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© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 3878-3887 doi:10.1242/jcs.169342

RESEARCH ARTICLE Galactosylsphingosine (psychosine)-induced demyelination is attenuated by 1-phosphate signalling Catherine O’Sullivan and Kumlesh K. Dev*

ABSTRACT almost complete loss of in the white matter, Globoid cell (Krabbe disease) is a rare infantile reactive astrocytosis and infiltration of numerous multinucleated ‘ ’ neurodegenerative disorder. Krabbe disease is caused by deficiency macrophages termed globoid cells . These globoid cells in the lysosomal galactocerebrosidase (GALC) resulting in accumulate around blood vessels and in the regions of accumulation, in the micromolar range, of the toxic metabolite demyelination and are a unique feature of Krabbe disease galactosylsphingosine (psychosine) in the . Here we find that (Suzuki, 2003). Progressive accumulation of psychosine in the psychosine induces human cell death probably via an of Krabbe disease patients is thought to be the critical apoptotic process in a concentration- and time-dependent manner pathogenic mechanism of this illness (Davenport et al., 2011). In (EC50∼15 μM at 4 h). We show these effects of psychosine are some cases the levels of psychosine rise more than 100-fold, from attenuated by pre-treatment with the sphingosine 1-phosphate sub-nanomolar concentrations to those in the micromolar range receptor agonist pFTY720 () (IC50∼100 nM). Psychosine (Svennerholm et al., 1980). In Krabbe disease, the high levels of (1 μM, 10 μM) also enhances LPS-induced (EC50∼100 ng/ml) psychosine escape from lysosomes and dying cells forming production of pro-inflammatory cytokines in mouse , aggregates (Orfi et al., 1997). Several reports have demonstrated which is also attenuated by pFTY720 (1 μM). Most notably, for the that psychosine causes direct cellular cytotoxicity by mechanisms first time, we show that psychosine, at a concentration found in the that include mitochondrial dysfunction (Haq et al., 2003), caspase brains of patients with Krabbe disease (EC50∼100 nM), directly activation, alteration of rafts and modulation of the protein κ induces demyelination in mouse organotypic cerebellar slices in a kinase C (PKC), Jun N-terminal kinase (JNK) and NF B signalling manner that is independent of pro-inflammatory cytokine response pathways (Davenport et al., 2011; Haq et al., 2003; Yamada et al., and that pFTY720 (0.1 nM) significantly inhibits. These results 1996). Inflammation is also now accepted to play an important role support the idea that psychosine is a pathogenic agent in Krabbe in the pathogenesis of Krabbe disease (LeVine and Brown, 1997). disease and suggest that sphingosine 1-phosphate signalling could Inflammatory molecules, such as AMP-activated protein kinase be a potential drug target for this disorder. (AMPK), prostaglandin D, inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines have all been implicated in Krabbe KEY WORDS: Krabbe disease, Astrocytes, Demyelination, disease as well as in the twi/twi mouse animal model of this disease Sphingosine 1-phosphate receptors, pFTY720 (Giri et al., 2008). Taken together, this increasing evidence now suggests that the loss of oligodendrocytes and widespread INTRODUCTION demyelination seen in Krabbe disease is due to apoptotic Globoid cell leukodystrophy (Krabbe disease) is a rare autosomal processes as well as aberrant inflammatory response (Giri et al., recessive neurodegenerative disorder affecting 1:100,000 live births 2008, 2006; Haq et al., 2003; Tohyama et al., 2001). in the United States (Wenger et al., 1997). This lysosomal disorder The GALC enzyme, mutated in Krabbe disease, is involved in the typically has an early onset, is rapidly progressing and is invariably complex pathway of metabolism, which includes fatal in infants. The vast majority (85–90%) of cases are of the bioactive such as , sphingosine and sphingosine infantile form, with the juvenile and adult-onset forms being 1-phosphate (S1P), all of them particularly important in regulating considered extremely rare (Wenger et al., 1997). The hallmark neural cell function. In particular, the family of S1P receptors symptoms of the infantile form include , hypersensitivity, (S1PRs) are G-protein coupled and expressed in a range of cells, psychomotor arrest and hypertonia. This is followed by rapid mental including those of the immune, cardiovascular and central nervous and motor deterioration, and optic atrophy. Death usually systems (Dev et al., 2008; Fyrst and Saba, 2010). These receptors ensues within the first two years of life and there is currently no are targets for the drug pFTY720 (fingolimod), which is the first (Davenport et al., 2011). Krabbe disease is caused by a in oral therapy for relapsing remitting (Kappos et al., the lysosomal enzyme (GALC) (Suzuki, 2010). The proposed mechanism of action for pFTY720 is reported 2003). This GALC deficiency results in the accumulation of a as being dependent on internalisation of S1PRs in T cells, which toxic lipid metabolite psychosine (galactosylsphingosine) and, to a limits their S1P-mediated egress from lymph nodes and thus lesser extent, β- (Giri et al., 2002). Pathological attenuates the inflammatory response in the brains of multiple features of Krabbe disease include profound demyelination and sclerosis patients (Adachi and Chiba, 2008). Importantly, a number of studies have now demonstrated that compounds such as pFTY720 can also regulate neuronal and glial cell function Drug Development, School of Medicine, Trinity College Dublin, Dublin D02 R590, (Balatoni et al., 2007; Choi et al., 2011; Fischer et al., 2011; Ireland. Osinde et al., 2007). Indeed, we and others have shown that S1PRs *Author for correspondence ([email protected]) regulate a number of intracellular signalling pathways in astrocytes and promote astrocyte migration (Mullershausen et al., 2007, 2009).

Received 26 January 2015; Accepted 4 September 2015 In addition, modulation of S1PRs also promotes Journal of Cell Science

