Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jun 1.
Published in final edited form as: J Neurochem. 2012 Apr 12;121(5):806–817. doi: 10.1111/j.1471-4159.2012.07723.x

Ethanol triggers sphingosine 1-phosphate elevation along with neuroapoptosis in the developing mouse brain

Goutam Chakraborty 1, Mitsuo Saito 2,3, Relish Shah 1, Rui-Fen Mao 1, Csaba Vadasz 1,3, Mariko Saito 1,3
PMCID: PMC3342397  NIHMSID: NIHMS364043  PMID: 22393932

Abstract

Our previous studies have indicated that de novo ceramide synthesis plays a critical role in ethanol-induced apoptotic neurodegeneration in the 7-day-old mouse brain. Here, we examined whether the formation of sphingosine 1-phosphate (S1P), a ceramide metabolite, is associated with this apoptotic pathway. Analyses of basal levels of S1P-related compounds indicated that S1P, sphingosine, sphingosine kinase 2, and S1P receptor 1 increased significantly during postnatal brain development. In the 7-day-old mouse brain, sphingosine kinase 2 was localized mainly in neurons. Subcellular fractionation studies of the brain homogenates showed that sphingosine kinase 2 was enriched in the plasma membrane and the synaptic membrane/synaptic vesicle fractions, but not in the nuclear and mitochondrial/lysosomal fractions. Ethanol exposure in 7-day-old mice induced sphingosine kinase 2 activation and increased the brain level of S1P transiently 2-4h after exposure, followed by caspase-3 activation that peaked around 8h after exposure. Treatment with dimethylsphingosine, an inhibitor of sphingosine kinases, attenuated the ethanol-induced caspase-3 activation and the subsequent neurodegeneration. These results indicate that ethanol activates sphingosine kinase 2, leading to a transient increase in S1P, which may be involved in neuroapoptotic action of ethanol in the developing brain.

Keywords: sphingosine 1-phosphate, sphingosine kinase 2, ethanol, apoptotic neurodegeneration, developing brain, plasma membrane

Introduction

Ethanol triggers apoptotic neurodegeneration in the newborn rodent brain during the period of rapid synaptogenesis that corresponds to the last trimester of pregnancy in humans (Ikonomidou et al., 2000; Olney et al., 2002), and causes long-lasting neuronal loss and impaired neurobehavior as observed in human fetal alcohol spectrum disorders (FASD). This rodent model for FASD has been widely utilized to elucidate mechanisms of ethanol-induced apoptotic neurodegeneration (Carloni et al., 2004; Han et al., 2006; Young et al., 2005). We have previously demonstrated that ethanol-induced neurodegeneration in 7-day-old (postnatal day 7; P7) mice is accompanied by increases in several brain lipids (Saito et al., 2007a). Among them, de novo ceramide synthesis appears to play a vital role in ethanol-induced apoptosis, because ceramide elevation is associated with ethanol-induced caspase-3 activation, and because inhibitors of serine palmitoyltransferase, a rate-limiting enzyme in sphingolipid synthesis, rescue ethanol-induced apoptosis (Saito et al., 2010a). However, we cannot rule out the possibility that ceramide metabolites, specifically sphingosine and sphingosine 1-phosphate (S1P), are also involved in the ethanol-induced apoptotic pathway because these lipids have been implicated as regulators of the cell survival/death pathways.

It is generally postulated that ceramide and sphingosine induce growth arrest or apoptosis (Ogretmen and Hannun, 2004), while S1P promotes cell proliferation and cell survival (Spiegel and Milstien, 2003), and the balance between these bioactive lipids, termed `sphingolipid rheostat', determines cell fate (Spiegel and Milstien, 2003). This sphingolipid rheostat is mainly regulated by two isoforms of sphingosine kinases, sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2), which phosphorylate sphingosine to form S1P. It has been indicated that SphK1, a cytosolic protein, is translocated to the plasma membrane after activation (Johnson et al., 2002; Pitson et al., 2005) and exerts a pro-survival influence, whereas SphK2, a predominantly nuclear protein, inhibits cell growth and enhances apoptosis (Igarashi et al., 2003). While most of the S1P effects are mediated by the interaction of S1P with five G-protein-coupled cell surface receptors termed S1P receptor 1–5 (Spiegel and Milstien, 2003; Snider et al., 2010), intracellular actions of S1P have also been reported (Olivera and Spiegel, 1993). Induction of apoptosis by overexpressed SphK2 is independent of activation of S1P receptors (Liu et al., 2003), and S1P produced by SphK2 in the nucleus (Igarashi et al., 2003) as well as S1P produced by SphK2 in the endoplasmic reticulum (ER) (Maceyka et al., 2005; Hagen et al., 2009) have been reported to exert apoptotic action.

In the nervous system, S1P has a critical role in neural development. Dysfunction of SphK1/2 in SphK1/2 double knockout mice leads to embryonic lethality (Mizugishi et al., 2005). S1P plays roles in neurogenesis, neurite formation, and neuroprotection (Shinpo, et al., 1999; Okada et al., 2009; Agudo-Lopez et al., 2010), and may also be involved in astrocyte proliferation (Pebay et al., 2001; Malchinkhuu et al., 2003; Sorensen et al., 2003;Yamagata et al., 2003; Lee et al., 2010) and microglial activation (Nayak et al., 2010). Most of the SphK activity in the brain appears to be due to SphK2, which is localized in neurons, while SphK1 is localized primarily in astrocytes (Blondeau et al., 2007). Whereas activation of the SphK1/S1P axis signaling appears to be related to proliferation of astrocytes (Wu et al., 2008; Lee et al., 2010), protection of oligodendrocyte progenitors from apoptosis (Saini et al., 2005), and microglial activation (Nayak et al., 2010), SphK2 has been implicated to cause apoptosis through intracellular targets in cerebellar granule neurons derived from S1P lyase-deficient mice (Hagen et al., 2009). However, in some animal models of brain ischemia, SphK2 activation is considered neuroprotective (Wacker et al., 2009; Hasegawa et al., 2010; Pfeilschifter et al., 2011; Yung et al., 2012).

While S1P is thought to play important roles in the developing brain, profiles and functions of the S1P system have not been well studied in the early postnatal brain. Here, we examined S1P metabolism with a particular focus on SphK2 under the basal and ethanol-treated conditions in the P7 mouse brain and evaluated the possibility that S1P is involved in ethanol-induced apoptotic neurodegeneration.

Materials and Methods

Animals

C57BL/6By mice were maintained at the Animal Facility of Nathan S. Kline Institute for Psychiatric Research. All procedures followed guidelines consistent with those developed by the National Institute of Health and the Institutional Animal Care and Use Committee of Nathan S. Kline Institute.

