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

Ethanol triggers apoptotic neurodegeneration in the newbornrodent brain during the period of rapid synaptogenesis thatcorresponds to the last trimester of pregnancy in humans(Ikonomidou et al. 2000; Olney et al. 2002), and causeslong-lasting neuronal loss and neurobehavioral impairmentas observed in human fetal alcohol spectrum disorders. Thisrodent model for fetal alcohol spectrum disorders has beenwidely utilized to elucidate mechanisms of ethanol-inducedapoptotic neurodegeneration (Carloni et al. 2004; Younget al. 2005; Han et al. 2006). We have previously demon-strated that ethanol-induced neurodegeneration in 7-day-old(postnatal day 7; P7) mice is accompanied by increases inseveral brain lipids (Saito et al. 2007a). Among them,de novo ceramide synthesis appears to play a vital role inethanol-induced apoptosis, because ceramide elevation is

associated with ethanol-induced caspase 3 activation,and because inhibitors of serine palmitoyltransferase, arate-limiting enzyme in sphingolipid synthesis, rescueethanol-induced apoptosis (Saito et al. 2010a). However,

Received November 1, 2011; revised manuscript received March 2,2012; accepted March 3, 2012.Address correspondence and reprint requests to Mariko Saito,

Division of Neurochemistry, Nathan S. Kline Institute for PsychiatricResearch, 140 Old Orangeburg Rd, Orangeburg, NY 10962, USA.E-mail: [email protected] used: CC3, cleaved caspase 3; Ctau, cleaved tau; DMS,

D-erythro-N,N-dimethylsphingosine; ER, endoplasmic reticulum;PSD95, postsynaptic density protein 95; S1P, sphingosine 1-phosphate;S1P1-R, sphingosine 1-phosphate receptor 1; SDS, sodium dodecylsulfate; SphK, sphingosine kinase.

,

, ,

*Division of Neurochemisty, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, New

York, USA

�Division of Analytical Psychopharmacology, Nathan S. Kline Institute for Psychiatric Research,

Orangeburg, New York, USA

�Department of Psychiatry, New York University Langone Medical Center, New York, New York, USA

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. In this

study, 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, sphin-

gosine 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 in-

duced sphingosine kinase 2 activation and increased the

brain level of S1P transiently 2–4 h after exposure, followed

by caspase 3 activation that peaked around 8 h after expo-

sure. Treatment with dimethylsphingosine, an inhibitor of

sphingosine kinases, attenuated the ethanol-induced cas-

pase 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: apoptotic neurodegeneration, ethanol, develop-

ing brain, plasma membrane, sphingosine 1-phosphate,

sphingosine kinase 2.

J. Neurochem. (2012) 121, 806–817.

JOURNAL OF NEUROCHEMISTRY | 2012 | 121 | 806–817 doi: 10.1111/j.1471-4159.2012.07723.x

806 Journal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 121, 806–817� 2012 The Authors

we cannot rule out the possibility that ceramide metabolites,specifically sphingosine and sphingosine 1-phosphate (S1P),are also involved in the ethanol-induced apoptotic pathwaybecause these lipids have been implicated as regulators of thecell survival/death pathways.

It is generally postulated that ceramide and sphingosineinduce growth arrest or apoptosis (Ogretmen and Hannun2004), while S1P promotes cell proliferation and cellsurvival (Spiegel and Milstien 2003), and the balancebetween these bioactive lipids, termed ‘sphingolipid rheo-stat’, determines cell fate (Spiegel and Milstien 2003). Thissphingolipid rheostat is mainly regulated by two isoforms ofsphingosine kinases, sphingosine kinase 1 (SphK1) andsphingosine kinase 2 (SphK2), which phosphorylate sphin-gosine to form S1P. It has been indicated that SphK1, acytosolic protein, is translocated to the plasma membraneafter activation (Johnson et al. 2002; Pitson et al. 2005) andexerts a pro-survival influence, whereas SphK2, a predom-inantly nuclear protein, inhibits cell growth and enhancesapoptosis (Igarashi et al. 2003). While most of the S1Peffects are mediated by the interaction of S1P with five G-protein-coupled cell surface receptors termed S1P receptor1–5 (Spiegel and Milstien 2003; Snider et al. 2010),intracellular actions of S1P have also been reported (Oliveraand Spiegel 1993). Induction of apoptosis by over-expressedSphK2 is independent of activation of S1P receptors (Liuet al. 2003), and S1P produced by SphK2 in the nucleus(Igarashi et al. 2003) as well as S1P produced by SphK2 inthe endoplasmic reticulum (ER) (Maceyka et al. 2005;Hagen et al. 2009) have been reported to exert apoptoticaction.