3878 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3878-3887 doi:10.1242/jcs.169342 differentiation and survival (Dev et al., 2008; Miron et al., 2008b). tetraethylbenzimidazolylcarbocyanine iodide (JC-1), which S1PRs have also been shown to limit events of demyelination and exhibits potential-dependent accumulation in mitochondria. At promote remyelination, which are probably mediated by the high ΔΨm this dye forms aggregates yielding an emission at dampening of pro-inflammatory cytokine levels (Miron et al., 590 nm (red) whereas at low ΔΨm JC1 is primarily in monomeric 2010; Sheridan and Dev, 2012). Overall, therefore, S1PRs represent form yielding an emission at 530 nm (green) (Fig. 3A). Cultured an important drug target that can be exploited for use in astrocytes were serum starved and pre-treated for 1 h with neuroinflammatory, demyelinating and neurodegenerative diseases, pFTY720 (1 µM) before treatment with psychosine (5 µM, as documented by a growing body of literature (Asle-Rousta et al., 10 µM, 15 µM, 20 µM). Cells were then loaded with 1 μMJC-1 2013; Deogracias et al., 2012). Here we investigate whether and after 30 min the emission spectra were measured. Psychosine regulation of S1P signalling alters psychosine-induced astrocyte treatment of mouse astrocytes decreased the aggregate:monomer dysfunction, pro-inflammatory cytokine release and demyelination. ratio of JC-1, indicating loss of ΔΨm compared with control (Fig. 3B). Notably, treatment with pFTY720 attenuated the RESULTS psychosine-induced decrease in aggregate:monomer ratio of Psychosine-induced human astrocyte cell death is JC-1, returning the ΔΨm close to control levels. These findings attenuated by pFTY720 were supported by use of the MTT colorimetric cell viability assay The modulation of psychosine on astrocyte cell function is less well (based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5- studied in Krabbe disease than the effect of psychosine on diphenyltetrazolium bromide (MTT) into formazan crystals by oligodendrocytes and myelination. We therefore first investigated metabolically active cells), which showed that psychosine caused a the effects of this toxin on human astrocyte survival and also significant concentration-dependent reduction of rat astrocyte demonstrated the protective effects of pFTY720. Cultured human viability (10 µM, 73.3±2.7%; 15 µM, 63.5±3.6%; and 20 µM, astrocytes were serum starved for 4 h and then pre-treated with 49.9±1.7%, compared with control; mean±s.e.m.). In agreement, pFTY720 for 1 h before treatment with psychosine, at the timepoints pFTY720 significantly attenuated this psychosine-induced cell and concentrations indicated (Fig. 1A). Psychosine reduced human death by 14.4±1.7% and 17.3±2.5% at 10 µM and 20 µM astrocyte numbers in a concentration-dependent manner: psychosine psychosine concentrations, respectively (Fig. 3C). treatment for 4 h significantly reduced astrocyte cell survival (10 µM, 55.4±3.8%; 15 µM, 44.3±2.7%; and 20 µM, 39.8±1.8%, Psychosine potentiates lipopolysaccharide (LPS)-induced compared with control; mean±s.e.m.). Importantly, pre-treatment levels of pro-inflammatory cytokines in mouse astrocytes with 1 µM pFTY720 significantly attenuated the psychosine- The expression of several pro-inflammatory cytokines and induced cell death (reduced by 9.8± 4.1%, 21.1±2.5% and chemokines has been observed in the twi/twi mouse model 18.4±2.2%, respectively) (Fig. 1B). The psychosine (10 µM)-induced (LeVine and Brown, 1997). Psychosine has also been seen decrease in survival of human astrocytes was also observed to be time in vitro to markedly potentiate the LPS-induced production of dependent (2 h, 79.2±3.2%; 4 h, 63.5±2.4%; 6 h, 37.1±3.3%) and pro-inflammatory cytokines in primary rat astrocyte cultures (Giri again significantly attenuated in the presence of pFTY720 (reduced by et al., 2002). Here, we investigated the effect of psychosine on the 17.4±4.6% at 4 h and 23.1±4.3% at 6 h) (Fig. 1C). These effects of levels of IL6, TNFα and IL1β in mouse astrocytes in the presence pFTY720 were also concentration dependent: 1 µM pFTY720 and absence of LPS. Firstly, LPS (1 ng/ml to 10 µg/ml) was shown significantly increased cell survival by 34.3±3.2%, compared with to induce a concentration-dependent increase in the levels of IL6 10 µM psychosine treatment alone (Fig. 1D). (Fig. 4A), TNFα (Fig. 4B) and IL1β (data not shown): 100 ng/ml During the course of our experiments demonstrating psychosine- LPS was selected as the optimal concentration for use in further induced astrocyte cell death and reversal by pFTY720 (Fig. 1A–D), experiments. Next, cultured mouse astrocytes were serum starved, we noted these effects were also dependent on the density of pre-treated with pFTY720 (1 µM for 1 h) and then treated with LPS cultured astrocytes. To quantify these observations, human (100 ng/ml) and/or psychosine (1 µM and 10 µM) for 3 h, 6 h and astrocytes were seeded at densities ranging from 1.2×105 to 12 h. No discernible release of IL6, TNFα or IL1β was observed 6×105. The cells were then pre-treated with 1 μM pFTY720 after 3 h treatment with LPS and/or psychosine (data not shown). In followed by 10 μM psychosine treatment for 2 h and imaged contrast, the treatment of astrocytes for 6 h with LPS induced at (Fig. 2A). Human astrocytes seeded at low densities of 1.2×105 and least a 2-fold increase in the levels of IL6 (5.1±0.9 pg/ml vs 2.4×105 were most sensitive to psychosine insult, with 81.5±6.2% 31.5±4.3 pg/ml; mean±s.e.m.) (Fig. 4C), TNFα (19.4±3.4 pg/ml vs and 68.5±5.1% cell death occurring after 2 h treatment. Astrocytes 48.3±2.1 pg/ml) (Fig. 4D) and IL1β (1.1±0.5 pg/ml vs 14.3± seeded at a density of 3.9×105 displayed 39.4±5.3% cell death for 5.5 pg/ml) (data not shown), compared with control. Moreover, psychosine treatment alone, which was significantly attenuated to psychosine (10 µM), while having little effect alone, significantly 20.6±3.6% in the presence of pFTY720 (Fig. 2B). At the two augmented the LPS-induced production of IL6 (31.5±4.3 pg/ml vs highest densities, 4.8×105 and 6×105,10μM psychosine treatment 44.8±4.2 pg/ml) (Fig. 4C) and TNFα (48.3±2.1 pg/ml vs 59.1± for 2 h did not induce overt astrocyte toxicity, with cell densities of 2.8 pg/ml) (Fig. 4D) (unpaired Student’s t-test P<0.05). 94.4±3.7% and 99.9±5.4%, respectively (Fig. 2C). Importantly, pFTY720 treatment significantly attenuated the increased levels of IL6 (44.8±4.2 pg/ml vs 25.6±4.9 pg/ml) pFTY720 attenuates psychosine-induced decrease of (Fig. 4C) and TNFα (59.1±2.8 pg/ml vs 42.4±4.4 pg/ml) mitochondrial membrane potential in astrocytes (Fig. 4D) induced by LPS or psychosine (10 µM). Similar Increasing evidence now suggests the involvement of apoptosis as findings were observed at 12 h: pFTY720 attenuated the LPS a mechanism underlying oligodendrocyte cell death seen in and/or psychosine (10 µM) mediated increase in levels of IL6 Krabbe disease, a disorder that involves mitochondrial (132.0±3.4 pg/ml vs 56.4±21.3 pg/ml) (Fig. 4E) and TNFα (183.8± cytochrome c release, alterations in electron transport and loss of 88.9 pg/ml vs 78.8±25.3 pg/ml) (Fig. 4F). Collectively, these mitochondrial membrane potential (ΔΨm) (Haq et al., 2003). Here, findings suggest that psychosine may enhance the LPS-induced the ΔΨm was measured using the membrane-permeant dye levels of pro-inflammatory cytokines in astrocytes as previously Journal of Cell Science