Experimental Procedure

C57BL/6By mice were subcutaneously injected with saline (control) or ethanol at P7 as described previously (Olney et al., 2002; Saito et al., 2007a, b) except that one-time injection with 25 μl/g body weight of ethanol (5.0 g/kg, 20% solution in saline) or saline was performed instead of two-time injections of 2.5 g/kg ethanol with a 2h-interval, because the present study included short-term (less than two hours) treatment conditions. It has been reported that blood ethanol levels obtained by this one-time injection protocol are similar to those of two-time injections (Ieraci and Herrera, 2006). The effects of d-erythro-N,N-dimethylsphingosine (DMS, a SphK inhibitor) on ethanol-induced caspase-3 activation and the subsequent neurodegeneration were examined using both one-time and two-time ethanol injection paradigms. In both cases, DMS (3 μg in 1.5 μl DMSO) was administered 0.5h before the first ethanol injection via intracerebroventricular injection as described (Sadakata et al., 2007). Ethanol-induced caspase-3 activation at 8h after the first ethanol injection was indirectly assessed by measuring increases in cleaved caspase-3 (CC3), and cleaved tau (Ctau) levels by immunoblotting. We have previously shown that Ctau formation is mainly catalyzed by caspase-3 and detected noticeably in degenerating axons/dendrites (Saito et al., 2010b). Ethanol-induced neurodegeneration was assessed by Fluoro-Jade C (Millipore, Billerica, MA, USA) staining using brain sections from mice perfusion-fixed 19h after the first ethanol injection. Except for brief periods of time for injections, mice were kept with dams until sacrificed 1–24h after the saline/ethanol injection. For developmental studies, P1, 4, 7, 10, 13, 16, 19, 25, and 31 naïve mice were used. Three to ten animals were used for each data point.

Lipid analysis

Ceramide and sphingomyelin were separated from total lipids using high performance thin layer chromatography, and the amounts were measured as described previously (Saito et al., 2010a). Determination of S1P and sphingosine content was performed according to the method of He et al. (2009), modified as described in detail in Appendix S1.

Immunohistochemistry

Eight and 19 hours after the ethanol injection, mice were perfusion-fixed with a 4% paraformaldehyde solution, and vibratome sections (50 μm) of the fixed brains were prepared and immunofluorescence-labeled as described previously (Saito et al., 2007b, 2010a) using antibodies against SphK2, NeuN, and Na+,K+-ATPase (see Table S1 for details of the antibodies used). In order to check the specificity of anti-SphK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the antibody was incubated with the blocking peptide (Santa Cruz) for 1h at room temperature prior to incubation with brain sections. For some experiments, the above SphK2 antibody was further affinity-purified as follows: P2 (mitochondria/synaptosome) fraction of P7 brain homogenates was isolated as described below in the subcellular fractionation section, and was separated on SDS gel electrophoresis and blotted on nitrocellulose membranes. A strip of the membrane containing a band corresponding to SphK2 was blocked with 5% BSA in Tris-buffered saline (pH 7.5) containing 0.1% Tween 20 and incubated with the antibody in the blocking buffer overnight at 4°C. After rinsing, the antibody was eluted with 50 mM glycine/150 mM NaCl (pH 2.4) and neutralized. The antibody thus purified gave a single band when analyzed by immunoblotting. For Fluoro-Jade C staining, brain sections were processed according to the manufacturer's instruction. The extent of neurodegeneration was expressed as the number of Fluoro-Jade C-positive cells per square millimeter in the cingulate and retrosplenial cortices as described previously (Saito et al., 2010a) using four brains per treatment group. Photomicrographs were taken through a 20X and a 100X objective with a Nikon Eclipse TE2000 inverted microscope attached to a digital camera DXM1200F.

Subcellular fractionation

Nuclei from the P7 mouse forebrain were isolated according to the method of Block et al. (1992) and Koppler et al. (1993) with modification by Wu et al. (1995). For separation of P1 (crude nucleus), P2 (mitochondria/lysosome/synaptosomal pellet), and P3 (microsomal pellet) fractions, and for further fractionation of P2, the combined method of Rajapakse et al. (2001) and Kiebish et al. (2008) was used. P3 was further fractionated using OptiPrep (60% w/v iodixanol in water, Sigma-Aldrich Chemical Company, St. Louis, MO, USA) density gradient centrifugation according to the methods of Araki et al. (2003), Li and Donowitz (2008), and OptiPrep instruction manual with some minor modifications. Additional detailed subcellular fractionation protocols are available in Appendix S1.

Immunoblotting

Immunoblotting was performed as described previously (Saito et al., 2010a). Tissue samples or subcellular fractions described above (30 μg of protein) were boiled in SDS-sample buffer, separated on 10% or 15% SDS-PAGE, and blotted onto nitrocellulose membranes. The membranes were then blocked with Odyssey blocking buffer containing 0.1% Tween 20 and probed with various antibodies (see Table S1 for details of the antibodies used). Either mouse monoclonal anti-β-actin antibody, or mouse monoclonal anti-β-tubulin antibody was included as loading control. Antigens were detected by the Odyssey infrared imaging system using secondary antibodies, IR dye 680 conjugated goat anti-rabbit IgG, IR dye 680 conjugated donkey anti-goat IgG, and IR dye 800 conjugated goat anti-mouse IgG, and analyzed by Multi Gauge ver.2.0 (Fujifilm USA Medical Systems). For the quantification analyses, the intensity of each protein band was normalized by the corresponding β-actin intensity. Due to the close molecular weights of S1P1-receptor (S1P1-R; ~44 kD) and β-actin (~43 kD), β-tubulin (~50 kD) was used for S1P1-R quantification. In the developmenal studies (Fig. 1), intensities of bands obtained from samples with the same protein amounts were directly compared among various developmental stages, because amounts of β-actin and β-tubulin changed significantly during development (data not shown). In order to check the specificities of anti-SphK1, anti-SphK2, and anti-S1P1-R antibody, antibodies were pre-incubated with corresponding peptides for 1h at room temperature prior to probing. Blocking peptides used were SphK2-Blocking Peptide (Cat No. SC-22704P, Santa Cruz Biotechnology), unphosphorylated SK1 (Ser-225) Peptide (Cat No. SX1645, ECM-Biosciences), and S1P1-Blocking Peptide (Cat No. 10006616, Cayman Chemicals). The amount of protein was measured by a BCA method (Pierce, Rockford, IL, USA).

Fig. 1.

Fig. 1

Open in a new tab

Developmental profiles of S1P1-R, SphK2, and SphK1 proteins. 50 μg of total protein from forebrain samples of P1, P4, P7, P10, P13, P16, P19, and P25 mice were loaded and separated on SDS-PAGE, and immunoblots were carried out as described in Materials and Methods. A: Representative immonoblots probed with anti-S1P1-R, anti-SphK2, and anti-SphK1 antibodies. * indicates a non-specific band. B: Quantitative analyses of immunoblots of S1P1-R, SphK2, and SphK1. Values [mean ± SEM (n=3–4)] are expressed as fold changes compared to the content of P1 mice. *Significantly (p<0.05) different from P4 mice by ANOVA with the Bonferroni's post hoc test.

Sphingosine kinase assay

SphK1 and SphK2 activities in the P7 forebrain were measured according to the methods of Billich and Ettmayer (2004), Wacker et al. (2009), and Liu et al. (2000) except that NBD [ω-(7-nitro-2-1,3-benoxadiazol-4-yl)-D-erythro]-S1P formed was extracted according to the method of Matyash et al. (2008) using methyl tert-butyl ether. Fluorescence was measured using a fluorometer (BioTek Instrument Inc, Winooski, VE, USA) at excitation wavelength 485 nm and emission wavelength 528 nm. Values were expressed as percent of control.

Statistics

Values in figures are expressed as mean ± Standard Error of Mean (SEM) obtained from 4–10 samples. Statistical analysis of the data was performed by two-tailed Student's t test, one sample t test, and ANOVA with Bonferroni's post hoc test using the SPSS 11.0 program. A p value of <0.05 was considered significant.