In the nervous system, S1P has a critical role in neuraldevelopment. 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 inastrocyte proliferation (Pebay et al. 2001; Malchinkhuuet 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 bedue to SphK2, which is localized in neurons, while SphK1 islocalized primarily in astrocytes (Blondeau et al. 2007).Whereas activation of the SphK1/S1P axis signaling appearsto be related to proliferation of astrocytes (Wu et al. 2008;Lee et al. 2010), protection of oligodendrocyte progenitorsfrom apoptosis (Saini et al. 2005), and microglial activation(Nayak et al. 2010), SphK2 has been implicated to causeapoptosis through intracellular targets in cerebellar granuleneurons derived from S1P lyase-deficient mice (Hagen et al.2009). However, in some animal models of brain ischemia,SphK2 activation is considered neuroprotective (Wackeret al. 2009; Hasegawa et al. 2010; Pfeilschifter et al. 2011;Yung et al. 2012).

While S1P is thought to play important roles in thedeveloping brain, profiles and functions of the S1P systemhave not been well studied in the early postnatal brain.Here, we examined S1P metabolism with a particular focuson SphK2 under the basal and ethanol-treated conditionsin the P7 mouse brain and evaluated the possibility thatS1P is involved in ethanol-induced apoptotic neurodegen-eration.

Materials and methods

AnimalsC57BL/6By mice were maintained at the Animal Facility of NathanS. Kline Institute for Psychiatric Research. All procedures followedguidelines consistent with those developed by the National Instituteof Health and the Institutional Animal Care and Use Committee ofNathan S. Kline Institute.

Experimental procedureC57BL/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 with25 lL/g body weight of ethanol (5.0 g/kg, 20% solution insaline) or saline was performed instead of two-time injections of2.5 g/kg ethanol with a 2-h interval, because the present studyincluded short-term (< 2 h) treatment conditions. It has beenreported that blood ethanol levels obtained by this one-timeinjection protocol are similar to those of two-time injections(Ieraci and Herrera 2006). The effects of D-erythro-N,N-dimeth-ylsphingosine (DMS, a SphK inhibitor) on ethanol-inducedcaspase 3 activation and the subsequent neurodegeneration wereexamined using both one-time and two-time ethanol injectionparadigms. In both cases, DMS (3 lg in 1.5 lL dimethylsulfoxide) was administered 0.5 h before the first ethanolinjection via intracerebroventricular injection as described (Sa-dakata et al. 2007). Ethanol-induced caspase 3 activation at 8 hafter the first ethanol injection was indirectly assessed bymeasuring increases in cleaved caspase 3 (CC3), and cleavedtau (Ctau) levels by immunoblotting. We have previously shownthat Ctau formation is mainly catalyzed by caspase 3 and detectednoticeably in degenerating axons/dendrites (Saito et al. 2010b).Ethanol-induced neurodegeneration was assessed by Fluoro-JadeC (Millipore, Billerica, MA, USA) staining using brain sectionsfrom mice perfusion-fixed 19 h after the first ethanol injection.Except for brief periods of time for injections, mice were keptwith dams until killed 1–24 h after the saline/ethanol injection.For developmental studies, P1, 4, 7, 10, 13, 16, 19, 25, and 31naıve mice were used. Three to 10 animals were used for eachdata point.

