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Sphingosine 1-phosphate receptor modulator fingolimod (FTY720) does not promote remyelination in vivo
Molecular and Cellular Neuroscience 48 (2011) 72–81
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Molecular and Cellular Neuroscience j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m c n e
Sphingosine 1-phosphate receptor modulator fingolimod (FTY720) does not promote remyelination in vivo☆ Yinghui Hu a, Xinhua Lee a, Benxiu Ji a, Kevin Guckian a, Daniel Apicco a, R. Blake Pepinsky a, Robert H. Miller b,⁎, Sha Mi a,⁎⁎ a b
Biogen Idec Inc., Neuro-Discovery Biology, 14 Cambridge Center, Cambridge, Massachusetts 02142, United States Case Western Reserve University, School of Medicine, E721, 10900 Euclid Avenue, Cleveland, Ohio 44106, United States
a r t i c l e
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Article history: Received 9 March 2011 Revised 19 May 2011 Accepted 8 June 2011 Available online 24 June 2011 Keywords: FTY720 Remyelination Oligodendrocyte
a b s t r a c t The sphingosine 1-phosphate (S1P) receptor modulators have emerged as a new therapeutic opportunity paradigm for the treatment of immune-mediated demyelinating diseases such as multiple sclerosis (MS). The S1P analog fingolimod (FTY720) has been shown to alleviate disease burden in immune-mediated animal models of MS, and has been approved for treatment in clinical trials in patients with MS in the United States. While the immunological effects of FTY720 are well established, there is controversy in the literature regarding the contribution of FTY720 on myelin repair. Here, we directly assessed the impact of FTY720 on myelin repair in cuprizone and lysolecithin (LPC) demyelination models that have a minimal immunological component. FTY720 failed to promote remyelination in either animal model. These studies suggest that while FTY720 may be effective at modulating the immunological attack in MS, it may benefit from an add-on therapy to enhance the myelin repair required for long-term functional restoration in MS. © 2011 Elsevier Inc. All rights reserved.
Introduction Multiple sclerosis (MS) is an autoimmune demyelinating disease characterized by areas of demyelination and loss of oligodendrocytes (Compston and Coles, 2008). A major effector of this central nervous system (CNS) damage is thought to be T cell infiltration across the blood brain barrier. These T cells target CNS antigens to damage myelin, resulting in demyelinated lesions throughout the brain and spinal cord (Compston and Coles, 2008). The subsequent inability of CNS processes to remyelinate these damaged lesions results in neurodegeneration and various cognitive and physical neurological deficits. As a result, effective functional restoration in MS may depend on the ability to remyelinate and thereby regenerate healthy axons and neurons. Any treatment effect on myelin repair is likely to affect oligodendrocyte precursor cell (OPC) maturation. Myelination is known to result from directed activation of complex cellular processes, including OPC proliferation, migration, adhesion, process extension/retraction, and differentiation (Miller and Mi, 2007; Miron et al., 2008b). Furthermore, detailed functional studies of repair can be
☆ Yinghui Hu, Xinhua Lee, Benxiu Ji, Kevin Guckian, R Blake Pepinsky, and Sha Mi are employees of Biogen Idec. Biogen Idec markets two products for the treatment of multiple sclerosis, which are competitors of FTY720. ⁎ Corresponding author. Fax: +1 216 368 4650. ⁎⁎ Corresponding author. Fax: +1 617 679 3148. E-mail addresses: [email protected] (R.H. Miller), [email protected] (S. Mi). 1044-7431/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2011.06.007
accomplished through the use of a range of approaches, including the analysis of oligodendrocyte differentiation in vitro and the use of ex vivo brain slice cultures that permit cellular and molecular perturbation of the environment of actively myelinating systems. In previous chemical-induced demyelination studies, we have used these approaches to demonstrate that inhibition of leucine-rich repeat- and Ig domain-containing Nogo receptor-interacting protein 1 (LINGO-1) function promotes myelin formation that is correlated with promotion of OPC differentiation and maturation of oligodendrocytes (Mi et al., 2007; Mi et al., 2009). Sphingosine 1-phosphate (S1P) is a signaling sphingolipid that mediates a variety of cellular responses through the endothelial differentiation G-protein coupled (EDG) receptors. S1P signaling plays a critical role in immunomodulation and has recently become a focus of attention in the treatment of immune-mediated demyelinating diseases such as MS (Brinkmann et al., 2010). One modulator of this system is the S1P analog fingolimod (FTY720), a non-specific agonist for S1P receptors that possesses clear anti-inflammatory properties (Brinkmann et al., 2010). FTY720 (Gilenya) has recently been approved as an oral agent for use in relapsing forms of MS in the United States (Cohen and Rieckmann, 2007; Hemmer and Hartung, 2007). Clinical studies have shown that Gilenya treatment reduces annual relapse rate and severity of symptoms (Cohen et al., 2010; Kappos et al., 2006, 2010; O'Connor et al., 2009). The primary mechanism of FTY720 efficacy is believed to be immunological, whereby FTY720 prevents lymphocyte egress from lymphoid organs, thus inhibiting the circulation of autoaggressive T cells (Compston and Coles, 2008).
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Experimental autoimmune encephalitis (EAE) is an animal model for studying inflammatory diseases of the central nervous system and has contributed valuable insights into the effects of immunologicallyinduced demyelination at the whole organism level (Furlan et al., 2009). FTY720 treatment has been shown to ameliorate symptoms of EAE disease and some studies even report evidence of enhanced myelination (Foster et al., 2007; Kataoka et al., 2005; Papadopoulos et al., 2010). However, it is unclear whether the increased myelination results from FTY720 indirectly inhibiting demyelination by blocking T cell infiltration into the CNS or from FTY720 directly promoting OPC maturation and remyelination. There are limited in vivo studies that directly address the role of FTY720 on OPC proliferation, differentiation and remyelination, which could be critical for understanding the long term effects of FTY720 on CNS remyelination and repair. A nonimmune-based animal model, one where the primary demyelination is not mediated by immunological attack, is necessary in order to determine whether FTY720 treatment promotes remyelination. In this study, we used two such models, cuprizone and lysolecithin (LPC), to study the specific effects of FTY720 on remyelination. These models allow clearer temporal delineation of the phases of demyelination and remyelination in the absence of primary involvement of the immune system. Our studies demonstrate that FTY720 does not promote remyelination in either model.
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Results Local delivery of FTY720 induces demyelination in the LPC model The lysolecithin-induced (LPC) demyelination model was utilized to assess the effects of FTY720 on remyelination in vivo. LPC was injected into rat dorsal columns to produce demyelinated lesions after a few hours. 3 days after LPC injection, animals were dosed with FTY720, antiLINGO-1 antibody, or vehicle control treatment by direct injection into the lesion. Remyelination was examined on day 10 post-LPC injection by luxol fast blue (LFB) staining (Fig. 1A, top panels). FTY720 treatment led to a two-fold increase in the size of demyelinated lesions compared to the vehicle control treatment group, as determined by 3-dimensional measurement (Fig. 1B; control n=5, FTY720 n=7). In contrast, antiLINGO-1 antibody treatment reduced the lesion size 18-fold compared with controls (Fig. 1A, B; n=8), consistent with our previous finding that inhibiting LINGO-1 promotes remyelination (Mi et al., 2009). The FTY720-treated animals contained non-LFB staining spots in the white matter perilesional areas with moth-eaten morphology (Bradl and Lassmann, 2010) characteristic of OPC death and demyelination (Fig. 1C, D). This morphology suggests that locally delivered FTY720 may diffuse into nearby white matter tissue to induce OPC death and demyelination. To clarify the direct effect of FTY720 on demyelination, FTY720 was
Fig. 1. Local delivery of FTY720 causes demyelination in the LPC model. (A) Luxol fast blue staining (LFB) of white matter in spinal cord dorsal column lesions following local injection of FTY720, vehicle control, or anti-LINGO-1 antibody (top panels). Bottom panels show three-dimensional reconstructions of the lesion areas (red, lesion volume). Scale bar = 100 μm. (B) Quantification of the lesion volumes of (A, bottom panels). (C) LFB staining of spinal cord tissue surrounding lesions from vehicle control- and FTY720-treated rats. Arrows denote moth-eaten morphology. (D) Enlargement of right panel from (C). (E) Black gold staining of brain slice cultures to visualize demyelination by FTY720P treatment. (F) Quantification of black gold staining intensity in (E).