Results

S1P metabolism in the developing brain

Prior to studies of the effects of ethanol on S1P metabolism in the P7 mouse brain, basal levels of S1P-related lipids and proteins (SphK1, SphK2, and S1P1-R) were examined during the early postnatal brain development. First, we measured changes in brain levels of ceramide, sphingosine and S1P. Results showed that levels of S1P gradually increased between P4 and P31, while sphingosine reached maximum at P19, and ceramide peaked around P13 (Fig. S1). Developmental changes in protein levels of S1P1-R, SphK2, and SphK1 in the forebrain were also examined by immunoblot analyses (Fig. 1). The band in Fig. 1A indicated by an asterisk (*) was considered non-specific because pre-incubation of anti-SphK1 antibody with a blocking peptide solution (as described in Materials and Methods) did not reduce the intensity of this band, while the band below disappeared completely. Bands shown for SphK2 and S1P1-R were considered specific to each antibody, based on experiments using corresponding blocking peptides (data not shown). Fig. 1B shows quantitative results expressed as fold changes compared to P1 values. Levels of SphK2 gradually increased between P1 and P25 in a manner similar to that of S1P (Fig. S1C), while the trace amounts of SphK1 found in the P1 forebrain did not increase during the developmental period. Fig. 1 also shows that S1P1-R, a major S1P receptor isoform in the brain (Brinkmann, 2007), increased during early postnatal days as observed in levels of S1P (Fig. S1C). Thus, the neonatal brain contains significant levels of S1P, sphingosine, SphK2, and S1P1-R, although the levels were lower than those at later developmental stages.

In Fig. 2, protein levels of SphK2, SphK1, and S1P1-R in the P7 brain were compared with those in other organs by immunoblot analyses. SphK2 protein was high in the forebrain and moderate in the liver, whereas SphK1 protein was very low in the forebrain, moderate in the brainstem, and high in the heart and the liver. S1P1-R protein was high in the brain, especially in the brainstem.

Fig. 2.

Fig. 2

Open in a new tab

Tissue/organ distribution of SphK1, SphK2, and S1P1-R. 50 μg of total protein from the forebrain, cerebellum, brain stem, heart, lung, liver, kidney, and spleen of P7 mice were analyzed by immunoblotting using anti-SphK1, anti-SphK2, and anti-S1P1-R antibodies as described in Materials and Methods. A: Representative immunoblots probed with anti-SphK1, anti-SphK2, and anti-S1P1-R antibody. * indicates a non-specific band. B–D: Quantitative analyses of immunoblots probed with anti-SphK1 (B), anti-SphK2 (C), and anti-S1P1-R (D) antibodies. Values [mean ± SEM (n=3)] are expressed as fold changes compared to the band intensities of forebrain samples after normalization with β-actin or β-tubulin.

We also examined the cellular localization of SphK2 in the P7 brain by immunohistochemistry. Brain sections from saline-treated (control) P7 mice were dual immunofluorescence-labeled with anti-SphK2 antibody and anti-NeuN antibody (Fig. 3B). The image shows the cingulate cortex region. In the lower panels, anti-SphK2 antibody was pretreated with blocking peptides. These results indicate that SphK2 is localized primarily in neurons. The affinity-purified anti-SphK2 antibody (prepared as described in Materials and Methods) also gave similar staining in neurons (data not shown). Immunohistochemistry using anti-SphK1 antibody gave faint staining mainly in astrocytes (data not shown). Also, S1P1-R expression was mostly limited to astrocytes (manuscript in preparation) in agreement with previous studies on the human brain (Nishimura et al., 2010).

Fig. 3.

Fig. 3

Open in a new tab

Cellular localization of SphK2. Images show the cingulate cortex region from brain coronal sections of control P7 mice dual-labeled with anti-SphK2 antibody and anti-NeuN antibody. In the lower panels, anti-SphK2 antibody was pretreated with the blocking peptide. The bar indicates 10 μm.

Ethanol-induced alterations in the levels of ceramide, sphingosine, and S1P

P7 mice were exposed to ethanol (5 g/kg) once as described in Materials and Methods. This treatment induced caspase-3 activation in the forebrain 8h after ethanol injection (Fig. 4). Caspase-3 activation was assessed by cleaved caspase-3 (CC3) and cleaved tau (Ctau) formation using immunoblotting. Under these ethanol treatment conditions, time course studies on the effects of ethanol on levels of ceramide, sphingosine, and S1P in the brain were performed (Fig. 5). Ethanol exposure in P7 mice significantly increased ceramide levels 8h after ethanol exposure, and the increase was maintained for at least another 16h (Fig. 5A). The result was similar to the effects of two-time ethanol injections (2.5 g/kg each) with a 2 h interval (Saito et al., 2007a). Sphingosine levels, which increased significantly 8h after ethanol injection, were also maintained for another 16h (Fig. 5B). In contrast, S1P increased 2.5 times 4h after ethanol injection and decreased to the basal level within the following 4h (Fig. 5C). The level of sphingomyelin, one of the potential precursors for ceramide/sphingosine/S1P, was not altered by ethanol treatment (data not shown).

Fig. 4.

Fig. 4

Open in a new tab

Ethanol-induced caspase-3 activation. Cleaved caspase-3 (CC3) and cleaved tau (Ctau) in forebrain samples of P7 mice were analyzed by immunoblots at varying times after ethanol injection. A: Representative immunoblots from P7 mice after 1, 2, 4, 8, and 24h of saline (Ctr) or ethanol (Eth) treatment probed with anti-CC3, anti-Ctau, and anti-β-actin antibodies as described in Materials and Methods. B: Quantitative analyses of amounts of CC3 and Ctau, expressed as ratios of CC3 or Ctau band intensities in the ethanol group to those in the saline (control) group after normalization with β-actin. Data are expressed as mean ± SEM, n =3 – 5. *Significantly (p< 0.05) different from the values of the 1h group by ANOVA with the Bonferroni's post hoc test.

Fig. 5.

Fig. 5

Open in a new tab

The effects of ethanol on levels of ceramide, sphingosine, and S1P in the brain. At the indicated time points after ethanol or saline (control) treatment, levels of ceramide (A), sphingosine (B), and S1P (C) in mouse brains were measured. Values, presented as ng/mg brain wet weight, are mean ± SEM for four to five animals. For all these lipids, there are significant differences between the control and ethanol groups by ANOVA. *Significantly (p< 0.05) different from values at 0h with the Bonferroni's post hoc test.

Ethanol transiently increased SphK2 activity

Ethanol transiently increased SphK2 enzyme activity (P<0.01 after Bonferroni's correction) at 2h (Fig. 6A). On the other hand, SphK1 enzyme activity, which was roughly one fourth of SphK2 enzyme activity, remained unaffected (data not shown). This transient increase of SphK2 activity after ethanol treatment was not due to an increase in the SphK2 level, because the level measured by immunoblot analyses was unaltered (Fig. 6B, C). The level of S1P1-R was also unchanged after ethanol treatment (Fig. 6B, C). Immunohistochemical analyses indicated that ethanol treatment did not change the cellular distribution of SphK2 (data not shown).

Fig. 6.

Fig. 6

Open in a new tab

The effects of ethanol on SphK2 and S1P1-R expression and SphK2 enzyme activity. A: SphK2 activity in the forebrain was measured at different time points after saline/ethanol treatment. Values are presented as % of control (saline treatment). Data presented are mean ± SEM (n = 3–4). *Significantly (p<0.05) different from control by one sample t-test with Bonferroni's correction. B: Forebrain samples were collected at different hours after saline (Ctr) or ethanol (Eth) treatment as indicated in the figure. 50 μg of protein was analyzed by immunoblotting. Representative immunoblots were probed with anti-S1P1-R, anti-β-tubulin, anti-SphK2, and anti-β-actin antibodies. C: Quantitative analysis of immunoblots. Data are expressed as ratios of band intensities in the ethanol group to those in the control group after normalizing SphK2 with β-actin and S1P1-R with β-tubulin. Ethanol treatment induced no significant changes when analyzed by one-sample t-test with Bonferroni's correction.

