to clinical outcome. These studies suggest that restricting activated lymphocyte entry into the CNS may contribute to the therapeutic effect of FTY720 in MS patients. 4. Effects on FTY720 within the CNS 4.1. In vivo studies Orally administered FTY720 readily accesses the CNS [3] where neural cells have endogenous sphingosine kinase activity to contribute to generating the active form of the drug [17,29]. Levels of both the parent drug and active metabolite are found in higher concentrations in the brain parenchyma versus the blood in treated animals. While blood levels reach a plateau by 7 days of treatment, brain concentrations of FTY720 further increase between 7 and 13 days of treatment. After this, the levels appear to stay constant (until 23 days of treatment) [30]. FTY720 in the brain co-localizes with myelin, suggesting that a majority of this lipophilic molecule is integrated into the myelin membranes [30]. Levels in the CSF are in the subnanomolar range, which is, however, sufficient to act on CNS cells in vitro [31]. In mice, S1P5 is primarily expressed in CNS white matter [32,33]. S1P5 receptor knockouts have no myelin pathology likely due to redundancy in receptor-associated signaling pathways [33]. In contrast, there is a defect in the CNS development in mice deficient in S1P1 receptors [34]. These receptors are ubiquitously expressed and play a major role in vascular development, which might be causative for the effects on neurogenesis. No pathological changes were observed in the CNS of mice in which S1P1 was specifically deleted in neural cells (Nes-Cre; Boisclair, personal communications). 4.2. In vitro studies Such studies permit analysis of the direct effects of FTY720 on neural cells, however contributions of the CNS microenvironment are not considered (including actions of the endogenous ligand S1P). The optimal concentration of FTY720 as an immuno-modulatory therapeutic agent in MS remains under clinical trial study. The concentrations of FTY720 used in in vitro studies (10 nM–10 μM)
will need to be considered in context of concentrations of FTY720 in the CNS of patients. 4.2.1. Astrocytes Astrocytes are glial cells that have supportive, metabolic, and homeostatic functions in the CNS. Upon injury, astrocytes adopt a hypertrophic phenotype, invade the lesion, and proliferate to create the glial scar. This is thought to create a barrier between damaged and healthy cells, but is also hypothesized to impair repair processes in MS lesions [35]. S1P treatment of astrocytes in vitro stimulates their proliferation and activation of the extracellular signal regulated kinases (ERK) 1/2, implicated in cell survival signaling [36]. S1P treatment of astrocytes and other glial cells also stimulates their production of growth factors [11,37] that can affect the proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) and support survival of neural cells. FTY720 can also induce ERK1/2 activation in cultured astrocytes and promotes their migration via S1P1 activation [31,38]. Others have suggested that S1P receptor activation on astrocytes may impact their role in BBB regulation [30,39]. These findings have implications for glial scar formation and repair processes in FTY720treated MS patients. 4.2.2. Microglia As the resident macrophages of the CNS, microglia have the capacity to phagocytose dying cells and debris. In addition, they have a major role in regulating the innate and adaptive immune responses in the CNS. Microglial reactivity in the MS brain has the potential to contribute both to tissue injury (e.g. by cytokine profiles) and repair (e.g. via growth factor production) [40,41]. In vitro studies using rat CNS-derived microglia have demonstrated that their S1P receptor levels are modulated in response to activation, and S1P treatment alters cytokine production [42]. This likely reflects a mechanism whereby microglia modulate their responses based on environmental signals (i.e. increased S1P levels following trauma/inflammation), but also highlights the importance of S1P signaling in this regulation. FTY720 is able to inhibit macrophage infiltration into inflammatory lesions [43]; whether this impacts microglial activation, migration, and phagocytic properties in MS brain remains to be determined. 