Lipid analysisCeramide and sphingomyelin were separated from total lipids usinghigh performance thin-layer chromatography, and the amounts weremeasured as described previously (Saito et al. 2010a). Determina-tion of S1P and sphingosine content was performed according to themethod of He et al. (2009), modified as described in detail inAppendix S1.

� 2012 The AuthorsJournal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 121, 806–817

Ethanol alters sphingosine 1-phosphate metabolism | 807

ImmunohistochemistryEight and 19 h after the ethanol injection, mice were perfusion-fixedwith a 4% paraformaldehyde solution, and vibratome sections(50 lm) of the fixed brains were prepared and immunofluorescence-labeled as described previously (Saito et al. 2007b, 2010a) usingantibodies against SphK2, NeuN, and Na+,K+-ATPase (seeTable S1 for details of the antibodies used). To check the specificityof anti-SphK2 antibody (Santa Cruz Biotechnology, Santa Cruz,CA, USA), the antibody was incubated with the blocking peptide(Santa Cruz) for 1 h at 23�C prior to incubation with brain sections.For some experiments, the above SphK2 antibody was furtheraffinity-purified as follows: P2 (mitochondria/lysosome/synapto-some) fraction of P7 brain homogenates was isolated as describedbelow in the subcellular fractionation section, and was separated onsodium dodecyl sulfate (SDS) gel electrophoresis and blotted onnitrocellulose membranes. A strip of the membrane containing aband corresponding to SphK2 was blocked with 5% bovine serumalbumin in Tris-buffered saline (pH 7.5) containing 0.1% Tween 20and incubated with the antibody in the blocking buffer overnight at4�C. After rinsing, the antibody was eluted with 50 mM glycine/150 mM NaCl (pH 2.4) and neutralized. The antibody thus purifiedgave a single band when analyzed by immunoblotting. For Fluoro-Jade C staining, brain sections were processed according to themanufacturer’s instruction. The extent of neurodegeneration wasexpressed as the number of Fluoro-Jade C-positive cells per squaremillimeter in the cingulate and retrosplenial cortices as describedpreviously (Saito et al. 2010a) using four brains per treatmentgroup. Photomicrographs were taken through a 20X and a 100Xobjective with a Nikon Eclipse TE2000 inverted microscopeattached to a digital camera DXM1200F.

Subcellular fractionationNuclei from the P7 mouse forebrain were isolated according to themethods of Block et al. (1992) and Koppler et al. (1993) withmodification by Wu et al. (1995). For separation of P1 (crudenucleus), P2 (mitochondria/lysosome/synaptosome), and P3 (micro-some) fractions, and for further fractionation of P2, the combinedmethod of Rajapakse et al. (2001) and Kiebish et al. (2008) wasused. P3 was further fractionated using OptiPrep (60% w/v iodixanolin water, Sigma-Aldrich Chemical Company, St Louis, MO, USA)density gradient centrifugation according to the methods of Arakiet al. (2003), Li and Donowitz (2008), and OptiPrep instructionmanual with some minor modifications. Additional detailed subcel-lular fractionation protocols are available in Appendix S1.

ImmunoblottingImmunoblotting was performed as described previously (Saito et al.2010a). Tissue samples (50 lg of protein) or subcellular fractionsdescribed above (30 lg of protein) were boiled in SDS–samplebuffer, separated on 10% or 15% SDS–polyacrylamide gelelectrophoresis, and blotted onto nitrocellulose membranes. Themembranes were then blocked with Odyssey blocking buffercontaining 0.1% Tween 20 and probed with various antibodies(see Table S1 for details of the antibodies used). Either mousemonoclonal anti-b-actin antibody, or mouse monoclonal anti-b-tubulin antibody was included as loading control. Antigens weredetected by the Odyssey infrared imaging system using secondaryantibodies, IR dye 680 conjugated goat anti-rabbit IgG, IR dye 680