Subcellular localization of SphK2

It has been reported that subcellular localization of SphK2 is important for exerting its cellular functions (Igarashi et al., 2003; Hait et al., 2009; Wattenberg, 2010; Strub et al., 2011). P7 mouse brains decapitated 2h after saline/ethanol injection were homogenized and fractionated into P1 (crude nucleus), P2 (mitochondria/lysosome/synaptosome), P3 (microsome), and soluble (cytosol) fractions, and SphK2 levels in these fractions were analyzed by immunoblotting (Fig. 7A). We observed that SphK2 was highly expressed in the P2 and P3 (Micro) fractions while it was not detected in the P1 and very low in the cytosol fraction. Subcellular distribution of SphK2 was not significantly different between saline and ethanol-treated brains. Because the presence of SphK2 in the nucleus has been reported previously (Igarashi et al., 2003; Sankala et al., 2007; Ding et al., 2007; Hait et al., 2009), P1 fraction was further purified as described in Appendix S1 to increase the ratio of SphK2 to other proteins, if SphK2 is enriched in the nucleus. However, SphK2 was not detected in the purified nuclear fraction [Nuc (P)] (Fig. 7B). The purity of the nuclear fraction was confirmed by the abundant presence of acetyl histone and the absence of other subcellular marker proteins, synaptophysin (synaptic vesicle), PSD95 (synaptic membrane), voltage-dependent anion channel (VDAC, mitochondria), Complex IV (COX IV, mitochondria), Na+,K+- ATPase (plasma membrane), and β-glucosidase (lysosome). In order to better understand the subcellular localization of SphK2 found in the P2 fraction, P2 was further fractionated by different density gradient centrifugations, followed by Western blot analyses of each fraction containing 30 μg of protein (Fig. 8A). As predicted from a previous report (Rajapakse et al., 2001), Mito (ns) and Mito (s) fractions were enriched in VDAC, a mitochondrial marker. Also, Mito (ns) contains LAMP1, a lysosomal/late endosomal marker. Synap1 and Synap2 were enriched in PSD95 (a synaptic membrane marker) and synaptophysin (a synaptic vesicle marker), respectively. These synaptosomal fractions also contained Na+,K+-ATPase (a plasma membrane marker) and flotillin-1 (a lipid raft marker). Fig. 8A indicates that SphK2 was absent from mitochondrial/lysosomal fractions and predominantly localized in synaptosomal vesicle- and synaptosomal membrane-enriched fractions. The results shown in Fig. 7 also indicated that SphK2 was present in the P3 (microsomal) fraction. To examine the subcellular localization of SphK2 in this fraction, components of the microsomal pellet were separated by iodixanol density gradient centrifugation and probed with antibodies against different organelle markers. As shown in Fig. 8B and C, SphK2 showed similar distribution to that of flotillin-1, Na+,K+-ATPase, and Rab5 (an early endosomal marker), indicating that SphK2 was enriched in plasma membrane regions. This notion also agrees with the enrichment of SphK2 in synaptic membrane and synaptic vesicle fractions in the P2 pellet (Fig. 8A). Syntaxin6, a Golgi marker, and ERp72, an endoplasmic reticulum (ER) marker, showed different distribution pattern from that of SphK2, although the presence of SphK2 in the ER cannot be excluded because of the small difference in the densities. In Fig. 8D, brain sections from saline-treated (control) P7 mice were dual immunofluorescence-labeled with anti-SphK2 antibody and anti-Na+,K+-ATPase antibody. The image shows the cingulate cortex region. The SphK2 antibody used here was affinity-purified as described in Materials and Methods. The results indicated partial co-localization of SphK2 with Na+,K+-ATPase, which was consistent with the subcellular fractionation results.

Fig. 7.

Fig. 7

Open in a new tab

Subcellular localization of SphK2 in the P7 mouse forebrain. Brain samples were collected 2h after either saline or ethanol treatment, and various subcellular fractions were prepared. 30 μg of protein from homogenate (Homog), P1, P2, P3 (Micro), cytosol, a subfraction of P1 [Nuc(U)], and nucleus purified [Nuc(P)] were analyzed by immunoblotting. A: A representative immunoblot of saline (S) and ethanol (E) samples was probed with anti-SphK2 antibody. B: Representative immunoblots of saline (S) and ethanol (E) samples were probed with anti-SphK2, anti-synaptophysin, anti-PSD-95, anti-VDAC, anti-COX IV, anti-Na+,K+-ATPase, anti-β-glucosidase, and anti-acetyl histone antibodies.

Fig. 8.

Fig. 8

Open in a new tab

Localization of SphK2 in fractions isolated from P2 and P3 in the P7 mouse forebrain. P2 and P3 fractions obtained from control mouse forebrain samples were further subfractionated as described in Materials and Methods. A: Fractionation of P2. A representative immunoblot of homogenate (Homog) and subfractions derived from P2: Mito1 (mitochondria1), Mito (ns) (non-synaptic mitochondria), Mito (s) (synaptic mitochondria), Synap1 (synaptic fraction 1), and Synap2 (synaptic fraction 2). Samples (30 μg of protein each) were probed with anti-SphK2, anti-Na+,K+- ATPase, anti-flotillin-1, anti-VDAC, anti-LAMP-1, anti PSD-95, anti-synaptophysin, and BiP78 antibodies. B: Fractionation of P3. The microsomal fraction was fractionated by iodixanol continuous density gradient centrifugation. 25 μl of each fraction was immunoblotted and probed with anti-SphK2, anti-Na+,K+-ATPase, anti-flotillin-1, anti-ERp78, anti-Rab 5, and anti-syntaxin 6 antibodies. Since no visible bands were detected with these antibodies in fractions 1 to 4, only fractions 5 to 20 are shown here. C: The figure illustrates semi-quantitative profiles of distribution of each marker protein described in B. D: Panels show images of the cingulate cortex region from brain sections of control P7 mice dual-labeled with anti-SphK2 and anti-Na+,K+-ATPase antibodies. The bar indicates 10 μm.

The effects of DMS on ethanol-induced caspase-3 activation and neurodegeneration in the P7 mouse brain

In order to evaluate the involvement of S1P in the ethanol-induced apoptotic pathway, DMS, a SphK inhibitor, was administered into P7 mice via intracerebroventricular injection 0.5h before the ethanol injection, and cleaved caspase-3 formation at 8h after ethanol injection was analyzed by Western blotting. Fig. 9 shows the effects of DMS on cleaved caspase-3 (CC3) formation induced by the two-time ethanol injections. The results indicated that DMS alone did not affect caspase-3 activation, but DMS attenuated ethanol-induced caspase-3 activation. Similar effects of DMS were observed using the one-time ethanol injection protocol (data not shown). It has been shown that caspase-3 activation in the P7 mouse brain that peaks around 8h after the ethanol injection leads to robust neurodegeneration detected by silver staining (Olney et al., 2002) and Fluoro-Jade staining (Ieraci and Herrera, 2006; Saito et al., 2010a). In order to assess if DMS attenuates ethanol-induced neurodegeneration, the effects of DMS on Fluoro-Jade C staining were examined in the cingulate and the retrosplenial cortex. Fig. 10A shows representative images of Fluoro-Jade C staining in the cingulate cortex region from control (Ctr), DMS, ethanol (Eth), ethanol+DMS (Eth+DMS) mice, and Fig. 10B shows the quantified results calculated from the images and expressed as Fluoro-Jade C-positive cell number per square millimeter. ANOVA with the Bonferroni's post hoc test showed that the “Eth+DMS” group was significantly different from all other groups, indicating that DMS treatment partially blocked ethanol-induced neurodegeneration assessed by Fluoro-Jade staining.