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Fig. 1 (continued).
locally injected into the spinal cord of normal animals without LPCinduced demyelination. Consistent with the LPC study, FTY720 injection alone produced demyelinated lesions (Supplementary Fig. 1). This result shows that the demyelinating effect of FTY720 is independent of LPCinduced demyelination, suggesting that FTY720 actively promotes demyelination when administered by local injection. Brain slice cultures provide an in vitro surrogate to in vivo animal models to study demyelination and remyelination. For these studies, phosphorylated FTY720 (FTY720P), the active form of the compound, was added to rat brain slice cultures (P17 + 3DIV). 10 nM and 100 nM FTY720P treatment led to a dosedependent reduction in myelination in the corpus callosum region compared with vehicle-treated slices (Fig. 1E, F), suggesting that FTY720P either induced demyelination or inhibited ongoing myelination of the brain slice cultures. To further assess the effect of FTY720 on remyelination, a LPC demyelination study was performed with FTY720 administered orally (1 mg/kg/day), a treatment regimen that is highly efficacious in EAE
models (Kataoka et al., 2005; Al-lzki et al., 2011). The lesion size was determined by toluidine blue staining (Fig. 2A, B). Unlike with local delivery, oral FTY720 treatment did not increase the lesion volume. Quantification of remyelinated axons in the lesion after examination by electron microscopy showed no difference in the proportion of myelinated axon fibers between FTY720-treated and control animals (Fig. 2C, D). This result suggests that FTY720 did not promote remyelination or acceleration of demyelination in these lesions. The discrepancy between the local and oral treatment results may be due to the higher concentration of FTY720 in the lesions following local administration. FTY720 does not promote remyelination in the cuprizone demyelination model The effects of FTY720 on remyelination were further investigated in the cuprizone-induced demyelination model (Fig. 3). In this model, demyelinated lesions can be detected in corpus callosum after
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4 weeks dietary ingestion of cuprizone, at which point animals were treated for 2 weeks with FTY720 (1 mg/kg oral daily) or vehicle control along with a normal cuprizone-free diet. At 6 weeks, animals were sacrificed and the remyelinating effect of FTY720 relative to control treatment was evaluated by histological methods. The cuprizone diet resulted in a 2-fold reduction in myelinated axons in corpus collosum compared to normal diet (Fig. 3C). Anti-MBP (myelin basic protein) immunohistochemistry (Fig. 3A) and EM (Fig. 3B) showed no difference in the number of myelinated axons between the FTY720- and control-treated animals, suggesting that FTY720 had no effect on remyelination. A significant effect of FTY720 on the model was identified by quantifying the number of CC1 + mature oligodendrocytes, NG2 + OPCs, GFAP + astrocytes, and CD68 + microglia/macrophages in the corpus callosum (Fig. 4). Cuprizone diet led to a 2-fold decrease in the number of CC1 + mature oligodendrocytes compared to normal diet (Fig. 4B). There was no difference in the number of CC1 + mature oligodendrocytes in the corpus callosum between the control-treated and FTY720-treated groups (Fig. 4B). However, there was a 2-fold increase in NG2 + OPCs (Fig. 4D) and a 44% increase in GFAP + astrocytes (Supplementary Fig. 2, n = 4 each). There was no difference in the number of CD68 + microglia/ macrophages in FTY720-treated mice compared with control mice (Supplementary Fig. 3). This result suggests that FTY720 promotes OPC and astrocyte proliferation in vivo while having no effect on microglial activation.
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FTY720P stimulates OPC proliferation but inhibits OPC maturation in vitro To directly evaluate the effect of FTY720 on OPC function, purified OPCs from rat P2 forebrain were cultured in the presence and absence of 10 nM FTY720P and live images were captured for 98 h (Fig. 5 and Supplementary Video 1). Over the course of the recording, vehicletreated OPCs formed extensive processes typical of differentiation (Fig. 5A–F). In contrast, OPCs treated with 10 nM FTY720P initially showed morphological changes characteristic of differentiation (Fig. 5G–I), but by 60 h the processes had retracted (Fig. 5J) and the cells started to proliferate, as evident by the increased cell number at the 60, 80, and 98 h time points (Fig. 5J–L). Supplementary Video 1 demonstrates that the two OPCs seen at time 24 h in Fig. 5I become 24 OPCs at time 98 h in Fig. 5L. The longer processes apparent at 40 h (Fig. 5I) are lost at the later time points (Fig. 5J–L). Quantification of the total number of OPCs per field at 98 h revealed about a 4-fold increase in FTY720P-treated compared to vehicle-treated cultures (Fig. 5M, n = 6). The proliferative effect of FTY720P on OPCs was confirmed in OPC cultures using BrdU labeling to monitor cell proliferation. Consistent with the live image results, 10 nM FTY720P treatment for 72 h led to less-developed processes for NG2 + cells (Fig. 6A) and a 4-fold increase in BrdU +NG2 + OPCs (Fig. 6A, B) compared with vehicletreated cultures, thus confirming the effects of FTY720P on OPC proliferation and differentiation.
Fig. 2. Toluidine blue staining of LPC-induced lesions following oral FTY720 treatment. (A) Treatment with dimethyl sulfoxide (DMSO). (B) Treatment with FTY720. Red lines denote demyelinated areas. Scale bar = 100 μm. (C) Ultrastructural visualization of lesions by EM (top panels, scale bar = 1 μm). Higher magnifications (bottom panels, scale bar = 500 nm) show the presence of naked demyelinated axons (asterisks). (D) Quantification of myelinated axons in EM images from (C). n = 30 fields/group from 3 rats.
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The reversal of OPC differentiation and induction of OPC proliferation was further studied by immunocytochemistry. A2B5 + OPCs were cultured for 3 days with or without 10 nM FTY720P in PDGF/FGF-containing media. Treatment with FTY720P resulted in dramatic morphological changes of A2B5 + OPCs, which showed a rounding and retraction of the processes (Fig. 6C), suggesting that FTY720P inhibits OPC process formation and maturation. Quantification of average process length demonstrates approximately a 4-fold reduction in FTY720P-treated compared to control-treated cultures (Fig. 6D). This effect was further confirmed by culturing OPCs in CNTF/ T3-containing differentiation media for 3 days. In the presence of 10 nM FTY720P, oligodendrocyte processes were approximately 2fold shorter compared with the vehicle-treated cultures (Fig. 6E, F). The total numbers of MBP + cells between the 10 nM FTY720P and control groups are similar. OPC cell death occurred when the concentration of FTY720P was increased to 1 μM (data not shown), consistent with the cytotoxic effect at high concentrations observed in vivo when LPC was delivered by local injection. Discussion We have demonstrated that FTY720 treatment has no direct effect on remyelination in two chemical-induced animal models of demyelination, LPC and cuprizone. Unlike the EAE model, the clear delineation of the phases of demyelination and remyelination in the LPC and cuprizone models allows for the effects of FTY720 on specific
processes such as remyelination to be examined in the absence of primary involvement of the immune system. xIn the LPC demyelination model, local injection of FTY720 enhanced demyelinated lesions and moth-eaten morphology suggesting oligodendrocyte death. This is consistent with our ex vivo brain slice cultures where FTY720P treatment decreased myelin intensity by 20% at 10 nM and by 50% at 100 nM. Since myelination is an ongoing process in P17 brain slice cultures, the observed decrease in myelination could be due to either FTY720P inhibiting ongoing myelination or inducing demyelination. Miron et al. have reported that FTY720P enhanced remyelination in LPCinduced demyelinated P0 cerebellum slice cultures (Miron et al., 2010); however, their study design differed significantly from ours. When FTY720 was administered orally in the LPC model, we did not see enhanced demyelinated lesions nor moth-eaten morphology after a 7 day treatment, suggesting that FTY720 only induces oligodendrocyte death at higher concentrations, such as by local injection. In the cuprizone demyelination model, no effect on remyelination was observed following 2 week oral FTY720 treatment despite a 44% increase in astrocyte number, consistent with a recent study by Kim et al. (Kim et al., 2011). Our data also demonstrate a 2-fold increase in NG2 + OPCs in the corpus callosum, suggesting that FTY720 promotes OPC proliferation. The effects of FTY720 on oligodendrocyte biology were further examined in vitro by OPC culture in order to comprehensively understand the specific effects of FTY720 on OPC maturation and remyelination. 10 nM FTY720P treatment first inhibited OPC
Fig. 3. Effects of FTY720 treatment in the cuprizone demyelination model. (A) Anti-MBP IHC to visualize demyelination in the corpus callosum. Normal (left, cuprizone-free diet) and cuprizone-fed vehicle control-treated (middle) and FTY720-treated (right) corpus collosum. Scale bar = 100 μm. (B) Detection of myelinated axons by EM in the corpus callosum. Scale bar = 2 μm. (C) Quantification of myelinated axons from the EM images in (B).