3879 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3878-3887 doi:10.1242/jcs.169342

Fig. 1. pFTY720 attenuates psychosine-mediated astrocyte cell death. (A) Diagram of experimental timeline and treatments. Human astrocytes were pre-treated with 1 µM pFTY720 (pFTY) for 1 h followedby 5 µM, 10 µM, 15 µM and 20 µM psychosine (Psy) for 1–6 h. Cellswere imaged under light microscopy. (B) Concentration- dependent psychosine-induced cell death is attenuated by pFTY. Human astrocytes were treated with 5 µM, 10 µM, 15 µM and 20 µM psychosine for 4 h±pFTY. (C) Time-dependent psychosine-induced cell death isattenuated by pFTY. Human astrocytes were pre-treated with 1 µM pFTYfor 1 h followedby10 µM psychosine for 1, 2, 4 and 6 h. (D) pFTY attenuates psychosine-induced astrocyte cell death in a concentration-dependent manner. Human astrocytes were pre-treated with 10 nM, 100 nM and 1 µM pFTY for 1 h followed by 10 µM psychosine for 2 h (black). Control incubations are shown in white. In all cases, image analysis was performed using ImageJ software and graphical data are presented as mean±s.e.m. (n=3–6). Representative images are also shown. Statistical analysis was performed using one-way ANOVA and Newman–Keuls multiple comparison post-test *P<0.05, **P<0.01 compared with pFTY; ##P<0.01, ###P<0.001 compared with control. reported (Giri et al., 2002), and that pFTY720 attenuates cerebellar slices and secondly examined whether pFTY720 these effects. attenuated this psychosine-induced demyelination. Organotypic cerebellar slices were exposed to LPC (0.5 mg/ml) or psychosine pFTY720 inhibits psychosine-induced demyelination in (100 nM, 1 µM, 20 μM) in the presence or absence of pFTY720 organotypic cerebellar slices (0.1 nM, 1 nM) for 18 h and treated for a further 30 h with pFTY720 Rapid and complete loss of and the myelin-forming (0.1 nM, 1 nM) or control medium (Fig. 5A). In agreement with our oligodendrocytes is one of the main pathological features of previous studies (Pritchard et al., 2014; Sheridan and Dev, 2012), Krabbe disease (Davenport et al., 2011). pFTY720 promotes pFTY720 attenuated LPC-induced demyelination compared with remyelination as well as limiting the demyelination induced by the control, as observed by expression of myelin oligodendrocyte bioactive lipid lysolecithin (lysophosphatidylcholine, LPC) (Miron glycoprotein (MOG) (Fig. 5B) and myelin basic protein (MBP) et al., 2010; Sheridan and Dev, 2012). With this in mind, we (Fig. 5C–D). Importantly, the exposure of the slice cultures first determined whether psychosine induced demyelination in to psychosine also decreased the expression of MOG (Fig. 5B, Journal of Cell Science

3880 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3878-3887 doi:10.1242/jcs.169342

Fig. 2. Psychosine-induced cell toxicity is dependent on astrocyte cell density. (A) Human astrocytes were seeded at densities ranging from 1.2×105 to 6×105 and pre-treated with 1 µM pFTY for 1 h followed by 10 µM psychosine for 2 h. (B) Image analysis was performed using ImageJ software. Data presented as mean±s.e.m. (n=4), one-way ANOVA and Newman–Keuls multiple comparison post-test ***P<0.001. (C) Representative images showing psychosine- induced cell loss, with or without pFTY treatment.

Fig. S1), MBP (33.4±3.8 vs 12.4±2.5, 1 µM psychosine; psychosine (100 nM) treatment did not induce the release of IL6, mean±s.e.m.) (Fig. 5C–D) and myelin proteolipid protein (PLP) TNFα or IL1β, in agreement with our data showing the treatment of (Fig. S2), as well as decreasing the expression of neurofilament H mouse astrocytes with psychosine alone had little effect on the (NFH) (29.9±5.4 vs 13.4±4.6, 1 µM psychosine). pFTY720 release of IL6 (Fig. 4C and E), TNFα (Fig. 4B and F) and IL1β (data (0.1 nM, 1 nM) prevented the psychosine-induced decrease in not shown). Furthermore, we did not observe significant effects of expression of MOG (Fig. 5B, Fig. S1), MBP (12.43±2.5 vs 31.6±4.9), psychosine on ionized calcium binding adaptor molecule 1 (Iba1) PLP (Fig. S2) and NFH (13.4±4.6 vs 40.7±10.3) (Fig. 5C-D). Taken (microglia) staining, nor did BV2 microglia cells treated with together, therefore, these results demonstrate that pFTY720 reverses psychosine show enhanced levels of IL6 cytokine release (Fig. S3). psychosine-induced demyelination and neuronal toxicity in cerebellar Taken together, these results suggest that, in organotypic cerebellar slice cultures. slices, psychosine-induced demyelination occurs via a mechanism that is probably independent from the release of pro-inflammatory Psychosine-induced demyelination in cerebellar slices cytokines. Moreover, pFTY720 inhibits this type of demyelination. occurs independently of pro-inflammatory cytokines The involvement of pro-inflammatory cytokines in the pathogenesis DISCUSSION of demyelination has been previously investigated (di Penta et al., Accumulation of psychosine, propagation of pro-inflammatory 2013), and a model in which S1PR activation may reduce cytokines, demyelination and the widespread loss of demyelination via a mechanism involving attenuation of cytokine/ oligodendrocytes are all hallmarks of the Krabbe disease brain chemokine release has been proposed (Sheridan and Dev, 2012). (Suzuki, 1998; Wenger et al., 2001). To date, little is known about Hence we investigated whether psychosine treatment would induce the role of astrocytes in Krabbe disease, where the majority of the release of the pro-inflammatory cytokines IL6, TNFα and IL1β studies have mainly been focused on the role oligodendrocytes in from organotypic cerebellar slices. As with the above protocol, this illness. Altered astrocytic function has gained recognition as a organotypic cerebellar slices were exposed to LPC (0.5 mg/ml) or major contributing factor to a growing number of neurological psychosine (100 nM) in the presence or absence of pFTY720 disorders (Claycomb et al., 2013) and the belief that astrocytic (0.1 nM) for 18 h and treated for a further 30 h with pFTY720 dysfunction significantly contributes to the development of (0.1 nM). After the 30 h incubation the medium was collected and inflammation in the CNS has gained traction in the past number analysed by ELISA. LPC induced at least a 4-fold increase in levels of years (Sharma et al., 2010). In addition, the immunomodulatory of IL6 (136.2±61.5 pg/ml vs 1543.2±89.4 pg/ml; mean±s.e.m.), functions of astrocytes are now being shown to actively participate TNFα (15.0±4.6 pg/ml vs 61.9±12.3 pg/ml) and IL1β (0.6± 0.6 pg/ in the pathogenesis of a number of demyelinating disorders (Sharma ml vs 178.8±32.7 pg/ml), compared with controls (Fig. 6A–C). et al., 2010). It has also been reported that astrocytic processes may pFTY720 treatment attenuated the LPC-induced release of IL-6 closely surround demyelinating fibres and that astrocytes release (1543.2±89.4 pg/ml vs 1273.1±22.1 pg/ml) (Fig. 6A), TNFα factors such leukaemia inhibitory factor (LIF) to promote (61.94±12.3 pg/ml vs 42.32±11.2 pg/ml) (Fig. 6B) and IL1β myelination. Remarkably, when oligodendrocytes from twi/twi