Fig. 9.

Fig. 9

Open in a new tab

Effects of DMS, a SphK inhibitor, on ethanol-induced caspase-3 activation. 0.5h before saline/ethanol injection, DMS (3 μg in 1.5 μl DMSO) or vehicle was administered to P7 mice via intracerebroventricular injection. Saline or ethanol was injected twice with a 2h interval, and forebrains were taken 8h after the first saline/ethanol injection. Forebrain samples from control (Ctr), DMS alone (DMS), ethanol (Eth), ethanol+DMS (Eth+DMS) groups were analyzed by immunoblotting using anti-CC3 antibody. A: A representative immunoblot probed with anti-CC3 antibody and anti-β-actin antibody. B: Quantitative analyses of immunoblots. Data [mean ± SEM (n = 4)] are expressed as ratios of treatment groups to the control group after normalization with β-actin. *Significantly different from all other groups by ANOVA with the Bonferroni's post hoc test.

Fig. 10.

Fig. 10

Open in a new tab

Effects of DMS, a SphK inhibitor, on ethanol-induced neurodegeneration. 0.5h before saline/ethanol injection, DMS (3 μg in 1.5 μl DMSO) or vehicle was administered to P7 mice via intracerebroventricular injection. Saline or ethanol was injected twice with a 2h interval, and the mice were perfusion-fixed19 h after the first saline/ethanol injection. Brain sections from control (Ctr), DMS alone (DMS), ethanol (Eth), ethanol+DMS (Eth+DMS) mice were stained with Fluoro-Jade C. A: The representative image here shows the cingulate cortex region. The bar indicates 50 μm. B: Fluoro-Jade C (FJ) positive cells were counted in the cingulate cortex (CX) and in the retrosplenial cortex (CX). Data [mean ± SEM (n = 4)] are expressed as the number of FJ-positive cells per square millimeter. *#Significantly different from all other groups by ANOVA with the Bonferroni's post hoc test.

Discussion

Our studies showed that ethanol treatment transiently increased SphK2 activity and S1P content in the P7 mouse brain prior to peak caspase-3 activation, and that pretreatment with DMS (an inhibitor of SphK) attenuated the caspase-3 activation and the subsequent neurodegeneration. As far as we know, this is the first report describing the effects of ethanol on the endogenous S1P metabolism in the brain. Under the present conditions, ethanol increased ceramide, sphingosine, and S1P in the P7 mouse brain (Fig. 5) along with inducing robust caspase-3 activation (Fig. 4). While the time course of sphingosine elevation was similar to that of ceramide, the elevation of S1P occurred transiently 4h after ethanol exposure. The similar transient activation of SphK2 by ethanol (Fig. 6A) shortly before the elevation of S1P levels strongly suggests that ethanol-induced SphK2 enzyme activation mediates S1P elevation. Since protein levels of SphK2 remained unaltered (Fig. 6B, C), post-translational modifications, such as phosphorylation described previously (Ding et al., 2007; Hait et al., 2007), may cause ethanol-induced SphK2 activation. The contribution of SphK1 to ethanol-induced elevation of S1P is expected to be minimal, because the SphK1 level was low in the brain (Fig. 1, 2), and the SphK1 enzyme activity, which was one fourth of the SphK2 activity, did not show any change by ethanol treatment. Our observation that SphK2 is the major SphK isoform in the mouse brain (Fig. 1, 2) agrees with previous studies (Blondeau et al., 2007, Pfeilschifter et al., 2011). The immunohistochemical data (Fig. 3) suggest that SphK2 is localized mainly in neurons as indicated in a previous study (Blondeau et al., 2007), while SphK1 seems mainly expressed in astrocytes (data not shown). The localization of SphK1 in astrocytes and microglia has also been reported by others (Lee at al., 2010; Nayak et al., 2010; Fischer et al., 2011). These results indicate that ethanol triggers S1P elevation via activation of SphK2 in neurons in the P7 mouse brain, although we cannot exclude the possibility that S1P is derived from other cell types, such as erythrocytes, microglia, and endothelial cells.

S1P regulates a wide variety of cellular processes, including growth, survival, differentiation, cytoskeletal rearrangements, angiogenesis, and immunity, and the level of S1P is mainly controlled by the SphK activity (Spiegel and Milstien, 2003). In contrast to the proliferative and anti-apoptotic effects of S1P produced by SphK1, S1P generated by SphK2 has been implicated to cause apoptosis and other cellular functions through intracellular targets in several cultured cells (Igarashi et al., 2003; Maceyka et al. 2005; Okada et al., 2009). Our subcellular fractionation studies showed that SphK2 was localized mainly in the P2 and P3 fractions but not in the nuclear fraction, and the localization was unchanged by ethanol treatment (Fig. 7). Further fractionation of P2 and P3 indicated that SphK2 was enriched in synaptic vesicles (identified by synaptophysin), synaptic membranes (identified by PSD95), and the plasma membrane (identified by Na+,K+-ATPase and flotillin) fractions (Fig. 8), although we cannot rule out the possibility that SphK2 was also localized in endosomes (identified by Rab5), endoplasmic reticulum (identified by ERp72), and other organelles which have similar densities. To the best of our knowledge, this is the first in depth subcellular fractionation study reporting the localization of SphK2 in the brain. It appears that the subcellular localization of SphK2 in the P7 brain is different from that reported mainly in cultured cells, where SphK2 is detected in the nucleus (Igarashi et al., 2003; Sankala et al., 2007; Ding et al., 2007; Hait et al., 2009), ER (Maceyka et al., 2005; Hagen et al., 2009), and mitochondria (Strub et al., 2011). Although the localization of SphK2 in the plasma membrane has been reported in some cell types (Maceyka et al., 2005), apoptotic functions of SphK2 are generally associated with the presence of SphK2 in the nucleus (Igarashi et al., 2003; Okada et al., 2005) and ER (Maceyka et al., 2005; Hagen et al., 2009). Interestingly, our results indicated that DMS, a SphK inhibitor, significantly attenuated ethanol-induced caspase-3 activation (Fig. 9) and neurodegeneration (Fig. 10), although further studies are necessary because DMS is not a specific inhibitor for SphK2 (French et al., 2010). Nonetheless, our results suggest that SphK2 primarily localized in or near the plasma membrane in neurons is activated by ethanol, produces S1P, and induces or enhances apoptotic action of ethanol. Because our previous studies have shown that inhibitors of serine palmitoyl transferase, a rate limiting enzyme for sphingolipid synthesis, attenuate ethanol-induced apoptosis (Saito et al., 2010a), de novo ceramide synthesis may be involved in the S1P formation catalyzed by SphK2. Also, the partial blocking of ethanol-induced neurodegeneration by DMS (Fig. 10) suggests that ceramide/sphingosine may enhance neurodegeneration independent of S1P action. It has been shown that S1P metabolism is affected under pathological and stressful conditions in the nervous system. For example, increases in the expression levels of SphK1 have been shown in kainic acid-treated hippocampal astrocytes (Lee et al., 2010) and lipopolysaccaride-treated cultured microglia (Nayak et al., 2010), whereas increases in SphK2 expression or activity have been reported in cerebral microvessels under hypoxic preconditioning (Wacker et al., 2009), in the ischemic brain (Blondeau et al., 2007), and in the brains of patients with Alzheimer's disease (Takasugi et al., 2011). While S1P elevation produced by SphK2 is considered apoptotic in the cerebellar granule neurons derived from S1P lyase-deficient mice (Hagen et al., 2009) as well as in other non-neural cells (Liu et al., 2003; Okada et al., 2005; Maceyka et al. 2005), SphK2 activation is found neuroprotective in some animal models of brain ischemia (Wacker et al., 2009; Hasegawa et al., 2010; Pfeilschifter et al., 2011; Yung et al., 2012). Whether SphK2 activation leads to neuroprotection or not may depend on the subcellular targets of S1P produced. The efficacy of S1P receptor agonists in neuroprotection in some of these studies (Wacker et al., 2009; Hasegawa et al., 2010; Pfeilschifter et al., 2011) suggests that S1P produced by SphK2 may activate S1P receptors, leading to neuroprotection, or S1P in mitochondria may exert cytoprotection as indicated in a myocardial injury model (Gomez et al., 2011). S1P increased by ethanol treatment in our study may have different targets, inducing or enhancing neuroapoptosis.