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Fig. 4. CC1 IHC staining showing the effects of FTY720 on oligodendrocyte number in the cuprizone model. (A) CC1 IHC staining of mature oligodendrocytes in corpus callosum. Scale bar = 25 μm. (B) Quantification of the number of CC1+ cells in (A). (C) NG2 IHC staining of OPCs in corpus callosum. Scale bar = 25 μm. (D) Quantification of the number of NG2+ cells in (C).
maturation as evident by the retraction of oligodendrocyte processes and then promoted cell proliferation as evident by the increase in cell number. Published reports have shown that FTY720 treatment inhibits oligodendrocyte differentiation by promoting process retraction in human (Miron et al., 2008a) and rat (Jung et al., 2007) OPCs. Miron et al. found that prolonged FTY720 treatment reversed the initial retraction of OPC processes by 48 h (Miron et al., 2008a, 2008b). In contrast, our time-lapse images show continued process retraction and cell proliferation through 98 h of treatment, suggesting that FTY720 inhibits OPC maturation both initially and long-term. Cell
proliferation following FTY720 treatment has also been reported in other cell types, including astrocytes (Pébay et al., 2001) and neural progenitor cells (Harada et al., 2004). The mechanism driving the retraction of OPC processes and subsequent cell proliferation may be a Rho kinase/collapsin response-mediated protein signaling pathway activated by FTY720 through the S1P5 receptor-signaling pathway, as reported previously (Jaillard et al., 2005; Miron et al., 2008a). Although it is well established that FTY720 treatment alleviates the immunological attack in MS, there are a number of reports highlighting its failure to promote neuroregenerative repair in non-
78 Y. Hu et al. / Molecular and Cellular Neuroscience 48 (2011) 72–81 Fig. 5. FTY720P treatment promotes OPC proliferation in OPC cultures. Treatment effects of FTY720P on OPC function were evaluated by time lapse images at the indicated time points for vehicle control-treated (A–F) and FTY720P-treated (G– L) OPCs. Scale bar = 10 μm. (M) Quantification of total numbers of OPCs at 98 h.
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immune-mediated models. Most recently, Rau et al. reported that FTY720 exerted significant anti-inflammatory effects during optic neuritis, but did not prevent apoptosis of retinal ganglion cells or improve visual function (Rau et al., 2011). In another study, long term FTY720 treatment (1 mg/kg, P.O. daily for 2 months) did not improve axon remyelination or prevent progressive neurodegeneration in the secondary progressive EAE model (Al-lzki et al., 2011) despite the claims by Choi et al. that FTY720 treatment decreased gliosis and protected neurons via down-regulation of S1P1 in astrocytes (Choi et al., 2011). In clinical trials, FTY720 was shown to have a strong effect on relapse rate reduction (60% versus placebo), but only a modest effect on disability progression (31% versus placebo) (Kappos et al., 2010). Our data, which shows no direct effect of FTY720 on remyelination, offers an explanation for this discrepancy since
Fig. 6 (continued).
slowing of disability progression may depend on the ability to promote myelin repair. The absence of remyelination in nonimmune-mediated models of demyelination suggests that FTY720 predominantly functions as an immune regulator, as previously reported in EAE studies and clinical trials (Cohen and Rieckmann, 2007; Foster et al., 2007; Hemmer and Hartung, 2007; Kataoka et al., 2005). In summary, through a comprehensive effort to understand the effects of FTY720 on oligodendrocyte proliferation, maturation, and myelination, we demonstrate that FTY720 does not promote remyelination following oral treatment in the two non-immunemediated demyelination models and induces oligodendrocyte death at higher concentrations, produced by local delivery in the LPC model and by direct addition of FTY720P to culture medium in brain slice cultures. While effective at modulating immunological attack in MS, our studies highlight the need for new and/or add-on therapies to promote the myelin repair necessary for long-term functional recovery of the CNS.
Experimental methods Formulation of FTY720
Fig. 6. IHC staining of cultured OPCs. (A) A2B5+ OPC proliferation visualized by antiBrdU (green), anti-NG2 (red), and co-labeling (yellow). (B) Quantification of BrdU+ NG2+ cells from (A). (C) A2B5+ OPCs treated with vehicle control or FTY720P in the presence of FGF/PDGF. Scale bar = 50 μm. (D) Quantification of the length of OPC processes in (C). (E) MBP+ OPCs treated with control or FTY720P in the presence of CNTF/T3. Scale bar = 50 μm. (F) Quantification of the length of OPC processes in (E).
FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol) was purchased from Cayman Chemical Corporation as the HCl salt (Catalog # 10006292 LOT 166442) and its identity was confirmed by NMR and LC/MS. (S)-FTY720-P ((2S)-amino-2-[2-(4-octylphenyl) ethyl]-1-(dihydrogen phosphate)-1,3-propanediol) was purchased from Cayman Chemical Corporation (Catalog # 10006407, LOT 191341) and its identity was confirmed by NMR, LC/MS and optical rotation. (S)-FTY720-P was further characterized by measurement in a functional cell assay (Millipore Corporation) and potencies were consistent with published values for (S)-FTY720-P at S1P1-S1P5. Working solutions of FTY720 were prepared at 2 mg/ml in water solution for local injections. FTY720 was used for all in vivo studies and its phosphorylated form, FTY720P, was used in all in vitro culture studies, except where noted otherwise. All procedures for animal use were performed in accordance with the US National Institute of
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Health guidelines and were approved by the Biogen Idec Institutional Committee for Animal Care. Oligodendrocyte culture Enriched populations of primary oligodendrocytes were grown from postnatal day 2 Sprague Dawley rats. The forebrain was dissected and placed in Hank's buffered saline solution (HBSS; Invitrogen, Grand Island, NY). The tissue was cut into 1 mm fragments and incubated at 37 °C for 15 min in 0.01% trypsin and 10 μg/mL deoxyribonuclease (DNase). Dissociated cells were plated on poly-Llysine-coated T75 tissue culture flasks and grown at 37 °C for 10 days in Dulbecco's Modified Eagle's Medium (DMEM) with 20% fetal calf serum (Invitrogen, Carlsbad, CA). OPCs were collected by shaking the flask overnight at 200 rpm and 37 °C, resulting in a more than 95% pure population of A2B5 + cells, as determined by anti-A2B5 immunocytochemistry co-stained with DAPI. To monitor OPC proliferation, OPCs were labeled with bromo-deoxyuridine (BrdU, 1:1000 dilution, GE Healthcare, Piscataway, NJ) and incubated overnight at 37 °C. After the incubation, cells were fixed with 4% paraformaldehyde for 30 min, and then treated with 4N HCl for 10 min. After three washes, cells were stained with anti-NG2 and anti-BrdU antibodies as described in the antibodies and immunocytochemistry methods. Live images and time-lapse analysis OPCs in high-glucose DMEM containing FGF/PDGF, were subjected to time-lapse recording for 98 h in the presence of 10 nM FTY720P or vehicle control. Cells were cultured in a 24-well plate mounted onto a motorized stage (Zeiss, Thornwood, NY) housed inside a sealed chamber and maintained at 37 °C in humidified air with 5% CO2. Wide-field microscopic images were acquired through the transmitted light channel on a Zeiss Axiovert microscope equipped with a digital camera and Axiovision software. Antibodies and immunocytochemistry Anti-A2B5 antibodies were obtained from Chemicon (Rosemont, IL cat#mAB312R), anti-MBP antibodies from Covance (Princeton, NJ cat#SM194 and cat#SM199), anti-NG2 antibodies from Chemicon (Rosemont, IL cat#AB5320), anti-CC1 antibodies from Calbiochem (Spring Valley, CA cat#OP80), and anti-BrdU monoclonal antibodies from Invitrogen (Carlsbad, CA A-21303). To visualize antibody labeling in tissue sections or cell cultures grown on chamber slides, samples were fixed in 4% paraformaldehyde, washed, and incubated with the indicated antibodies using standard protocols. Tissue sections were incubated in primary antibodies overnight at 4 °C, washed thoroughly, incubated with the appropriate Alexa-labeled secondary antibody (Invitrogen, Carlsbad, CA) for 2 h, then mounted in ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA) and visualized by fluorescence microscopy. Cultures were incubated for 2 h in primary and 1 h in Alexa-labeled secondary antibodies, and visualized by fluorescence microscopy. For quantitative analysis immunoreactive cells were counted at 20× magnification with tissue areas measured by openlab software. Rat forebrain slice cultures Postnatal, day 17 Sprague Dawley rats were purchased from Charles River (Wilmington, MA) and the brains were surgically removed. Four consecutive 300 μm slices were taken from the junction of the corpus callosum to the hippocampus in each. Slices were cultured in basal DMEM supplemented with 25% horse serum for 3 days before any treatment. For demyelination studies, cultured slices were treated with 10 nM or 100 nM FTY720P for 3 days followed by black gold staining to visualize myelination (Millipore,