4.2.3. Mature oligodendrocytes (OLGs) The mature OLGs in the adult human CNS are post-mitotic cells that are involved in maintenance and regular turnover of the myelin membrane. Initial studies studying S1P receptor activity in mature rat OLGs were conducted by differentiating OPCs in vitro. These demonstrate that S1P treatment promotes their survival in deathpromoting environments via S1P5 activation of Akt, but does not have any notable effects on cytoskeletal dynamics [33,44]. Similar conclusions were reached with exposure of these cells to FTY720 [44,45]. Adult human CNS-derived OLGs respond to FTY720 by modulating their process outgrowth in a dose- and treatment duration-dependent manner [46]. These mature OLGs express high levels of S1P5 and relatively lower levels of S1P3 and S1P5 [46], contrasting with OPCs that predominantly express the receptors in relative abundance of S1P1, S1P5 and S1P3 [47]. 4.2.4. Oligodendrocyte progenitor cells (OPCs) Remyelination has been documented to occur in MS lesions [48,49] and in animal models of immune-(EAE) [50] and toxin-induced demyelination [51]. Remyelination requires OPC proliferation and migration into demyelinated lesion sites, and subsequent differentiation into mature myelinating phenotypes [52]. 4.2.4.1. Survival. Overnight FTY720 treatment (1 μM) of rodentderived OPCs rescues these cells from death induced by growth
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factor withdrawal, treatment with cytokines, and exposure to activated microglia conditioned media via ERK1/2 and Akt signaling [44]. In addition, synergistic effects on survival are observed when rat OPCs are co-treated with the growth factor neurotrophic factor 3 (NT-3). However, this rescue effect with FTY720 (1 μM) is abrogated with more prolonged growth factor deprivation (2 days) [45]. Human CNS-derived OPCs are rescued from prolonged (2 day)growth factor withdrawal-induced apoptosis via S1P1-dependent ERK1/2 activation by FTY720 [47]. This is in contrast with the S1P5dependent rescue of rodent OLG from death-promoting environments with S1P treatment [33], and may represent a species-specific survival response, differences between S1P and FTY720 receptor binding, or a developmental switch in S1P-receptor mediated cell survival. 4.2.4.2. Proliferation. Although S1P treatment of neural progenitor cells induces their proliferation [53], FTY720 does not have any effect on mitotic properties of OPCs [44,45,47]. However, FTY720 can synergistically increase platelet-derived growth factor (PDGF)-dependent cell cycle progression of rat OPCs [45]. PDGF can also modulate S1P transcript levels in OPCs [45], thereby potentially modulating responses to FTY720 and S1P. 4.2.4.3. Migration. Treatment with either S1P or FTY720 can hamper OPC spontaneous migration via S1P5 activation [54]. Nevertheless, FTY720 does not have any effect on directed migration towards PDGF [45]. Whether OPC migration in response to demyelination in MS is predominantly spontaneous or directed is unknown. 4.2.4.4. Process dynamics. S1P induces transient process retraction in rodent pre-OLGs in vitro via S1P5-dependent activation of Rho GTPase and the microtubule-associated collapsing response mediator protein 2 (CRMP2) [33]. This contrasts with the aforementioned S1P5driven effects on mature OLGs, suggesting that with rodent oligodendroglial maturation, there is either a switch in the G proteinassociated pathways downstream of S1P5, or changes in G protein coupling efficiency due to alternative splicing of receptor transcripts [55]. The former hypothesis is supported by S1P5 having the potential to be associated with both Gαi and G12 [55]. Whereas a high dose of FTY720 does not modulate rat OPC cytoskeleton, simultaneous exposure to FTY720 and NT-3 for less than 24 h causes an increase in branching of processes [44]. Conversely, FTY720 treatment alone of human OPCs for 1 day induces an initial process retraction, reversed by uncoupling S1P3/5 from their G protein, and associated with RhoA GTPase cytoskeletal signaling (myosin light chain II and cofilin) [47]. Although some degree of process extension is required for remyelination, process retraction is critical in the regular course of terminal oligodendroglial differentiation [56]. Prolonged treatment with higher doses induces process extension associated with Rac1-linked cytoskeletal signaling cascades (ERK1/2), mimicked with an agonist of S1P1 [47]. This may highlight a fundamental difference between human and rodent S1P receptor signaling in OPCs, although potential technical contributions are also considered (sample age, culture conditions, etc.). 4.2.4.5. Differentiation. Low nanomolar doses of FTY720 promote rat OPC differentiation, whereas an S1P1 agonist does not enhance maturation [45]. However, higher doses of FTY720 inhibit the differentiation of these cells into mature OLGs [44,45,47]. In human OPCs, both low and high doses of FTY720 inhibit differentiation, which is rescued by blocking S1P3/5 signaling with suramin cotreatment. Again in contrast to the rodent counterparts, human OPCs demonstrate enhanced differentiation when treated with an S1P1 agonist [47]. The inhibitory effect of FTY720 on progenitor differentiation is reversed by co-treatment of rat OPCs with NT-3