Our observation that S1P1-R, a major S1P receptor in the brain (Brinkmann, 2007), was mainly localized in astrocytes as reported previously (Nishimura et al., 2010) suggests that S1P produced by SphK2 in neurons may exert its function independent of S1P1-R. S1P may interact with other S1P receptor isoforms or may have other targets, such as Na+,K+-ATPase as recently reported (Dakroub and Kreydiyyeh et al., 2012).

In summary, we demonstrated that ethanol transiently elevated SphK2 activity and S1P levels in the P7 mouse brain, which may be related to ethanol-induced apoptotic neurodegeneration.

Supplementary Material

Supp Table S1, Fig S1 & App S1

Acknowledgments

This work was supported by an NIH/NIAAA grant R01 AA015355 (to MS). The authors have no conflict of interest to disclose.

Abbreviations used

S1P

sphingosine 1-phosphate

FASD

fetal alcohol spectrum disorders

P7

postnatal day 7

SphK

sphingosine kinase

DMS

d-erythro-N,N-dimethylsphingosine

CC3

cleaved caspase-3

Ctau

cleaved tau

S1P1-R

sphingosine 1-phosphate receptor 1

SEM

Standard Error of Mean

References

  1. Agudo-Lopez A, Miguel BG, Fernandez I, Martinez AM. Involvement of mitochondria on neuroprotective effect of sphingosine-1-phosphate in cell death in an in vitro model of brain ischemia. Neurosci Lett. 2010;470:130–133. doi: 10.1016/j.neulet.2009.12.070. [DOI] [PubMed] [Google Scholar]
  2. Araki Y, Tomita S, Yamaguchi H, Miyagi N, Sumioka A, Kirino Y, Suzuki T. Novel cadherin-related membrane proteins, Alcadeins, enhance the X11-like protein-mediated stabilization of amyloid beta-protein precursor metabolism. J Biol Chem. 2003;278:49448–49458. doi: 10.1074/jbc.M306024200. [DOI] [PubMed] [Google Scholar]
  3. Billich A, Ettmayer P. Fluorescence-based assay of sphingosine kinases. Anal Biochem. 2004;326:114–119. doi: 10.1016/j.ab.2003.11.018. [DOI] [PubMed] [Google Scholar]
  4. Block C, Freyermuth S, Beyersmann D, Malviya AN. Role of cadmium in activating nuclear protein kinase C and the enzyme binding to nuclear protein. J Biol Chem. 1992;267:19824–19828. [PubMed] [Google Scholar]
  5. Blondeau N, Lai Y, Tyndall S, Popolo M, Topalkara K, Pru JK, Zhang L, Kim H, Liao JK, Ding K, Waeber C. Distribution of sphingosine kinase activity and mRNA in rodent brain. J Neuochem. 2007;103:509–517. doi: 10.1111/j.1471-4159.2007.04755.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brinkmann V. Sphingosine 1-phosphate receptors in health and disease: Mechanistic insights from gene deletion studies and reverse pharmacology. Pharmacol Ther. 2007;115:84–105. doi: 10.1016/j.pharmthera.2007.04.006. [DOI] [PubMed] [Google Scholar]
  7. Carloni S, Mazzoni E, Balduini W. Caspase-3 and calpain activities after acute and repeated ethanol administration during the rat brain growth spurt. J Neurochem. 2004;89:197–203. doi: 10.1111/j.1471-4159.2004.02341.x. [DOI] [PubMed] [Google Scholar]
  8. Dakroub Z, Kreydiyyeh SI. Sphingosine-1-phosphate is a mediator of TNF-α action on the Na+/K+ ATPase in HepG2 cells. J Cell Biochem. 2012 doi: 10.1002/jcb.24079. in press. [DOI] [PubMed] [Google Scholar]
  9. Ding G, Sonoda H, Yu H, Kajimoto T, Goparaju SK, Jahangeer S, Okada T, Nakamura S. Protein kinase D-mediated phophorylation and nuclear export of sphingosine kinase 2. J Biol Chem. 2007;282:27493–27502. doi: 10.1074/jbc.M701641200. [DOI] [PubMed] [Google Scholar]
  10. Fischer I, Alliod C, Martinier N, Newcombe J, Brana C, Pouly S. Sphingosine Kinase 1 and Sphingosine 1-Phosphate Receptor 3 Are Functionally Upregulated on Astrocytes under Pro-Inflammatory Conditions. PLoS One. 2011;6(8):e23905. doi: 10.1371/journal.pone.0023905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. French KJ, Zhuang Y, Maines LW, Gao P, Wang W, Beljanski V, Upson JJ, Green CL, Keller SN, Smith CD. Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2. J Pharmacol Exp Ther. 2010;333:129–139. doi: 10.1124/jpet.109.163444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gomez L, Paillard M, Price M, Chen Q, Teixeira G, Spiegel S, Lesnefsky EJ. A novel role for mitochondrial sphingosine-1-phosphate produced by sphingosine kinase-2 in PTP-mediated cell survival during cardioprotection. Basic Res Cardiol. 2011;106:1341–1353. doi: 10.1007/s00395-011-0223-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hagen N, Van Veldhoven PP, Proia RL, Park H, Merrill AH, Jr, van Echten-Deckert G. Subcellular origin of sphingosine 1-phosphate is essential for its toxic effect in lyase-deficient neurons. J Biol Chem. 2009;284:11346–11353. doi: 10.1074/jbc.M807336200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hait NC, Bellamy A, Milstien S, Kordula T, Spiegel S. Sphingosine kinase type 2 activation by ERK-mediated phosphorylation. J Biol Chem. 2007;282:12058–12065. doi: 10.1074/jbc.M609559200. [DOI] [PubMed] [Google Scholar]
  15. Hait NC, Allegood J, Maceyka M, Strub GM, Harikumar KB, Singh SK, Luo C, Marmorstein R, Kordula T, Milstien S, Spiegel S. Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate. Science. 2009;325:1254–1257. doi: 10.1126/science.1176709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Han JY, Jeong JY, Lee YK, Roh GS, Kim HJ, Kang SS, Cho GJ, Choi WS. Suppression of survival kinases and activation of JNK mediate ethanol-induced cell death in the developing rat brain. Neurosci Lett. 2006;398:113–117. doi: 10.1016/j.neulet.2005.12.065. [DOI] [PubMed] [Google Scholar]
  17. Hasegawa Y, Suzuki H, Sozen T, Rolland W, Zhang JH. Activation of sphingosine 1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats. Stroke. 2010;41:368–374. doi: 10.1161/STROKEAHA.109.568899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. He X, Huang CL, Schuchman EH. Quantitiative analysis of sphingosine-1-phosphate by HPLC after napthalene-2,3-dicarboxaldehyde (NDA) derivatization. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877:983–990. doi: 10.1016/j.jchromb.2009.02.048. [DOI] [PubMed] [Google Scholar]