Bedford, MA). Images were acquired using a Leica M420 microscope (Bannockburn, IL) and the staining intensity of the corpus callosum was analyzed using Metamorph software from Molecular Devices (Downingtown, PA). 4 slices for each treatment group were analyzed and two independent experiments were conducted to generate data (8 total slices per group). Histology and microscopy For immunohistochemistry studies, animals were terminally anesthetized and transcardiac perfusion was performed using phosphate buffered saline (PBS) followed by fixation with 4% paraformaldehyde. Tissue was removed, post-fixed overnight, and then cryoprotected in 20% sucrose in 0.1 M PBS for 48 h. Tissue was embedded in Optimal Cutting Temperature (O.C.T.) compound and cryo-sectioned. For luxol fast blue (LFB) staining, 100 μm transverse sections from LPC-treated rat spinal cord were stained with 0.5% LFB, cleared, dehydrated, and mounted. Lesion volumes were determined using three-dimensional reconstruction from serial sections. For toluidine blue staining and electron microscopy (EM), animals were transcardiac perfused with 4% paraformaldehyde plus 2.5% glutaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Spinal cords or brains were dissected and immersed in 2% osmium tetroxide, dehydrated through a graded ethanol series, and Epon™-embedded following procedures described previously (Mi et al., 2007). Semi-thin sections of 1 μm thickness were stained with toluidine blue and images were captured by light microscopy. The samples were then processed by EM analysis as described previously (Mi et al., 2007). Lysolecithin demyelination model Nine-week-old Sprague–Dawley rats received a 1.5 μL direct injection of 1% LPC into the spinal cord dorsal column, followed 2 days later by oral treatment with 1 mg/kg FTY720 or vehicle control daily for 7 days (toluidine blue and electron microscopy studies). Oral dose of 1 mg/kg FTY720 was selected based on the published literature (Kataoka et al., 2005; Kim et al., 2011; Al-lzki et al., 2011). In a separate experiment, nine-week-old Sprague–Dawley rats received local 2 μL injection of 2 mg/mL FTY720, vehicle control or 0.75 mg/mL anti-LINGO-1 antibody 3 days after LPC injection. Animals were sacrificed on day 9 (oral treatment) or day 10 (local delivery) after LPC injection. To determine the direct effect on demyelination, FTY720P or vehicle (2 μL of 2 mg/mL) were injected into the normal rat spinal cord dorsal column (without LPC-induced demyelination). The animals were sacrificed 3 days later for histology study. All sections were stained with LFB for examination of lesion size. Myelinated axons in lesion areas were quantified from EM images (10 fields were counted for each animal, three rats per group were analyzed). Cuprizone demyelination model Nine-week-old C57/B6 mice were fed 0.3% cuprizone diet (chow pellets, Harlan) for 4 weeks along with intraperitoneal injection of 10 mg/kg rapamycin for 5 days per week (Narayanan et al., 2009). Animals were then treated with 1 mg/kg FTY720 or vehicle control by oral gavage for an additional 2 weeks with normal diet. The plasma concentration of FTY720 was measured 24 h after last dose to confirm accurate dosing. At the end of the 6 week cuprizone study (4 weeks cuprizone diet plus 2 weeks FTY720 treatment with normal diet), animals were perfused and brains were dissected for immunohistochemistry and EM analysis. Coronal slices (septostriatal section, 1 mm thick) through the corpus callosum were cut in midsagittal plane and embedded in epon, oriented to visualize the entire cross-section of the midsagittal corpus callosum. The myelinated axons were compared by
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electron microscopy from at least 30 different non-overlapping fields from 3 mice in each group. Statistical methods GraphPad Prism software was used for statistical analysis. Data are shown as mean and standard error of the mean. In all studies, comparison of mean values was conducted using unpaired student's t-tests or one-way ANOVA analysis of variance with Tukey correction. In all analyses, statistical significance was determined at the 5% level (p b 0.05). Supplementary materials related to this article can be found online at doi:10.1016/j.mcn.2011.06.007. References Al-lzki, S., Pryce, G., Jackson, S.J., Giovannoni, G., Baker, D., 2011. Immunosuppression with FTY720 is insufficient to prevent secondary progressive neurodegeneration in experimental autoimmue encephalomyelitis. Mult. Scle. J. 1–10. Bradl, M., Lassmann, H., 2010. Oligodendrocytes: biology and pathology. Acta Neuropathol. 119, 37–53. Brinkmann, V., Billich, A., Baumruker, T., Heining, P., Schmouder, R., Francis, G., Aradhye, S., Burtin, P., 2010. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discov. 9, 883–897. Choi, J.W., Gardell, S.E., Herr, D.R., Rivera, R., Lee, C.W., Noguchi, K., Teo, S.T., Yung, Y.C., Lu, M., Kennedy, G., Chun, J., 2011. FTY720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1P1) modulation. Proc. Natl. Acad. Sci. U. S. A. 108, 751–756. Cohen, B.A., Rieckmann, P., 2007. Emerging oral therapies for multiple sclerosis. Int. J. Clin. Pract. 61, 1922–1930. Cohen, J.A., Barkhof, F., Comi, G., Hartung, H.P., Khatri, B.O., Montalban, X., Pelletier, J., Capra, R., Gallo, P., Izquierdo, G., Tiel-Wilck, K., de Vera, A., Jin, J., Stites, T., Wu, S., Aradhye, S., Kappos, L., 2010. Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis. N. Engl. J. Med. 362, 402–415. Compston, A., Coles, A., 2008. Multiple sclerosis. Lancet 372, 1502–1517. Foster, C.A., Howard, L.M., Schweitzer, A., Persohn, E., Hiestand, P.C., Balatoni, B., Reuschel, R., Beerli, C., Schwartz, M., Billich, A., 2007. Brain penetration of the oral immunomodulatory drug FTY720 and its phosphorylation in the central nervous system during experimental autoimmune encephalomyelitis: consequences for mode of action in multiple sclerosis. J. Pharmacol. Exp. Ther. 323, 469–475. Furlan, R., Cuomo, C., Martino, G., 2009. Animal models of multiple sclerosis. Methods Mol. Biol. 549, 157–173. Harada, J., Foley, M., Moskowitz, M.A., Waeber, C., 2004. Sphingosine-1-phosphate induces proliferation and morphological changes of neural progenitor cells. J. Neurochem. 88, 1026–1039. Hemmer, B., Hartung, H.P., 2007. Toward the development of rational therapies in multiple sclerosis: what is on the horizon? Ann. Neurol. 62, 314–326. Jaillard, C., Harrison, S., Stankoff, B., Aigrot, M.S., Calver, A.R., Duddy, G., Walsh, F.S., Pangalos, M.N., Arimura, N., Kaibuchi, K., Zalc, B., Lubetzki, C., 2005. Edg8/S1P5: an
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oligodendroglial receptor with dual function on process retraction and cell survival. J. Neurosci. 25, 1459–1469. Jung, C., Kim, H., Miron, V., Cook, S., Kennedy, T., Foster, C., Antel, J., Soliven, B., 2007. Functional consequences of S1P receptor modulation in rat oligodendroglial lineage cells. Glia 55, 1656–1667. Kappos, L., Antel, J., Comi, G., Montalban, X., O'Connor, P., Polman, C.H., Haas, T., Korn, A.A., Karlsson, G., Radue, E.W., 2006. Oral fingolimod (FTY720) for relapsing multiple sclerosis. N. Engl. J. Med. 355, 1124–1140. Kappos, L., Radue, E.W., O'Connor, P., Polman, C., Hohlfeld, R., Calabresi, P., Selmaj, K., Agoropoulou, C., Leyk, M., Zhang-Auberson, L., Burtin, P., 2010. A placebocontrolled trial of oral fingolimod in relapsing multiple sclerosis. N. Engl. J. Med. 362, 387–401. Kataoka, H., Sugahara, K., Shimano, K., Teshima, K., Koyama, M., Fukunari, A., Chiba, K., 2005. FTY720, sphingosine 1-phosphate receptor modulator, ameliorates experimental autoimmune encephalomyelitis by inhibition of T cell infiltration. Cell. Mol. Immunol. 2, 439–448. Kim, H.J., Miron, V.E., Dukala, D., Proia, R.L., Ludwin, S.K., Traka, M., Antel, J.P., Soliven, B., 2011. Neurobiological effects of sphingosine 1-phosphate receptor modulation in the cuprizone model. FASEB J. 25, 1509–1518. Mi, S., Hu, B., Hahm, K., Luo, Y., Kam Hui, E.S., Yuan, Q., Wong, W.M., Wang, L., Su, H., Chu, T.H., Guo, J., Zhang, W., So, K.F., Pepinsky, B., Shao, Z., Graff, C., Garber, E., Jung, V., Wu, E.X., Wu, W., 2007. LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis. Nat. Med. 13, 1228–1233. Mi, S., Miller, R.H., Tang, W., Lee, X., Hu, B., Wu, W., Zhang, Y., Shields, C.B., Zhang, Y., Miklasz, S., Shea, D., Mason, J., Franklin, R.J., Ji, B., Shao, Z., Chedotal, A., Bernard, F., Roulois, A., Xu, J., Jung, V., Pepinsky, B., 2009. Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells. Ann. Neurol. 65, 304–315. Miller, R.H., Mi, S., 2007. Dissecting demyelination. Nat. Neurosci. 10, 1351–1354. Miron, V., Jung, C., Kim, H., Kennedy, T., Soliven, B., Antel, J., 2008a. FTY720 modulates human oligodendrocyte progenitor process extension and survival. Ann. Neurol. 63, 61–71. Miron, V.E., Schubart, A., Antel, J.P., 2008b. Central nervous system-directed effects of FTY720 (fingolimod). J. Neurol. Sci. 274, 13–17. Miron, V.E., Ludwin, S.K., Darlington, P.J., Jarjour, A.A., Soliven, B., Kennedy, T.E., Antel, J.P., 2010. Fingolimod (FTY720) enhances remyelination following demyelination of organotypic cerebellar slices. Am. J. Pathol. 176, 2682–2694. Narayanan, S.P., FLores, A.I., Wang, F., Macklin, W.B., 2009. Akt signals through the mammalian target of rapamycin pathway to regulate CNS myelination. J. Neurosci. 29, 6860–6870. O'Connor, P., Comi, G., Montalban, X., Antel, J., Radue, E.W., de Vera, A., Pohlmann, H., Kappos, L., 2009. Oral fingolimod (FTY720) in multiple sclerosis: two-year results of a phase II extension study. Neurology 72, 73–79. Papadopoulos, D., Rundle, J., Patel, R., Marshall, I., Stretton, J., Eaton, R., Richardson, J.C., Gonzalez, M.I., Philpott, K.L., Reynolds, R., 2010. FTY720 ameliorates MOG-induced experimental autoimmune encephalomyelitis by suppressing both cellular and humoral immune responses. J. Neurosci. Res. 88, 346–359. Pébay, A., Toutant, M., Prémont, J., Calvo, C.-F., Venance, L., Cordier, J., Glowinski, J., Tencé, M., 2001. Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades. Eur. J. Neurosci. 13, 2067–2076. Rau, R.C., Hein, K., Sattler, B.M., Kretzschmar, B., Hillgruber, C., McRae, L.B., Diem, R., Bahr, M., 2011. Anti-inflammatory effects of FTY720 do not prevent neuronal cell loss in a rat model of optic neuritis. Neurobio. 178, 1770–1781.