(178.8±32.7 pg/ml vs 14.0±14.1 pg/ml) (Fig. 6C). Interestingly, mice are transplanted into the shiverer mouse model of Journal of Cell Science

3881 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3878-3887 doi:10.1242/jcs.169342

Sugama et al., 1990). In agreement with the current literature (Davenport et al., 2011; Haq et al., 2003; Jatana et al., 2002), our JC- 1 experiments show that psychosine induces mitochondrial dysfunction. Hence, we suggest, the astrocytic cell death induced by psychosine is likely to occur via an apoptotic process. Importantly, pFTY720 attenuated psychosine-induced cell death as well as restoring psychosine-induced mitochondrial dysfunction. Moreover, we found that although psychosine itself did not induce increased levels of pro-inflammatory cytokines in mouse astrocytes, it did enhance LPS-mediated release of IL6, TNFα and IL1β, and these effects were again reduced by pFTY720. We also report here, for the first time, that direct application of psychosine to organotypic slice cultures induces demyelination in a manner that does not include enhanced pro-inflammatory cytokine release. These data corroborate the idea that psychosine in the brains of Krabbe disease patients may directly induce demyelination. In these set of experiments, pFTY720 attenuated LPC-induced demyelination, which was shown to include enhanced levels of pro-inflammatory cytokines, as we have reported before (Sheridan and Dev, 2012). Of most interest, pFTY720 also reduced the demyelination caused by psychosine, in a manner that did not include enhanced levels of IL6, TNFα and IL1β. Overall, these studies suggest that S1PRs may regulate myelination state in both inflammatory and non- inflammatory paradigms. Inflammatory processes have been implicated in the pathogenesis of Krabbe disease and the expression of pro-inflammatory cytokines have been reported in the twi/twi mouse brain (Claycomb et al., 2013; LeVine and Brown, 1997). However, the mechanism governing psychosine-mediated cell toxicity and the direct role of pro- inflammatory cytokines in the degeneration of astrocytes and/or oligodendrocytes is still not fully understood. Here we found that psychosine treatment itself did not alter pro-inflammatory cytokine Fig. 3. pFTY720 attenuates psychosine-induced decrease of levels, and therefore such an effect on cytokines is unlikely to explain mitochondrial membrane potential in astrocytes. (A) Schematic shows that the decrease in astrocyte cell numbers that we observed after at high mitochondrial membrane potentials JC-1 forms red aggregates psychosine treatment. Instead, psychosine may induce astrocyte cell (590 nm) in mitochondria whereas at low mitochondrial membrane potentials the JC-1 is predominately found as a green monomer (535 nm) in the cytosol. death by altering mitochondrial function and electron transport, as (B) Treatment of mouse astrocytes with psychosine (2 h) resulted in a determined by increased JC1 levels in the cytosol and decreased decrease in the ratio of red aggregates to green monomers to ∼75% of controls, NAD(P)H oxidase function measured by MTT, respectively. while pre-treatment with pFTY (1 µM, 1 h before addition of psychosine) In agreement with this idea, previous studies have demonstrated that attenuated this decrease. Data are presented as mean±s.e.m. (n=3), unpaired psychosine alters mitochondrial function and electron transfer, Student’s t-test *P<0.05, **P<0.01 comparing psychosine±pFTY. (C) Graph probably via a mechanism involving changes in the lipid shows MTT assays performed on rat astrocytes treated with psychosine for environment of the membrane (Cooper et al., 1993; Tapasi et al., 2 h±pFTY. MTT absorbance read at 540 nm. Data presented as mean±s.e.m. (n=3), one-way ANOVA and Newman–Keuls multiple comparison post-test 1989). Moreover, studies have also reported that pFTY720 can *P<0.05, **P<0.01 comparing psychosine±pFTY; ##P<0.01, ###P<0.001 stabilise mitochondrial function, supporting our findings comparing control±psychosine. that pFTY720 rescues mitochondrial dysfunction induced by psychosine. Interestingly, in this current study, although psychosine demyelination, these oligodendrocytes are capable of myelinating alone had no effect on pro-inflammatory cytokine levels, it augmented the shiverer axons (Kondo et al., 2005). This indicates that the LPS-induced release of IL6 from mouse astrocytes, with a similar demyelination in Krabbe disease may not be solely attributed to trend for the levels of TNFα and IL1β. This finding is comparable to oligodendrocyte dysfunction and that given correct environmental those reported previously where psychosine potentiated LPS-induced support these twi/twi oligodendrocytes may function normally. production of TNFα, IL6, IL1β andNOinprimaryratastrocytes, Thus, astrocytic reactivity in Krabbe disease may not represent a which in turn was suggested to induce oligodendrocyte cell death secondary response to demyelination, but may possibly be a (Giri et al., 2002, 2006). These enhanced levels of cytokines primary response to psychosine and in turn astrocytes may were reduced by pFTY720, in agreement with previous studies from significantly contribute to the pathogenesis of Krabbe disease ourand other groups demonstrating that S1PRs playa role in regulating (Claycomb et al., 2013). the levels of cytokines in a number of immune and glial cells In our current study, we obtained data that demonstrated a direct (Choi et al., 2011; Sheridan and Dev, 2012; Wang et al., 2007; effect of psychosine on astrocyte cell death and attenuation by Zhang et al., 2008). Thus, in astrocytes, it appears that psychosine pFTY720. We investigated the effect of the cytotoxic lipid directly modulates mitochondrial function to induce cell death, metabolite psychosine on cultures of human astrocytes. while in parallel enhancing cytokine levels under conditions of LPS- Psychosine caused a time- and concentration-dependent decrease induced inflammation, and that pFTY720 can attenuate these effects in astrocyte cell numbers, as previously reported (Giri et al., 2002; of psychosine. Journal of Cell Science