  19. Ieraci A, Herrera DG. Nicotinamide protects against ethanol-induced apoptotic neurodegeneration in the developing mouse brain. PLoS Med. 2006;3(4):e101. doi: 10.1371/journal.pmed.0030101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Igarashi N, Okada T, Hayashi S, Fujita T, Jahangeer S, Nakamura S. Sphingosine kinase 2 is a nuclear protein and inhibits DNA synthesis. J Biol Chem. 2003;278:46832–46839. doi: 10.1074/jbc.M306577200. [DOI] [PubMed] [Google Scholar]
  21. Ikonomidou C, Bittigau P, Ishimaru MJ, Wozniak DF, Koch C, Genz K, Price MT, Stefovska V, Horster F, Tenkova T, Dikranian K, Olney JW. Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science. 2000;287:1056–1060. doi: 10.1126/science.287.5455.1056. [DOI] [PubMed] [Google Scholar]
  22. Johnson KR, Becker KP, Facchinetti MM, Hannun YA, Obeid LM. PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phorbol 12-myristate 13-acetate (PMA) J Biol Chem. 2002;277:35257–35262. doi: 10.1074/jbc.M203033200. [DOI] [PubMed] [Google Scholar]
  23. Kiebish MA, Han X, Cheng H, Lunceford A, Clarke CF, Moon H, Chuang JH, Seyfried TN. Lipidomic analysis and electron transport chain activities in C57BL/6J mouse brain mitochondria. J Neurochem. 2008;106:299–312. doi: 10.1111/j.1471-4159.2008.05383.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Koppler P, Matter N, Malviya AN. Evidence for stereospecific inositol 1,3,4,5-[3H]tetrakisphosphate binding sites on rat liver nuclei. Delineating inositol 1,3,4,5-tetrakisphosphate interaction in nuclear calcium signaling process. J Biol Chem. 1993;268:26248–26252. [PubMed] [Google Scholar]
  25. Lee DH, Jeon BT, Jeong EA, Kim JS, Cho YW, Kim HJ, Kang SS, Cho GJ, Choi WS, Roh GS. Altered expression of sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 in mouse hippocampus after kainic acid treatment. Biochem Biophys Res Commun. 2010;393:476–480. doi: 10.1016/j.bbrc.2010.02.027. [DOI] [PubMed] [Google Scholar]
  26. Li X, Donowitz M. Fractionation of subcellular membrane vesicles of epithelial and nonepithelial cells by OptiPrep density gradient ultracentrifugation. Methods Mol Biol. 2008;440:97–110. doi: 10.1007/978-1-59745-178-9_8. [DOI] [PubMed] [Google Scholar]
  27. Liu H, Sugiura M, Nava VE, Edsall LC, Kono K, Poulton S, Milstien S, Kohama T, Spiegel S. Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. J Biol Chem. 2000;275:19513–19520. doi: 10.1074/jbc.M002759200. [DOI] [PubMed] [Google Scholar]
  28. Liu H, Toman RE, Goparaju SK, Maceyka M, Nava VE, Sankala H, Payne SG, Bektas M, Ishii I, Chun J, Milstien S, Spiegel S. Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis. J Biol Chem. 2003;278:40330–40336. doi: 10.1074/jbc.M304455200. [DOI] [PubMed] [Google Scholar]
  29. Maceyka M, Sankala H, Hait NC, Le Stunff H, Liu H, Toman R, Collier C, Zhang M, Satin LS, Merrill AH, Jr, Milstien S, Spiegel S. SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism. J Biol Chem. 2005;280:37118–37129. doi: 10.1074/jbc.M502207200. [DOI] [PubMed] [Google Scholar]
  30. Malchinkhuu E, Sato K, Muraki T, Ishikawa K, Kuwabara A, Okajima F. Assessment of the role of sphingosine 1-phosphate and its receptors in high-density lipoprotien-induced stimulation of astroglial cell function. Biochem J. 2003;370:817–827. doi: 10.1042/BJ20020867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lipid Res. 2008;49:1137–1146. doi: 10.1194/jlr.D700041-JLR200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mizugishi K, Yamashita T, Olivera A, Miller GF, Spiegel S, Proia RL. Essential role for sphingosine kinases in neural and vascular development. Mol Cell Biol. 2005;25:11113–11121. doi: 10.1128/MCB.25.24.11113-11121.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nayak D, Huo Y, Kwang WX, Pushparaj PN, Kumar SD, Ling EA, Dheen ST. Sphingosine kinase 1 regulates the expression of proinflammatory cytokines and nitric oxide in activated microglia. Neuroscience. 2010;166:132–144. doi: 10.1016/j.neuroscience.2009.12.020. [DOI] [PubMed] [Google Scholar]
  34. Nishimura H, Akiyama T, Irei I, Hamazaki S, Sadahira Y. Cellular localization of sphingosine-1-phosphate receptor 1 expression in the human central nervous system. J Histochem Cytochem. 2010;58:847–856. doi: 10.1369/jhc.2010.956409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ogretmen B, Hannun YA. Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer. 2004;4:604–616. doi: 10.1038/nrc1411. [DOI] [PubMed] [Google Scholar]
  36. Okada T, Ding G, Sonoda H, Kajimoto T, Haga Y, Khosrowbeygi A, Gao S, Miwa N, Jahangeer S, Nakamura S. Involvement of N-terminal-extended form of sphingosine kinase 2 in serum-dependent regulation of cell proliferation and apoptosis. J Biol Chem. 2005;280:36318–36325. doi: 10.1074/jbc.M504507200. [DOI] [PubMed] [Google Scholar]
  37. Okada T, Kajimoto T, Jahangeer S, Nakamura S. Sphiongosine kinase/sphingosine 1-phosphate signalling in central nervous system. Cell Signalling. 2009;21:7–13. doi: 10.1016/j.cellsig.2008.07.011. [DOI] [PubMed] [Google Scholar]
  38. Olivera A, Spiegel S. Sphingosine-1-phosphate as a second messenger in cell proliferation induced by PDGF and FCS mitogens. Nature. 1993;365:557–560. doi: 10.1038/365557a0. [DOI] [PubMed] [Google Scholar]
  39. Olney JW, Tenkova T, Dikranian K, Qin YQ, Labruyere J, Ikonomidou C. Ethanol-induced apoptotic neurodegeneration in the developing C57BL/6 mouse brain. Brain Res Dev Brain Res. 2002;133:115–126. doi: 10.1016/s0165-3806(02)00279-1. [DOI] [PubMed] [Google Scholar]
  40. Pebay A, Toutant M, Premont J, Calvo CF, Venance L, Cordier J, Glowinski J, Tence M. Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades. Eur J Neurosci. 2001;13:2067–76. [PubMed] [Google Scholar]
  41. Pfeilschifter W, Czech-Zechmeister B, Sujak M, Mirceska A, Koch A, Rami A, Steinmetz H, Foerch C, Huwiler A, Pfeilschifter J. Activation of sphingosine kinase 2 is an endogenous protective mechanism in cerebral ischemia. Biochem Biophys Res Commun. 2011;413:212–217. doi: 10.1016/j.bbrc.2011.08.070. [DOI] [PubMed] [Google Scholar]