Contents lists available at ScienceDirect
Molecular and Cellular Neuroscience j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m c n e
Sphingosine 1-phosphate receptor modulator fingolimod (FTY720) does not promote remyelination in vivo☆ Yinghui Hu a, Xinhua Lee a, Benxiu Ji a, Kevin Guckian a, Daniel Apicco a, R. Blake Pepinsky a, Robert H. Miller b,⁎, Sha Mi a,⁎⁎ a b
Biogen Idec Inc., Neuro-Discovery Biology, 14 Cambridge Center, Cambridge, Massachusetts 02142, United States Case Western Reserve University, School of Medicine, E721, 10900 Euclid Avenue, Cleveland, Ohio 44106, United States
a r t i c l e
i n f o
Article history: Received 9 March 2011 Revised 19 May 2011 Accepted 8 June 2011 Available online 24 June 2011 Keywords: FTY720 Remyelination Oligodendrocyte
a b s t r a c t The sphingosine 1-phosphate (S1P) receptor modulators have emerged as a new therapeutic opportunity paradigm for the treatment of immune-mediated demyelinating diseases such as multiple sclerosis (MS). The S1P analog fingolimod (FTY720) has been shown to alleviate disease burden in immune-mediated animal models of MS, and has been approved for treatment in clinical trials in patients with MS in the United States. While the immunological effects of FTY720 are well established, there is controversy in the literature regarding the contribution of FTY720 on myelin repair. Here, we directly assessed the impact of FTY720 on myelin repair in cuprizone and lysolecithin (LPC) demyelination models that have a minimal immunological component. FTY720 failed to promote remyelination in either animal model. These studies suggest that while FTY720 may be effective at modulating the immunological attack in MS, it may benefit from an add-on therapy to enhance the myelin repair required for long-term functional restoration in MS. © 2011 Elsevier Inc. All rights reserved.
Introduction Multiple sclerosis (MS) is an autoimmune demyelinating disease characterized by areas of demyelination and loss of oligodendrocytes (Compston and Coles, 2008). A major effector of this central nervous system (CNS) damage is thought to be T cell infiltration across the blood brain barrier. These T cells target CNS antigens to damage myelin, resulting in demyelinated lesions throughout the brain and spinal cord (Compston and Coles, 2008). The subsequent inability of CNS processes to remyelinate these damaged lesions results in neurodegeneration and various cognitive and physical neurological deficits. As a result, effective functional restoration in MS may depend on the ability to remyelinate and thereby regenerate healthy axons and neurons. Any treatment effect on myelin repair is likely to affect oligodendrocyte precursor cell (OPC) maturation. Myelination is known to result from directed activation of complex cellular processes, including OPC proliferation, migration, adhesion, process extension/retraction, and differentiation (Miller and Mi, 2007; Miron et al., 2008b). Furthermore, detailed functional studies of repair can be
☆ Yinghui Hu, Xinhua Lee, Benxiu Ji, Kevin Guckian, R Blake Pepinsky, and Sha Mi are employees of Biogen Idec. Biogen Idec markets two products for the treatment of multiple sclerosis, which are competitors of FTY720. ⁎ Corresponding author. Fax: +1 216 368 4650. ⁎⁎ Corresponding author. Fax: +1 617 679 3148. E-mail addresses: [email protected] (R.H. Miller), [email protected] (S. Mi). 1044-7431/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2011.06.007
accomplished through the use of a range of approaches, including the analysis of oligodendrocyte differentiation in vitro and the use of ex vivo brain slice cultures that permit cellular and molecular perturbation of the environment of actively myelinating systems. In previous chemical-induced demyelination studies, we have used these approaches to demonstrate that inhibition of leucine-rich repeat- and Ig domain-containing Nogo receptor-interacting protein 1 (LINGO-1) function promotes myelin formation that is correlated with promotion of OPC differentiation and maturation of oligodendrocytes (Mi et al., 2007; Mi et al., 2009). Sphingosine 1-phosphate (S1P) is a signaling sphingolipid that mediates a variety of cellular responses through the endothelial differentiation G-protein coupled (EDG) receptors. S1P signaling plays a critical role in immunomodulation and has recently become a focus of attention in the treatment of immune-mediated demyelinating diseases such as MS (Brinkmann et al., 2010). One modulator of this system is the S1P analog fingolimod (FTY720), a non-specific agonist for S1P receptors that possesses clear anti-inflammatory properties (Brinkmann et al., 2010). FTY720 (Gilenya) has recently been approved as an oral agent for use in relapsing forms of MS in the United States (Cohen and Rieckmann, 2007; Hemmer and Hartung, 2007). Clinical studies have shown that Gilenya treatment reduces annual relapse rate and severity of symptoms (Cohen et al., 2010; Kappos et al., 2006, 2010; O'Connor et al., 2009). The primary mechanism of FTY720 efficacy is believed to be immunological, whereby FTY720 prevents lymphocyte egress from lymphoid organs, thus inhibiting the circulation of autoaggressive T cells (Compston and Coles, 2008).
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Experimental autoimmune encephalitis (EAE) is an animal model for studying inflammatory diseases of the central nervous system and has contributed valuable insights into the effects of immunologicallyinduced demyelination at the whole organism level (Furlan et al., 2009). FTY720 treatment has been shown to ameliorate symptoms of EAE disease and some studies even report evidence of enhanced myelination (Foster et al., 2007; Kataoka et al., 2005; Papadopoulos et al., 2010). However, it is unclear whether the increased myelination results from FTY720 indirectly inhibiting demyelination by blocking T cell infiltration into the CNS or from FTY720 directly promoting OPC maturation and remyelination. There are limited in vivo studies that directly address the role of FTY720 on OPC proliferation, differentiation and remyelination, which could be critical for understanding the long term effects of FTY720 on CNS remyelination and repair. A nonimmune-based animal model, one where the primary demyelination is not mediated by immunological attack, is necessary in order to determine whether FTY720 treatment promotes remyelination. In this study, we used two such models, cuprizone and lysolecithin (LPC), to study the specific effects of FTY720 on remyelination. These models allow clearer temporal delineation of the phases of demyelination and remyelination in the absence of primary involvement of the immune system. Our studies demonstrate that FTY720 does not promote remyelination in either model.