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Fig. 4. Psychosine potentiates LPS-induced production of pro-inflammatory cytokines in primary mouse astrocytes. Mouse astrocytes were serum starved for 4 h and pre-treated for 1 h with 1 µM pFTY before treatment with LPS and/or 1 µM or 10 µM psychosine. The supernatant was collected and an ELISA performed. Treatment of mouse astrocytes with LPS (1 ng/ml–10 µg/ml) induced a concentration-dependent increase in levels of (A) IL6, (B) TNFα. Mouse astrocytes treated with 100 ng/ml LPS±psychosine treatment for (C,D) 6 h or (E,F) 12 h showed an increase in (C,E) IL6 or (D,F) TNFα (white bars) that was attenuated by pre-treatment with 1 µM pFTY (black bars) . Data are presented as mean±s.e.m. (n=3–4), one-way ANOVA and Newman–Keuls multiple comparison post-test *P<0.05, ***P<001; unpaired Student’s t-test #P<0.05 compared with LPS alone. Profound demyelination and almost complete loss of generation of LPC and arachidonic acid, and inhibition of secreted oligodendrocytes are two of the major pathological features of phospholipase A2 (sPLA2) attenuates psychosine-induced Krabbe disease. The hypothesis that supraphysiological levels increases in both LPC and arachidonic acid as well as attenuating of psychosine kill oligodendrocytes and result in widespread psychosine-induced cell death (Giri et al., 2006). Furthermore, demyelination is now widely accepted. The mechanisms by which psychosine accumulation has been reported to induce psychosine induces demyelination remains unclear at present. phosphorylation of neurofilament proteins, resulting in reduced Increasing evidence however now suggests that the widespread radial growth of axons in the twi/twi mouse model and to induce demyelination and loss of oligodendrocytes seen in Krabbe disease axonal defects and cell death in isolated neuronal cultures (Cantuti- and induced by psychosine is due to apoptotic processes, probably Castelvetri et al., 2012; Castelvetri et al., 2011). S1PRs are known to via caspase dependent pathways (Zaka and Wenger, 2004; Tohyama play many roles in the regulation of differentiation, cell survival and et al., 2001; Giri et al., 2006; Giri et al., 2008; Haq et al., 2003). apoptosis of oligodendrocytes, astrocytes and microglia (Dev et al., Another proposed mechanism by which psychosine induces 2008; Miron et al., 2008b). In addition, previous studies have also toxicity involves the preferential accumulation of the molecule in shown that S1PRs are expressed on Schwann cells and that lipid rafts, associated with regional cholesterol increases and treatment with compounds such as pFTY720 can promote Schwann inhibition of PKC activity (White et al., 2011; Davenport et al., cell survival and regulate peripheral myelination (Kim et al., 2011; Yamada et al., 1996; Hannun and Bell, 1987). Psychosine 2011). Moreover studies have also shown that compounds such as treatment of oligodendrocytes has also been shown to induce the pFTY720 can significantly improve motor function recovery in Journal of Cell Science

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Fig. 5. pFTY720 treatment inhibits psychosine-induced demyelination of cerebellar slices. (A) Organotypic slice cultures were prepared from the cerebellum of P10 mice and grown in culture for 12 days. Slices were treated with LPC, psychosine and/or pFTY for 18 h. The medium was then changed and pFTY treatment continued for a further 30 h, or control medium was added for this time. Cerebellar culture were then processed for western blotting or immunocytochemistry. (B) LPC (0.4 mg/ml) and psychosine (100 nM or 1 μM) treatment induced a reduction in MOG expression, which was rescued by pFTY720 (0.1 nM) treatment. + with pFTY; −, without pFTY. (C) Representative confocal images displaying MBP (MBP, green) and neurofilament (NFH, red) immunostaining under treatment conditions indicated. Confocal images captured at ×10 magnification. Treatment with pFTY (0.1 nm) attenuates demyelination induced by LPC (0.4 mg/ml) and psychosine (100 nM or 1 μM). ML, molecular layer; WM, white matter; PCL, Purkinje cell layer; GCL, granule cell layer. (D) Bar graph illustrating changes in MBP and NFH staining after LPC (0.4 mg/ml) and psychosine (100 nM, 1 μM)±pFTY (0.1 nM) treatments. Mean fluorescence was calculated using a total of 25–36 independent ROI observations in each experiment. Data are presented as mean±s.e.m. (n=3), one-way ANOVA and Newman–Keuls multiple comparison post-test *P<0.05; #P<0.05 comparing control±psychosine. animal models of spinal cord injury (Lee et al., 2009). Therefore, we inflammatory cytokines and most importantly that pFTY720 can used organotypic slice cultures as a more complex cellular model to inhibit demyelination independently from the regulation of pro- investigate the effect of psychosine on demyelination. Here, for the inflammatory cytokines. Taking these current findings and previous first time, we showed that psychosine can directly induce a studies that have demonstrated pFTY720 can promote neuronal and concentration-dependent demyelination, as expressed by a decrease oligodendrocyte survival (Dev et al., 2008; Miron et al., 2008b) into in the levels of MOG, PLP and MBP, and that pFTY720 can account, we suggest that pFTY720 may rescue the toxic effects attenuate these effects. These findings are in agreement with our and induced by psychosine by having multimodal effects on both glial other previous reports demonstrating that pFTY720 rescues and neuronal cells. However, we acknowledge the need for further myelination (Coelho et al., 2007; Jung et al., 2007; Mattes et al., studies, including: (1) ultrastructural EM analysis of remyelination 2010; Miron et al., 2008a,b; Sheridan and Dev, 2012). Interestingly, to support our immunostaining data, which is not a direct unlike LPC, psychosine treatment alone did not induce the release of measurement of enhanced myelination per se, (2) testing the IL6, TNFα or IL1β from cerebellar slices, which was in agreement effects of pFTY720 on myelination in twi/twi cerebellar slice with our astrocyte data. These findings suggest that psychosine cultures to further test the hypothesis of psychosine-induced induces demyelination independently from the release of pro- demyelination and (3) most importantly, testing the effects of Journal of Cell Science

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split into 24-well plates to be treated. In all cases, before treatment cells were serum starved for 3–4 h by incubating in serum-free DMEM/F12 (Fisher) at 37°C and 5% CO2. Specific treatment details are indicated in the figure legends. Organotypic cerebellar slice cultures were prepared exactly as we have described previously (Pritchard et al., 2014; Sheridan and Dev, 2012). In brief, tissue isolated from postnatal day 10 (P10) C57BL/6 mice and 400 µm parasagittal slices of cerebellum were grown on cell culture inserts (5–6 slices each) (Millicell PICMORG50 Millipore). Slices were cultured using an interface method with 1 ml of medium per 35 mm well. For the first 3daysin vitro (DIV), slices were grown in serum-based medium (50% Opti- Mem (Invitrogen), 25% Hanks’ buffered salt solution (HBSS) (Gibco), 25% heat-inactivated horse serum and supplemented with 2 mM Glutamax, 28 mM D-glucose, 100 U/ml penicillin/streptomycin (Sigma) and 25 mM HEPES (Sigma), at 35.5°C and 5% CO2. After 3 DIV, slices were transferred to serum-free medium (98% Neurobasal-A and 2% B-27 (Invitrogen), supplemented with 2 mM Glutamax, 28 mM D-glucose, 100 U/ml penicillin/streptomycin and 25 mM HEPES). Demyelination was induced at 12 DIV and examined at 14 DIV. All tissue was isolated in accordance with EU guidelines and protocols approved by the Trinity College Dublin ethics committee.