  42. Pitson SM, Xia P, Leclercq TM, Moretti PA, Zebol JR, Lynn HE, Wattenberg BW, Vadas MA. Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling. J Exp Med. 2005;201:49–54. doi: 10.1084/jem.20040559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rajapakse N, Shimizu K, Payne M, Busija D. Isolation and characterization of intact mitochondria from neonatal rat brain. Brain Res Brain Res Protoc. 2001;8:176–83. doi: 10.1016/s1385-299x(01)00108-8. [DOI] [PubMed] [Google Scholar]
  44. Sadakata T, Washida M, Iwayama Y, Shoji S, Sato Y, Ohkura T, Katoh-Semba R, Nakajima M, Sekine Y, Tanaka M, Nakamura K, Iwata Y, Tsuchiya KJ, Mori N, Detera-Wadleigh SD, Ichikawa H, Itohara S, Yoshikawa T, Furuichi T. Autistic-like phenotypes in Cadps2-knockout mice and aberrant CADPS2 splicing in autistic patients. J Clin Invest. 2007;117:931–943. doi: 10.1172/JCI29031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Saini HS, Coelho RP, Goparaju SK, Jolly PS, Maceyka M, Spiegel S, Sato-Bigbee C. Novel role of sphingosine kinase 1 as a mediator of neurotrophin-3 action in oligodendrocyte progenitors. J Neurochem. 2005;95:1298–1310. doi: 10.1111/j.1471-4159.2005.03451.x. [DOI] [PubMed] [Google Scholar]
  46. Saito M, Chakraborty G, Hegde M, Ohsie J, Paik SM, Vadasz C, Saito M. Involvement of ceramide in ethanol-induced apoptotic neurodegeneration in the neonatal mouse brain. J Neurochem. 2010a;115:168–177. doi: 10.1111/j.1471-4159.2010.06913.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Saito M, Chakraborty G, Mao RF, Paik SM, Vadasz C, Saito M. Tau phosphorylation and cleavage in ethanol-induced neurodegeneration in the developing mouse brain. Neurochem Res. 2010b;35:651–659. doi: 10.1007/s11064-009-0116-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Saito M, Chakraborty G, Mao RF, Wang R, Cooper TB, Vadasz C, Saito M. Ethanol alters lipid profiles and phosphorylation status of AMP-activated protein kinase in the neonatal mouse brain. J Neurochem. 2007a;103:1208–1218. doi: 10.1111/j.1471-4159.2007.04836.x. [DOI] [PubMed] [Google Scholar]
  49. Saito M, Mao RF, Wang R, Vadasz C, and Saito M. Effects of gangliosides on ethanol-induced neurodegeneration in the developing mouse brain. Alcohol Clin Exp Res. 2007b;31:665–674. doi: 10.1111/j.1530-0277.2007.00351.x. [DOI] [PubMed] [Google Scholar]
  50. Sankala HM, Hait NC, Paugh SW, Shida D, Lépine S, Elmore LW, Dent P, Milstien S, Spiegel S. Involvement of sphingosine kinase 2 in p53-independent induction of p21 by the chemotherapeutic drug doxorubicin. Cancer Res. 2007;67:10466–10474. doi: 10.1158/0008-5472.CAN-07-2090. [DOI] [PubMed] [Google Scholar]
  51. Shinpo K, Kikuchi S, Moriwaka F, Tashiro K. Protective effects of the TNF-ceramide pathway against glutamate neurotoxicity on cultured mesencephalic neurons. Brain Res. 1999;819:170–173. doi: 10.1016/s0006-8993(98)01354-7. [DOI] [PubMed] [Google Scholar]
  52. Snider AJ, Orr Gandy KA, Obeid LM. Spingosine kinase: Role in regulation of bioactive sphingolipid mediators in inflammation. Biochimie. 2010;92:707–715. doi: 10.1016/j.biochi.2010.02.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sorensen SD, Nicole O, Peavy RD, Montoya LM, Lee CJ, Murphy TJ, Traynelis SF, Hepler JR. Common signaling pathways link activation of murine PAR-1, LPA, and S1P receptors to proliferation of astrocytes. Mol Pharmacol. 2003;64:1199–1209. doi: 10.1124/mol.64.5.1199. [DOI] [PubMed] [Google Scholar]
  54. Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol. 2003;4:397–407. doi: 10.1038/nrm1103. [DOI] [PubMed] [Google Scholar]
  55. Strub GM, Paillard M, Liang J, Gomez L, Allegood JC, Hait NC, Maceyka M, Price MM, Chen Q, Simpson DC, Kordula T, Milstien S, Lesnefsky EJ, Spiegel S. Sphingosine-1-phosphate produced by sphingosine kinase 2 in mitochondria interacts with prohibitin 2 to regulate complex IV assembly and respiration. FASEB J. 2011;25:600–612. doi: 10.1096/fj.10-167502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Takasugi N, Sasaki T, Suzuki K, Osawa S, Isshiki H, Hori Y, Shimada N, Higo T, Yokoshima S, Fukuyama T, Lee VM, Trojanowski JQ, Tomita T, Iwatsubo T. BACE1 activity is modulated by cell-associated sphingosine-1-phosphate. J Neurosci. 2011;31:6850–6857. doi: 10.1523/JNEUROSCI.6467-10.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wacker BK, Park TS, Gidday JM. Hypoxic preconditioning-induced cerebral ischemic tolerance: Role for microvasclar sphingosine kinase 2. Stroke. 2009;40:3342–3348. doi: 10.1161/STROKEAHA.109.560714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wattenberg BW. Role of sphingosine kinase localization in sphingolipid signaling. World J Biol Chem. 2010;1:362–368. doi: 10.4331/wjbc.v1.i12.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wu G, Lu ZH, Ledeen RW. Induced and spontaneous neuritogenesis are associated with enhanced expression of ganglioside GM1 in the nuclear membrane. J Neurosci. 1995;15:3739–3746. doi: 10.1523/JNEUROSCI.15-05-03739.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wu YP, Mizugishi K, Bektas M, Sandhoff R, Proia RL. Sphingosine kinase 1/S1P receptor signaling axis controls glial proliferation in mice with Sandhoff disease. Hum Mol Genet. 2008;17:2257–2264. doi: 10.1093/hmg/ddn126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yamagata K, Tagami M, Torii Y, Takenaga F, Tsumagari S, Itoh S, Yamori Y, Nara Y. Sphingosine 1-phosphate induces the production of glial cell line-derived neurotrophic factor and cellular proliferation in astrocytes. Glia. 2003;41:199–206. doi: 10.1002/glia.10180. [DOI] [PubMed] [Google Scholar]
  62. Young C, Roth KA, Klocke BJ, West T, Holtzman DM, Labruyere J, Qin YQ, Dikranian K, Olney JW. Role of caspase-3 in ethanol-induced developmental neurodegeneration. Neurobiol Dis. 2005;20:608–614. doi: 10.1016/j.nbd.2005.04.014. [DOI] [PubMed] [Google Scholar]
  63. Yung LM, Wei Y, Qin T, Wang Y, Smith CD, Waeber C. Sphingosine Kinase 2 Mediates Cerebral Preconditioning and Protects the Mouse Brain Against Ischemic Injury. Stroke. 2012;43(1):199–204. doi: 10.1161/STROKEAHA.111.626911. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Table S1, Fig S1 & App S1
NIHMS364043-supplement-Supp_Table_S1__Fig_S1___App_S1.doc (55.5KB, doc)

RESOURCES