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[44], or by co-treating human OPCs with the S1P3/5 pathway inhibitor, suramin [47]. These studies may reveal dose-dependent affinities to different S1P receptors with opposing effects on differentiation, and may also indicate differences between human and rodent OPC signaling in response to FTY720 treatment. These studies indicate the importance of S1P receptor signaling in oligodendroglial functions, and highlight the potential effects of FTY720 on myelin maintenance and remyelination in MS. 4.2.5. Neurons The endogenous ligand S1P is shown to have multiple effects on neurons in vitro. S1P is critical in potentiating growth factorregulated neuronal differentiation, survival, and process extension [57,58]. Prolonged S1P treatment of hippocampal neurons results in apoptosis [59]. S1P binding to S1P1 has a synergistic effect on NGFinduced neurite extension via Rac1 GTPase [58]. However, S1P can also cause neurite retraction in primary neuronal cultures and neuronal cell lines via S1P3/5- and Rho GTPase-signaling cascades [60,61]. Neurotransmitter release and modulation of neuronal excitability can be regulated by S1P receptor activity [62–64]. Interestingly, desensitization to chronic S1P exposure in these cells suggests that neurons would only respond to changes in S1P levels, for instance under inflammatory conditions. Although FTY720 is distributed to the brain, it does not seem to be primarily located in neurons suggesting that impacts on neuronal function may be limited. Accordingly, in vitro studies indicate that only supra-physiological micromolar concentrations of FTY720 can lead to apoptosis in rat cerebellar neurons [65]. 5. Effects of FTY720 in an animal model of MS (EAE) Many studies have shown that FTY720 is highly effective in different EAE models, both prophylactically and therapeutically [65–69]. In both mouse and rat models, treatment with FTY720 is associated with a pronounced reduction in inflammatory infiltrates (T cells, B cells and macrophages), demyelination and axonal loss. A reduction of macrophage recruitment into the brain is also observed by MRI-based in vivo tracking using USPIOs [67]. This is paralleled by reduced levels of transcripts of for pro-inflammatory cytokines and chemokines [69,70] and increased levels of transcripts for myelin genes [70]. Despite the clear effects on astrocyte cultures in vitro, there is no indication for glial abnormalities in the EAE models. Even a treatment initiation of animals entering a chronic disease stage of EAE results in almost complete suppression of clinical symptoms as well as a normalization of electrophysiological responses and visual/somatosensory evoked potentials in EAE-afflicted animals [69]. Also, a partial reduction in clinical disease is observed after therapeutic administration with very low doses of FTY720 that are ineffective prophylactically [30], indicative of a neuroprotective effect. 6. Conclusion Together, the above outlined studies indicate the importance of S1P receptor signaling cascades in regulating neural cell functions. Fully understanding the net effect of FTY720 on the CNS is a daunting task given the complexity of S1P receptor-associated signaling and regulation, and the ubiquitous expression of these receptors on most cell types. The impact of FTY720 on neural cells in the MS brain will depend on the relative S1P receptor levels of a given cell type, modulation of these receptors in response to the drug and other environmental cues, and putative counteracting signals from the inflammatory environment. The efficacy of FTY720 in late stages of EAE and in the absence of adverse neurologic effects in animal models and in phase II clinical trials, suggest neuroprotective rather than
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