Biochemical analysis For cytokine analysis supernatants from cell culture were removed and examined for their cytokine content using ELISA kits for IL6 (DY406), TNFα (DY410) and IL-1β (DY401) according to the manufacturer’s instructions (R&D Systems), and exactly as we have described before (Pritchard et al., 2014). For western blotting, cerebellar slices were scraped from the culture membrane and suspended in PTxE buffer (PBS, 1% Triton- x, 1 mM EDTA) using mechanical homogenisation and sonication. Samples were denatured and electrophoresis carried out on 10% SDS-polyacrylamide gels exactly as we have previously reported (Pritchard et al., 2014). Primary antibodies used were: anti-MOG (1:2000; Millipore: MAB5680) and anti- tubulinβ (1:5000; Millipore MAB3408). Secondary antibody used was: HRP-conjugated mouse (1:10,000; Sigma: A8924).

JC-1 and MTT assays JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) exhibits potential- dependent accumulation in mitochondria. Measurements of mitochondrial membrane potential (ΔΨm) using membrane-permeant dyes such as JC-1 are therefore widely used in apoptosis studies to monitor mitochondrial health. Mouse astrocytes were plated into a black 96-well plate in 100 μl DMEM/F12 and incubated for 72 h at 37°C and 5% CO2. On the day of the Fig. 6. Psychosine treatment does not induce the release of pro- assay, the cells were incubated for 3 h in serum-free DMEM/F12 at 37°C inflammatory cytokines from cerebellar slice cultures. Organotypic slice and 5% CO2 before receiving a pre-treatment of pFTY720 (1 µM) for 1 h. cultures prepared from the cerebellum of P10 mice were grown in culture for Cells were then treated with psychosine (5 µM, 10 µM, 15 µM, 20 µM) for 12 days before treatment with LPC (0.4 mg/ml), psychosine (100 nM) and/or 2 h. All wells were then loaded with 1 μM JC-1 for 30 min. After this pFTY (0.1 nM) for 18 h. Medium was then changed and pFTY treatment incubation the plate was centrifuged for 5 min at 400 g at room temperature. continued for a further 30 h. The media was collected and cytokine analysis μ performed by ELISA. Psychosine treatment did not induce the release of The supernatant was aspirated and the wells washed with 200 l of PBS. (A) IL6, (B) TNFα and (C) IL1β from cerebellar slice cultures. In contrast, This wash step was repeated and the emission spectra at 535 nm and treatment with LPC induced the release of all three cytokines analysed, and 590 nm was measured (SpectraMAX Gemini XS). A colorimetric 3-(4,5- pFTY attenuated this LPC-induced release of (A) IL6 (n=3), (B) TNFα (n=3) dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was and (C) IL1β (n=2). Data are presented as mean±s.e.m., one-way ANOVA and used to assess cell viability as per manufacturer’s instructions (Invitrogen, Newman–Keuls multiple comparison post-test *P<0.05, ***P<0.001. M6494), similar to our previous studies (Mullershausen et al., 2009; Rutkowska et al., 2015). pFTY720 on disease progression in twi/twi mice. Although these studies remain outstanding and while recent clinical data shows a Light and fluorescence microscopy All images of human astrocytes were taken using a CKX41 Olympus lack of efficacy for pFTY720 in progressive forms of multiple inverted microscope (Mason Technologies) at 10× magnification. ImageJ sclerosis, the cellular data presented in the current study goes some software was used to calculate the cell density of the surviving cells by way in suggesting that S1PRs may be useful targets in measuring the percentage of empty space compared with their own controls. demyelinating illnesses such as Krabbe disease. For organotypic cerebellar slice cultures, immunostaining was performed as we have described previously (Sheridan and Dev, 2012). Primary antibodies MATERIALS AND METHODS used were: anti-MBP (1:1000; Abcam: ab40390), anti-NFH (1:1000; Astrocyte and cerebellar slice cultures Millipore: MAB5539), anti-PLP (1:500; Millipore: MAB388), anti-MOG Human, rat and mouse astrocytes were cultured as we have described before (1:1000; Millipore: MAB5680) and anti-Iba1 (1:1000; Wako: 019-1974). (Healy et al., 2013; Rutkowska et al., 2015). BV2 microglia were cultured Secondary antibodies used were: anti-chicken 633 (1:1000; Invitrogen in DMEM supplemented with 2% FBS (Labtech) and 1% penicillin/ Alexa: A21103), anti-rabbit 488 (1:2000; Invitrogen Alexa: A27034) and streptomycin (Sigma) at 37°C and 5% CO2. When 80% confluent cells were anti-mouse Dylight 549 (1:2000; Jackson ImmunoResearch: 715-505-020). Journal of Cell Science