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Results Local delivery of FTY720 induces demyelination in the LPC model The lysolecithin-induced (LPC) demyelination model was utilized to assess the effects of FTY720 on remyelination in vivo. LPC was injected into rat dorsal columns to produce demyelinated lesions after a few hours. 3 days after LPC injection, animals were dosed with FTY720, antiLINGO-1 antibody, or vehicle control treatment by direct injection into the lesion. Remyelination was examined on day 10 post-LPC injection by luxol fast blue (LFB) staining (Fig. 1A, top panels). FTY720 treatment led to a two-fold increase in the size of demyelinated lesions compared to the vehicle control treatment group, as determined by 3-dimensional measurement (Fig. 1B; control n=5, FTY720 n=7). In contrast, antiLINGO-1 antibody treatment reduced the lesion size 18-fold compared with controls (Fig. 1A, B; n=8), consistent with our previous finding that inhibiting LINGO-1 promotes remyelination (Mi et al., 2009). The FTY720-treated animals contained non-LFB staining spots in the white matter perilesional areas with moth-eaten morphology (Bradl and Lassmann, 2010) characteristic of OPC death and demyelination (Fig. 1C, D). This morphology suggests that locally delivered FTY720 may diffuse into nearby white matter tissue to induce OPC death and demyelination. To clarify the direct effect of FTY720 on demyelination, FTY720 was
Fig. 1. Local delivery of FTY720 causes demyelination in the LPC model. (A) Luxol fast blue staining (LFB) of white matter in spinal cord dorsal column lesions following local injection of FTY720, vehicle control, or anti-LINGO-1 antibody (top panels). Bottom panels show three-dimensional reconstructions of the lesion areas (red, lesion volume). Scale bar = 100 μm. (B) Quantification of the lesion volumes of (A, bottom panels). (C) LFB staining of spinal cord tissue surrounding lesions from vehicle control- and FTY720-treated rats. Arrows denote moth-eaten morphology. (D) Enlargement of right panel from (C). (E) Black gold staining of brain slice cultures to visualize demyelination by FTY720P treatment. (F) Quantification of black gold staining intensity in (E).
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Fig. 1 (continued).
locally injected into the spinal cord of normal animals without LPCinduced demyelination. Consistent with the LPC study, FTY720 injection alone produced demyelinated lesions (Supplementary Fig. 1). This result shows that the demyelinating effect of FTY720 is independent of LPCinduced demyelination, suggesting that FTY720 actively promotes demyelination when administered by local injection. Brain slice cultures provide an in vitro surrogate to in vivo animal models to study demyelination and remyelination. For these studies, phosphorylated FTY720 (FTY720P), the active form of the compound, was added to rat brain slice cultures (P17 + 3DIV). 10 nM and 100 nM FTY720P treatment led to a dosedependent reduction in myelination in the corpus callosum region compared with vehicle-treated slices (Fig. 1E, F), suggesting that FTY720P either induced demyelination or inhibited ongoing myelination of the brain slice cultures. To further assess the effect of FTY720 on remyelination, a LPC demyelination study was performed with FTY720 administered orally (1 mg/kg/day), a treatment regimen that is highly efficacious in EAE
models (Kataoka et al., 2005; Al-lzki et al., 2011). The lesion size was determined by toluidine blue staining (Fig. 2A, B). Unlike with local delivery, oral FTY720 treatment did not increase the lesion volume. Quantification of remyelinated axons in the lesion after examination by electron microscopy showed no difference in the proportion of myelinated axon fibers between FTY720-treated and control animals (Fig. 2C, D). This result suggests that FTY720 did not promote remyelination or acceleration of demyelination in these lesions. The discrepancy between the local and oral treatment results may be due to the higher concentration of FTY720 in the lesions following local administration. FTY720 does not promote remyelination in the cuprizone demyelination model The effects of FTY720 on remyelination were further investigated in the cuprizone-induced demyelination model (Fig. 3). In this model, demyelinated lesions can be detected in corpus callosum after
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4 weeks dietary ingestion of cuprizone, at which point animals were treated for 2 weeks with FTY720 (1 mg/kg oral daily) or vehicle control along with a normal cuprizone-free diet. At 6 weeks, animals were sacrificed and the remyelinating effect of FTY720 relative to control treatment was evaluated by histological methods. The cuprizone diet resulted in a 2-fold reduction in myelinated axons in corpus collosum compared to normal diet (Fig. 3C). Anti-MBP (myelin basic protein) immunohistochemistry (Fig. 3A) and EM (Fig. 3B) showed no difference in the number of myelinated axons between the FTY720- and control-treated animals, suggesting that FTY720 had no effect on remyelination. A significant effect of FTY720 on the model was identified by quantifying the number of CC1 + mature oligodendrocytes, NG2 + OPCs, GFAP + astrocytes, and CD68 + microglia/macrophages in the corpus callosum (Fig. 4). Cuprizone diet led to a 2-fold decrease in the number of CC1 + mature oligodendrocytes compared to normal diet (Fig. 4B). There was no difference in the number of CC1 + mature oligodendrocytes in the corpus callosum between the control-treated and FTY720-treated groups (Fig. 4B). However, there was a 2-fold increase in NG2 + OPCs (Fig. 4D) and a 44% increase in GFAP + astrocytes (Supplementary Fig. 2, n = 4 each). There was no difference in the number of CD68 + microglia/ macrophages in FTY720-treated mice compared with control mice (Supplementary Fig. 3). This result suggests that FTY720 promotes OPC and astrocyte proliferation in vivo while having no effect on microglial activation.
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FTY720P stimulates OPC proliferation but inhibits OPC maturation in vitro To directly evaluate the effect of FTY720 on OPC function, purified OPCs from rat P2 forebrain were cultured in the presence and absence of 10 nM FTY720P and live images were captured for 98 h (Fig. 5 and Supplementary Video 1). Over the course of the recording, vehicletreated OPCs formed extensive processes typical of differentiation (Fig. 5A–F). In contrast, OPCs treated with 10 nM FTY720P initially showed morphological changes characteristic of differentiation (Fig. 5G–I), but by 60 h the processes had retracted (Fig. 5J) and the cells started to proliferate, as evident by the increased cell number at the 60, 80, and 98 h time points (Fig. 5J–L). Supplementary Video 1 demonstrates that the two OPCs seen at time 24 h in Fig. 5I become 24 OPCs at time 98 h in Fig. 5L. The longer processes apparent at 40 h (Fig. 5I) are lost at the later time points (Fig. 5J–L). Quantification of the total number of OPCs per field at 98 h revealed about a 4-fold increase in FTY720P-treated compared to vehicle-treated cultures (Fig. 5M, n = 6). The proliferative effect of FTY720P on OPCs was confirmed in OPC cultures using BrdU labeling to monitor cell proliferation. Consistent with the live image results, 10 nM FTY720P treatment for 72 h led to less-developed processes for NG2 + cells (Fig. 6A) and a 4-fold increase in BrdU +NG2 + OPCs (Fig. 6A, B) compared with vehicletreated cultures, thus confirming the effects of FTY720P on OPC proliferation and differentiation.
Fig. 2. Toluidine blue staining of LPC-induced lesions following oral FTY720 treatment. (A) Treatment with dimethyl sulfoxide (DMSO). (B) Treatment with FTY720. Red lines denote demyelinated areas. Scale bar = 100 μm. (C) Ultrastructural visualization of lesions by EM (top panels, scale bar = 1 μm). Higher magnifications (bottom panels, scale bar = 500 nm) show the presence of naked demyelinated axons (asterisks). (D) Quantification of myelinated axons in EM images from (C). n = 30 fields/group from 3 rats.