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Confocal images were captured using a LSM 510 Meta microscope at 10× or Deogracias, R., Yazdani, M., Dekkers, M. P. J., Guy, J., Ionescu, M. C. S., Vogt, 20× magnification. These resulting images were analysed using ImageJ K. E. and Barde, Y.-A. (2012). Fingolimod, a sphingosine-1 phosphate receptor software. A total of 5–6 slices were used per condition and the fluorescence modulator, increases BDNF levels and improves symptoms of a mouse model of – Rett syndrome. Proc. Natl. Acad. Sci. USA 109, 14230-14235. of each cerebellar slice was captured using 5 6 independent regions of Dev, K. K., Mullershausen, F., Mattes, H., Kuhn, R. R., Bilbe, G., Hoyer, D. and interest (ROI). The ROI were selected randomly to cover the whole slice and Mir, A. (2008). Brain sphingosine-1-phosphate receptors: implication for FTY720 the mean fluorescence was calculated using a total of 25–36 independent in the treatment of multiple sclerosis. Pharmacol. Ther. 117, 77-93. ROI observations for each independent experiment. di Penta, A., Moreno, B., Reix, S., Fernandez-Diez, B., Villanueva, M., Errea, O., Escala, N., Vandenbroeck, K., Comella, J. X. and Villoslada, P. (2013). Oxidative stress and proinflammatory cytokines contribute to demyelination and Statistical analysis axonal damage in a cerebellar culture model of neuroinflammation. PLoS ONE 8, All statistical analysis was performed using Prism 5 GraphPad Software e54722. package. A one-way ANOVA with Newman–Keuls post-hoc test was used Fischer, I., Alliod, C., Martinier, N., Newcombe, J., Brana, C. and Pouly, S. to compare groups and an unpaired Student’s t-test was used to compare two (2011). Sphingosine kinase 1 and sphingosine 1-phosphate receptor 3 are sets of data with each other. Individual statistical tests are described in text functionally upregulated on astrocytes under pro-inflammatory conditions. PLoS ONE 6, e23905. and figure legends. The significance levels (or alpha levels) were set at P P P Fyrst, H. and Saba, J. D. (2010). An update on sphingosine-1-phosphate and other <0.05, <0.01 and <0.001. sphingolipid mediators. Nat. Chem. Biol. 6, 489-497. Giri, S., Jatana, M., Rattan, R., Won, J.-S., Singh, I. and Singh, A. K. (2002). Acknowledgements Galactosylsphingosine (psychosine)-induced expression of cytokine-mediated We are grateful to Dr Anis Mir (Novartis Pharma, Basel, Switzerland) for supply of inducible nitric oxide synthases via AP-1 and C/EBP: implications for Krabbe pFTY720 and to Prof. Veronica Campbell (School of Medicine, Trinity College disease. FASEB J. 16, 661-672. Dublin) for supply of BV2 microglia. Giri, S., Khan, M., Rattan, R., Singh, I. and Singh, A. K. (2006). Krabbe disease: psychosine-mediated activation of phospholipase A2 in oligodendrocyte cell death. J. Lipid Res. 47, 1478-1492. Competing interests Giri, S., Khan, M., Nath, N., Singh, I. and Singh, A. K. (2008). The role of AMPK in The authors declare no competing or financial interests. psychosine mediated effects on oligodendrocytes and astrocytes: implication for Krabbe disease. J. Neurochem. 105, 1820-1833. Author contributions Hannun, Y. A. and Bell, R. M. (1987). Lysosphingolipids inhibit protein kinase C: K.K.D. conceived, supervised and co-ordinated the study. C.O. performed the implications for the . Science 235, 670-674. experiments and analysed the data. K.K.D. and C.O. wrote the manuscript. Haq, E., Giri, S., Singh, I. and Singh, A. K. (2003). Molecular mechanism of psychosine-induced cell death in human oligodendrocyte cell line. J. Neurochem. Funding 86, 1428-1440. This work was supported by The Higher Education Authority Ireland [Programme for Healy, L. M., Sheridan, G. K., Pritchard, A. J., Rutkowska, A., Mullershausen, F. Research in Third Level Institutions (PRTLI)] and co-funded by the European and Dev, K. K. (2013). Pathway specific modulation of S1P1 receptor signalling in Regional Development Fund and the HEA. C.O. is a PRTLI funded PhD scholar. rat and human astrocytes. Br. J. Pharmacol. 169, 1114-1129. Jatana, M., Giri, S. and Singh, A. K. (2002). Apoptotic positive cells in Krabbe brain and induction of apoptosis in rat C6 glial cells by psychosine. Neurosci. Lett. 330, Supplementary information 183-187. Supplementary information available online at Jung, C. G., Kim, H. J., Miron, V. E., Cook, S., Kennedy, T. E., Foster, C. A., Antel, http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.169342/-/DC1 J. P. and Soliven, B. (2007). Functional consequences of S1P receptor modulation in rat oligodendroglial lineage cells. 55, 1656-1667. References Kappos, L., Radue, E.-W., O’Connor, P., Polman, C., Hohlfeld, R., Calabresi, P., Adachi, K. and Chiba, K. (2008). FTY720 story. Its discovery and the following Selmaj, K., Agoropoulou, C., Leyk, M., Zhang-Auberson, L. et al. (2010). A accelerated development of sphingosine 1-phosphate receptor agonists as placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. immunomodulators based on reverse pharmacology. Perspect. Med. Chem. 1, N. Engl. J. Med. 362, 387-401. 11-23. Kim, H. J., Miron, V. E., Dukala, D., Proia, R. L., Ludwin, S. K., Traka, M., Antel, J. Asle-Rousta, M., Kolahdooz, Z., Oryan, S., Ahmadiani, A. and Dargahi, L. P. and Soliven, B. (2011). Neurobiological effects of sphingosine 1-phosphate (2013). FTY720 (Fingolimod) Attenuates Beta-amyloid Peptide (Aβ42)-induced receptor modulation in the cuprizone model. FASEB J. 25, 1509-1518. Kondo, Y., Wenger, D. A., Gallo, V. and Duncan, I. D. (2005). impairment of spatial learning and memory in rats. J. Mol. Neurosci. 50, 524-532. Galactocerebrosidase-deficient oligodendrocytes maintain stable central myelin Balatoni, B., Storch, M. K., Swoboda, E.-M., Schönborn, V., Koziel, A., by exogenous replacement of the missing enzyme in mice. Proc. Natl. Acad. Sci. Lambrou, G. N., Hiestand, P. C., Weissert, R. and Foster, C. A. (2007). USA 102, 18670-18675. FTY720 sustains and restores neuronal function in the DA rat model of MOG- Lee, K. D., Chow, W. N., Sato-Bigbee, C., Graf, M. R., Graham, R. S., Colello, R. induced experimental autoimmune encephalomyelitis. Brain Res. Bull. 74, J., Young, H. F. and Mathern, B. E. (2009). FTY720 reduces inflammation and 307-316. promotes functional recovery after spinal cord injury. J. Neurotrauma 26, Cantuti-Castelvetri, L., Zhu, H., Givogri, M. I., Chidavaenzi, R. L., Lopez-Rosas, 2335-2344. A. and Bongarzone, E. R. (2012). Psychosine induces the dephosphorylation of LeVine, S. M. and Brown, D. C. (1997). IL-6 and TNFα expression in brains of neurofilaments by deregulation of PP1 and PP2A phosphatases. Neurobiol. Dis. twitcher, quaking and normal mice. J. Neuroimmunol. 73, 47-56. 46, 325-335. Mattes, H., Dev, K. K., Bouhelal, R., Barske, C., Gasparini, F., Guerini, D., Castelvetri, L. C., Givogri, M. I., Zhu, H., Smith, B., Lopez-Rosas, A., Qiu, X., Mir,A.K.,Orain,D.,Osinde,M.,Picard,A.etal.(2010). Design and van Breemen, R. and Bongarzone, E. R. (2011). Axonopathy is a compounding synthesis of selective and potent orally active S1P5 agonists. ChemMedChem factor in the pathogenesis of Krabbe disease. Acta Neuropathol. 122, 35-48. 5, 1693-1696. Choi, J. W., Gardell, S. E., Herr, D. R., Rivera, R., Lee, C.-W., Noguchi, K., Miron, V. E., Hall, J. A., Kennedy, T. E., Soliven, B. and Antel, J. P. (2008a). Teo, S. T., Yung, Y. C., Lu, M., Kennedy, G. et al. (2011). FTY720 (fingolimod) Cyclical and dose-dependent responses of adult human mature oligodendrocytes efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine to fingolimod. Am. J. Pathol. 173, 1143-1152. 1-phosphate receptor 1 (S1P1) modulation. Proc. Natl. Acad. Sci. USA 108, Miron, V. E., Jung, C. G., Kim, H. J., Kennedy, T. E., Soliven, B. and Antel, J. P. (2008b). FTY720 modulates human oligodendrocyte progenitor process 751-756. extension and survival. Ann. Neurol. 63, 61-71. Claycomb, K., Johnson, K., Winokur, P., Sacino, A. and Crocker, S. (2013). Miron, V. E., Ludwin, S. K., Darlington, P. J., Jarjour, A. A., Soliven, B., Astrocyte regulation of CNS inflammation and remyelination. Brain Sci. 3, Kennedy, T. E. and Antel, J. P. (2010). Fingolimod (FTY720) enhances 1109-1127. remyelination following demyelination of organotypic cerebellar slices. Coelho, R. P., Payne, S. G., Bittman, R., Spiegel, S. and Sato-Bigbee, C. (2007). Am. J. Pathol. 176, 2682-2694. The immunomodulator FTY720 has a direct cytoprotective effect in Mullershausen, F., Craveiro, L. M., Shin, Y., Cortes-Cros, M., Bassilana, F., oligodendrocyte progenitors. J. Pharmacol. Exp. Ther. 323, 626-635. Osinde, M., Wishart, W. L., Guerini, D., Thallmair, M., Schwab, M. E. et al. Cooper, C. E., Markus, M., Seetulsingh, S. P. and Wrigglesworth, J. M. (1993). (2007). Phosphorylated FTY720 promotes astrocyte migration through Kinetics of inhibition of purified and mitochondrial cytochrome c oxidase by sphingosine-1-phosphate receptors. J. Neurochem. 102, 1151-1161. psychosine (beta-galactosylsphingosine). Biochem. J. 290, 139-144. Mullershausen, F., Zecri, F., Cetin, C., Billich, A., Guerini, D. and Seuwen, K. Davenport, A., Williamson, P. and Taylor, R. (2011). Pathophysiology of Krabbe (2009). Persistent signaling induced by FTY720-phosphate is mediated by

disease. Orbit 2, 1-20. internalized S1P1 receptors. Nat. Chem. Biol. 5, 428-434. Journal of Cell Science

3886 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3878-3887 doi:10.1242/jcs.169342

Orfi, L., Larive, C. K. and LeVine, S. M. (1997). Physicochemical characterization Tapasi, S., Padma, P. and Setty, O. H. (1998). Effect of psychosine on of psychosine by 1H nuclear magnetic resonance and electron microscopy. Lipids mitochondrial function. Indian J. Biochem. Biophys. 35, 161-165. 32, 1035-1040. Tohyama, J., Matsuda, J. and Suzuki, K. (2001). Psychosine is as potent an Osinde, M., Mullershausen, F. and Dev, K. K. (2007). Phosphorylated FTY720 inducer of cell death as C6-ceramide in cultured fibroblasts and in MOCH-1 cells. stimulates ERK phosphorylation in astrocytes via S1P receptors. Neurochem. Res. 26, 667-671. Neuropharmacology 52, 1210-1218. Wang, W., Huang, M.-C. and Goetzl, E. J. (2007). Type 1 sphingosine 1-phosphate Pritchard, A. J., Mir, A. K. and Dev, K. K. (2014). Fingolimod attenuates G protein-coupled receptor (S1P1) mediation of enhanced IL-4 generation by splenocyte-induced demyelination in cerebellar slice cultures. PLoS ONE 9, CD4T cells from S1P1 transgenic mice. J. Immunol. 178, 4885-4890. e99444. Wenger, D. A., Rafi, M. A. and Luzi, P. (1997). Molecular genetics of Krabbe Rutkowska, A., Preuss, I., Gessier, F., Sailer, A. W. and Dev, K. K. (2015). EBI2 regulates intracellular signaling and migration in human astrocyte. Glia 63, disease (globoid cell leukodystrophy): diagnostic and clinical implications. Hum. 341-351. Mutat. 10, 268-279. Sharma, R., Fischer, M.-T., Bauer, J., Felts, P. A., Smith, K. J., Misu, T., Fujihara, Wenger, D. A., Suzuki, K., Suzuki, Y. and Suzuki, K. (2001). Galactosylceramide K., Bradl, M. and Lassmann, H. (2010). Inflammation induced by innate Lipidosis: Globoid Cell Leukodystrophy (Krabbe Disease), pp. 3669-3694. immunity in the leads to primary astrocyte dysfunction New York. McGraw-Hill. followed by demyelination. Acta Neuropathol. 120, 223-236. White, A. B., Galbiati, F., Givogri, M. I., lopez rosas, A., Qiu, X., Van breemen, R. Sheridan, G. K. and Dev, K. K. (2012). S1P1 receptor subtype inhibits and Bongarzone, E. R. (2011). Persistence of psychosine in brain lipid rafts is a demyelination and regulates chemokine release in cerebellar slice cultures. Glia limiting factor in the therapeutic recovery of a mouse model for Krabbe disease. J. 60, 382-392. Neurosci. Res. 89, 352-364. Sugama, S., Kim, S. U., Ida, H. and Eto, Y. (1990). Psychosine cytotoxicity in rat Yamada, H., Martin, P. and Suzuki, K. (1996). Impairment of protein kinase C neural cell cultures and protection by phorbol ester and dimethyl sulfoxide. activity in twitcher Schwann cells in vitro. Brain Res. 718, 138-144. Pediatr. Res. 28, 473-476. Zaka, M. and Wenger, D. A. (2004). Psychosine-induced apoptosis in a mouse Suzuki, K. (1998). Twenty five years of the ‘Psychosine Hypothesis’: a personal perspective of its history and present status. Neurochem. Res. 23, 251-259. oligodendrocyte progenitor cell line is mediated by caspase activation. Neurosci. Suzuki, K. (2003). Globoid cell leukodystrophy (Krabbe’s disease): update. J. Child Lett. 358, 205-209. Neurol. 18, 595-603. Zhang, Z., Fauser, U. and Schluesener, H. J. (2008). Early attenuation of lesional Svennerholm, L., Vanier, M. T. and Månsson, J. E. (1980). Krabbe disease: a interleukin-16 up-regulation by dexamethasone and FTY720 in experimental galactosylsphingosine (psychosine) lipidosis. J. Lipid Res. 21, 53-64. traumatic brain injury. Neuropathol. Appl. Neurobiol. 34, 330-339. Journal of Cell Science

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