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The reversal of OPC differentiation and induction of OPC proliferation was further studied by immunocytochemistry. A2B5 + OPCs were cultured for 3 days with or without 10 nM FTY720P in PDGF/FGF-containing media. Treatment with FTY720P resulted in dramatic morphological changes of A2B5 + OPCs, which showed a rounding and retraction of the processes (Fig. 6C), suggesting that FTY720P inhibits OPC process formation and maturation. Quantification of average process length demonstrates approximately a 4-fold reduction in FTY720P-treated compared to control-treated cultures (Fig. 6D). This effect was further confirmed by culturing OPCs in CNTF/ T3-containing differentiation media for 3 days. In the presence of 10 nM FTY720P, oligodendrocyte processes were approximately 2fold shorter compared with the vehicle-treated cultures (Fig. 6E, F). The total numbers of MBP + cells between the 10 nM FTY720P and control groups are similar. OPC cell death occurred when the concentration of FTY720P was increased to 1 μM (data not shown), consistent with the cytotoxic effect at high concentrations observed in vivo when LPC was delivered by local injection. Discussion We have demonstrated that FTY720 treatment has no direct effect on remyelination in two chemical-induced animal models of demyelination, LPC and cuprizone. Unlike the EAE model, the clear delineation of the phases of demyelination and remyelination in the LPC and cuprizone models allows for the effects of FTY720 on specific
processes such as remyelination to be examined in the absence of primary involvement of the immune system. xIn the LPC demyelination model, local injection of FTY720 enhanced demyelinated lesions and moth-eaten morphology suggesting oligodendrocyte death. This is consistent with our ex vivo brain slice cultures where FTY720P treatment decreased myelin intensity by 20% at 10 nM and by 50% at 100 nM. Since myelination is an ongoing process in P17 brain slice cultures, the observed decrease in myelination could be due to either FTY720P inhibiting ongoing myelination or inducing demyelination. Miron et al. have reported that FTY720P enhanced remyelination in LPCinduced demyelinated P0 cerebellum slice cultures (Miron et al., 2010); however, their study design differed significantly from ours. When FTY720 was administered orally in the LPC model, we did not see enhanced demyelinated lesions nor moth-eaten morphology after a 7 day treatment, suggesting that FTY720 only induces oligodendrocyte death at higher concentrations, such as by local injection. In the cuprizone demyelination model, no effect on remyelination was observed following 2 week oral FTY720 treatment despite a 44% increase in astrocyte number, consistent with a recent study by Kim et al. (Kim et al., 2011). Our data also demonstrate a 2-fold increase in NG2 + OPCs in the corpus callosum, suggesting that FTY720 promotes OPC proliferation. The effects of FTY720 on oligodendrocyte biology were further examined in vitro by OPC culture in order to comprehensively understand the specific effects of FTY720 on OPC maturation and remyelination. 10 nM FTY720P treatment first inhibited OPC
Fig. 3. Effects of FTY720 treatment in the cuprizone demyelination model. (A) Anti-MBP IHC to visualize demyelination in the corpus callosum. Normal (left, cuprizone-free diet) and cuprizone-fed vehicle control-treated (middle) and FTY720-treated (right) corpus collosum. Scale bar = 100 μm. (B) Detection of myelinated axons by EM in the corpus callosum. Scale bar = 2 μm. (C) Quantification of myelinated axons from the EM images in (B).
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Fig. 4. CC1 IHC staining showing the effects of FTY720 on oligodendrocyte number in the cuprizone model. (A) CC1 IHC staining of mature oligodendrocytes in corpus callosum. Scale bar = 25 μm. (B) Quantification of the number of CC1+ cells in (A). (C) NG2 IHC staining of OPCs in corpus callosum. Scale bar = 25 μm. (D) Quantification of the number of NG2+ cells in (C).
maturation as evident by the retraction of oligodendrocyte processes and then promoted cell proliferation as evident by the increase in cell number. Published reports have shown that FTY720 treatment inhibits oligodendrocyte differentiation by promoting process retraction in human (Miron et al., 2008a) and rat (Jung et al., 2007) OPCs. Miron et al. found that prolonged FTY720 treatment reversed the initial retraction of OPC processes by 48 h (Miron et al., 2008a, 2008b). In contrast, our time-lapse images show continued process retraction and cell proliferation through 98 h of treatment, suggesting that FTY720 inhibits OPC maturation both initially and long-term. Cell
proliferation following FTY720 treatment has also been reported in other cell types, including astrocytes (Pébay et al., 2001) and neural progenitor cells (Harada et al., 2004). The mechanism driving the retraction of OPC processes and subsequent cell proliferation may be a Rho kinase/collapsin response-mediated protein signaling pathway activated by FTY720 through the S1P5 receptor-signaling pathway, as reported previously (Jaillard et al., 2005; Miron et al., 2008a). Although it is well established that FTY720 treatment alleviates the immunological attack in MS, there are a number of reports highlighting its failure to promote neuroregenerative repair in non-
78 Y. Hu et al. / Molecular and Cellular Neuroscience 48 (2011) 72–81 Fig. 5. FTY720P treatment promotes OPC proliferation in OPC cultures. Treatment effects of FTY720P on OPC function were evaluated by time lapse images at the indicated time points for vehicle control-treated (A–F) and FTY720P-treated (G– L) OPCs. Scale bar = 10 μm. (M) Quantification of total numbers of OPCs at 98 h.
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immune-mediated models. Most recently, Rau et al. reported that FTY720 exerted significant anti-inflammatory effects during optic neuritis, but did not prevent apoptosis of retinal ganglion cells or improve visual function (Rau et al., 2011). In another study, long term FTY720 treatment (1 mg/kg, P.O. daily for 2 months) did not improve axon remyelination or prevent progressive neurodegeneration in the secondary progressive EAE model (Al-lzki et al., 2011) despite the claims by Choi et al. that FTY720 treatment decreased gliosis and protected neurons via down-regulation of S1P1 in astrocytes (Choi et al., 2011). In clinical trials, FTY720 was shown to have a strong effect on relapse rate reduction (60% versus placebo), but only a modest effect on disability progression (31% versus placebo) (Kappos et al., 2010). Our data, which shows no direct effect of FTY720 on remyelination, offers an explanation for this discrepancy since
Fig. 6 (continued).
slowing of disability progression may depend on the ability to promote myelin repair. The absence of remyelination in nonimmune-mediated models of demyelination suggests that FTY720 predominantly functions as an immune regulator, as previously reported in EAE studies and clinical trials (Cohen and Rieckmann, 2007; Foster et al., 2007; Hemmer and Hartung, 2007; Kataoka et al., 2005). In summary, through a comprehensive effort to understand the effects of FTY720 on oligodendrocyte proliferation, maturation, and myelination, we demonstrate that FTY720 does not promote remyelination following oral treatment in the two non-immunemediated demyelination models and induces oligodendrocyte death at higher concentrations, produced by local delivery in the LPC model and by direct addition of FTY720P to culture medium in brain slice cultures. While effective at modulating immunological attack in MS, our studies highlight the need for new and/or add-on therapies to promote the myelin repair necessary for long-term functional recovery of the CNS.
Experimental methods Formulation of FTY720
Fig. 6. IHC staining of cultured OPCs. (A) A2B5+ OPC proliferation visualized by antiBrdU (green), anti-NG2 (red), and co-labeling (yellow). (B) Quantification of BrdU+ NG2+ cells from (A). (C) A2B5+ OPCs treated with vehicle control or FTY720P in the presence of FGF/PDGF. Scale bar = 50 μm. (D) Quantification of the length of OPC processes in (C). (E) MBP+ OPCs treated with control or FTY720P in the presence of CNTF/T3. Scale bar = 50 μm. (F) Quantification of the length of OPC processes in (E).
FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol) was purchased from Cayman Chemical Corporation as the HCl salt (Catalog # 10006292 LOT 166442) and its identity was confirmed by NMR and LC/MS. (S)-FTY720-P ((2S)-amino-2-[2-(4-octylphenyl) ethyl]-1-(dihydrogen phosphate)-1,3-propanediol) was purchased from Cayman Chemical Corporation (Catalog # 10006407, LOT 191341) and its identity was confirmed by NMR, LC/MS and optical rotation. (S)-FTY720-P was further characterized by measurement in a functional cell assay (Millipore Corporation) and potencies were consistent with published values for (S)-FTY720-P at S1P1-S1P5. Working solutions of FTY720 were prepared at 2 mg/ml in water solution for local injections. FTY720 was used for all in vivo studies and its phosphorylated form, FTY720P, was used in all in vitro culture studies, except where noted otherwise. All procedures for animal use were performed in accordance with the US National Institute of
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Health guidelines and were approved by the Biogen Idec Institutional Committee for Animal Care. Oligodendrocyte culture Enriched populations of primary oligodendrocytes were grown from postnatal day 2 Sprague Dawley rats. The forebrain was dissected and placed in Hank's buffered saline solution (HBSS; Invitrogen, Grand Island, NY). The tissue was cut into 1 mm fragments and incubated at 37 °C for 15 min in 0.01% trypsin and 10 μg/mL deoxyribonuclease (DNase). Dissociated cells were plated on poly-Llysine-coated T75 tissue culture flasks and grown at 37 °C for 10 days in Dulbecco's Modified Eagle's Medium (DMEM) with 20% fetal calf serum (Invitrogen, Carlsbad, CA). OPCs were collected by shaking the flask overnight at 200 rpm and 37 °C, resulting in a more than 95% pure population of A2B5 + cells, as determined by anti-A2B5 immunocytochemistry co-stained with DAPI. To monitor OPC proliferation, OPCs were labeled with bromo-deoxyuridine (BrdU, 1:1000 dilution, GE Healthcare, Piscataway, NJ) and incubated overnight at 37 °C. After the incubation, cells were fixed with 4% paraformaldehyde for 30 min, and then treated with 4N HCl for 10 min. After three washes, cells were stained with anti-NG2 and anti-BrdU antibodies as described in the antibodies and immunocytochemistry methods. Live images and time-lapse analysis OPCs in high-glucose DMEM containing FGF/PDGF, were subjected to time-lapse recording for 98 h in the presence of 10 nM FTY720P or vehicle control. Cells were cultured in a 24-well plate mounted onto a motorized stage (Zeiss, Thornwood, NY) housed inside a sealed chamber and maintained at 37 °C in humidified air with 5% CO2. Wide-field microscopic images were acquired through the transmitted light channel on a Zeiss Axiovert microscope equipped with a digital camera and Axiovision software. Antibodies and immunocytochemistry Anti-A2B5 antibodies were obtained from Chemicon (Rosemont, IL cat#mAB312R), anti-MBP antibodies from Covance (Princeton, NJ cat#SM194 and cat#SM199), anti-NG2 antibodies from Chemicon (Rosemont, IL cat#AB5320), anti-CC1 antibodies from Calbiochem (Spring Valley, CA cat#OP80), and anti-BrdU monoclonal antibodies from Invitrogen (Carlsbad, CA A-21303). To visualize antibody labeling in tissue sections or cell cultures grown on chamber slides, samples were fixed in 4% paraformaldehyde, washed, and incubated with the indicated antibodies using standard protocols. Tissue sections were incubated in primary antibodies overnight at 4 °C, washed thoroughly, incubated with the appropriate Alexa-labeled secondary antibody (Invitrogen, Carlsbad, CA) for 2 h, then mounted in ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA) and visualized by fluorescence microscopy. Cultures were incubated for 2 h in primary and 1 h in Alexa-labeled secondary antibodies, and visualized by fluorescence microscopy. For quantitative analysis immunoreactive cells were counted at 20× magnification with tissue areas measured by openlab software. Rat forebrain slice cultures Postnatal, day 17 Sprague Dawley rats were purchased from Charles River (Wilmington, MA) and the brains were surgically removed. Four consecutive 300 μm slices were taken from the junction of the corpus callosum to the hippocampus in each. Slices were cultured in basal DMEM supplemented with 25% horse serum for 3 days before any treatment. For demyelination studies, cultured slices were treated with 10 nM or 100 nM FTY720P for 3 days followed by black gold staining to visualize myelination (Millipore,
Bedford, MA). Images were acquired using a Leica M420 microscope (Bannockburn, IL) and the staining intensity of the corpus callosum was analyzed using Metamorph software from Molecular Devices (Downingtown, PA). 4 slices for each treatment group were analyzed and two independent experiments were conducted to generate data (8 total slices per group). Histology and microscopy For immunohistochemistry studies, animals were terminally anesthetized and transcardiac perfusion was performed using phosphate buffered saline (PBS) followed by fixation with 4% paraformaldehyde. Tissue was removed, post-fixed overnight, and then cryoprotected in 20% sucrose in 0.1 M PBS for 48 h. Tissue was embedded in Optimal Cutting Temperature (O.C.T.) compound and cryo-sectioned. For luxol fast blue (LFB) staining, 100 μm transverse sections from LPC-treated rat spinal cord were stained with 0.5% LFB, cleared, dehydrated, and mounted. Lesion volumes were determined using three-dimensional reconstruction from serial sections. For toluidine blue staining and electron microscopy (EM), animals were transcardiac perfused with 4% paraformaldehyde plus 2.5% glutaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Spinal cords or brains were dissected and immersed in 2% osmium tetroxide, dehydrated through a graded ethanol series, and Epon™-embedded following procedures described previously (Mi et al., 2007). Semi-thin sections of 1 μm thickness were stained with toluidine blue and images were captured by light microscopy. The samples were then processed by EM analysis as described previously (Mi et al., 2007). Lysolecithin demyelination model Nine-week-old Sprague–Dawley rats received a 1.5 μL direct injection of 1% LPC into the spinal cord dorsal column, followed 2 days later by oral treatment with 1 mg/kg FTY720 or vehicle control daily for 7 days (toluidine blue and electron microscopy studies). Oral dose of 1 mg/kg FTY720 was selected based on the published literature (Kataoka et al., 2005; Kim et al., 2011; Al-lzki et al., 2011). In a separate experiment, nine-week-old Sprague–Dawley rats received local 2 μL injection of 2 mg/mL FTY720, vehicle control or 0.75 mg/mL anti-LINGO-1 antibody 3 days after LPC injection. Animals were sacrificed on day 9 (oral treatment) or day 10 (local delivery) after LPC injection. To determine the direct effect on demyelination, FTY720P or vehicle (2 μL of 2 mg/mL) were injected into the normal rat spinal cord dorsal column (without LPC-induced demyelination). The animals were sacrificed 3 days later for histology study. All sections were stained with LFB for examination of lesion size. Myelinated axons in lesion areas were quantified from EM images (10 fields were counted for each animal, three rats per group were analyzed). Cuprizone demyelination model Nine-week-old C57/B6 mice were fed 0.3% cuprizone diet (chow pellets, Harlan) for 4 weeks along with intraperitoneal injection of 10 mg/kg rapamycin for 5 days per week (Narayanan et al., 2009). Animals were then treated with 1 mg/kg FTY720 or vehicle control by oral gavage for an additional 2 weeks with normal diet. The plasma concentration of FTY720 was measured 24 h after last dose to confirm accurate dosing. At the end of the 6 week cuprizone study (4 weeks cuprizone diet plus 2 weeks FTY720 treatment with normal diet), animals were perfused and brains were dissected for immunohistochemistry and EM analysis. Coronal slices (septostriatal section, 1 mm thick) through the corpus callosum were cut in midsagittal plane and embedded in epon, oriented to visualize the entire cross-section of the midsagittal corpus callosum. The myelinated axons were compared by
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electron microscopy from at least 30 different non-overlapping fields from 3 mice in each group. Statistical methods GraphPad Prism software was used for statistical analysis. Data are shown as mean and standard error of the mean. In all studies, comparison of mean values was conducted using unpaired student's t-tests or one-way ANOVA analysis of variance with Tukey correction. In all analyses, statistical significance was determined at the 5% level (p b 0.05). Supplementary materials related to this article can be found online at doi:10.1016/j.mcn.2011.06.007. References Al-lzki, S., Pryce, G., Jackson, S.J., Giovannoni, G., Baker, D., 2011. Immunosuppression with FTY720 is insufficient to prevent secondary progressive neurodegeneration in experimental autoimmue encephalomyelitis. Mult. Scle. J. 1–10. Bradl, M., Lassmann, H., 2010. Oligodendrocytes: biology and pathology. Acta Neuropathol. 119, 37–53. Brinkmann, V., Billich, A., Baumruker, T., Heining, P., Schmouder, R., Francis, G., Aradhye, S., Burtin, P., 2010. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discov. 9, 883–897. Choi, J.W., Gardell, S.E., Herr, D.R., Rivera, R., Lee, C.W., Noguchi, K., Teo, S.T., Yung, Y.C., Lu, M., Kennedy, G., Chun, J., 2011. FTY720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1P1) modulation. Proc. Natl. Acad. Sci. U. S. A. 108, 751–756. Cohen, B.A., Rieckmann, P., 2007. Emerging oral therapies for multiple sclerosis. Int. J. Clin. Pract. 61, 1922–1930. Cohen, J.A., Barkhof, F., Comi, G., Hartung, H.P., Khatri, B.O., Montalban, X., Pelletier, J., Capra, R., Gallo, P., Izquierdo, G., Tiel-Wilck, K., de Vera, A., Jin, J., Stites, T., Wu, S., Aradhye, S., Kappos, L., 2010. Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis. N. Engl. J. Med. 362, 402–415. Compston, A., Coles, A., 2008. Multiple sclerosis. Lancet 372, 1502–1517. Foster, C.A., Howard, L.M., Schweitzer, A., Persohn, E., Hiestand, P.C., Balatoni, B., Reuschel, R., Beerli, C., Schwartz, M., Billich, A., 2007. Brain penetration of the oral immunomodulatory drug FTY720 and its phosphorylation in the central nervous system during experimental autoimmune encephalomyelitis: consequences for mode of action in multiple sclerosis. J. Pharmacol. Exp. Ther. 323, 469–475. Furlan, R., Cuomo, C., Martino, G., 2009. Animal models of multiple sclerosis. Methods Mol. Biol. 549, 157–173. Harada, J., Foley, M., Moskowitz, M.A., Waeber, C., 2004. Sphingosine-1-phosphate induces proliferation and morphological changes of neural progenitor cells. J. Neurochem. 88, 1026–1039. Hemmer, B., Hartung, H.P., 2007. Toward the development of rational therapies in multiple sclerosis: what is on the horizon? Ann. Neurol. 62, 314–326. Jaillard, C., Harrison, S., Stankoff, B., Aigrot, M.S., Calver, A.R., Duddy, G., Walsh, F.S., Pangalos, M.N., Arimura, N., Kaibuchi, K., Zalc, B., Lubetzki, C., 2005. Edg8/S1P5: an
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