conjugated donkey anti-goat IgG, and IR dye 800 conjugated goatanti-mouse IgG, and analyzed by Multi Gauge ver.2.0 (FujifilmMedical Systems USA, Inc. Stamford, CT, USA). For thequantification analyses, the intensity of each protein band wasnormalized by the corresponding b-actin intensity. Because of theclose molecular weights of S1P1-receptor (S1P1-R; �44 kD) and b-actin (�43 kD), b-tubulin (�50 kD) was used for S1P1-R quanti-fication. In the developmental studies (Fig. 1), intensities of bandsobtained from samples with the same protein amounts were directlycompared among various developmental stages, because amounts ofb-actin and b-tubulin changed significantly during development(data not shown). To check the specificities of anti-SphK1, anti-SphK2, and anti-S1P1-R antibody, antibodies were pre-incubatedwith corresponding peptides for 1 h at 23�C 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, Versailles, KY,USA), and S1P1-Blocking Peptide (Cat No. 10006616; CaymanChemicals, Ann Arbor, MI, USA). The amount of protein wasmeasured by a BCA method (Pierce, Rockford, IL, USA).

Sphingosine kinase assaySphK1 and SphK2 activities in the P7 forebrain were measuredaccording to the methods of Billich and Ettmayer (2004), Wackeret al. (2009), and Liu et al. (2000) except that NBD [x-(7-nitro-2-1,3-benoxadiazol-4-yl)-D-erythro]-S1P formed was extractedaccording to the method of Matyash et al. (2008) using methyltert-butyl ether. Fluorescence was measured using a fluorometer(BioTek Instrument Inc., Winooski, VE, USA) at excitationwavelength 485 nm and emission wavelength 528 nm. Values wereexpressed as percent of control.

StatisticsValues in figures are expressed as mean ± standard error of meanobtained from 4 to 10 samples. Statistical analysis of the data wasperformed by two-tailed Student’s t test, one sample t-test, andANOVA 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 brainPrior to studies of the effects of ethanol on S1P metabolismin the P7 mouse brain, basal levels of S1P-related lipids andproteins (SphK1, SphK2, and S1P1-R) were examinedduring the early postnatal brain development. First, wemeasured changes in brain levels of ceramide, sphingosineand S1P. Results showed that levels of S1P graduallyincreased between P4 and P31, while sphingosine reachedmaximum at P19, and ceramide peaked around P13(Figure S1). Developmental changes in protein levels ofS1P1-R, SphK2, and SphK1 in the forebrain were alsoexamined by immunoblot analyses (Fig. 1). The band inFig. 1a indicated by an asterisk (*) was considered non-specific because pre-incubation of anti-SphK1 antibodywith a blocking peptide solution (as described in Materials

Journal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 121, 806–817� 2012 The Authors

808 | G. Chakraborty et al.

and methods) did not reduce the intensity of this band,while the band below disappeared completely. Bandsshown for SphK2 and S1P1-R were considered specific toeach antibody, based on experiments using correspondingblocking peptides (data not shown). Figure 1b showsquantitative results expressed as fold changes comparedwith P1 values. Levels of SphK2 gradually increasedbetween P1 and P25 in a manner similar to that of S1P(Figure S1c), while the trace amounts of SphK1 found inthe P1 forebrain did not increase during the developmentalperiod. Figure 1 also shows that S1P1-R, a major S1Preceptor isoform in the brain (Brinkmann 2007), increasedduring early postnatal days as observed in levels of S1P(Figure S1c). Thus, the neonatal brain contains significantlevels of S1P, sphingosine, SphK2, and S1P1-R, althoughthe levels were lower than those at later developmentalstages.

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

ForebrainCerebellum

Brainstem Heart Lung LiverKidney

Spleen

Pro

tein

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(fold

of f

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rain

)

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SphK1

Forebra

in

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llum

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tem

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Lung

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SphK1SphK2

S1P1-R

ForebrainCerebellum


Spleen

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(fold

of f

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)

0

1

2

3

4

S1P1-R

*

(a)

(b)

(c)

(d)

Fig. 2 Tissue/organ distribution of SphK1, SphK2, and S1P1-R. 50 lg

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 de-

scribed in Materials and methods. (a) Representative immunoblots

probed with anti-SphK1, anti-SphK2, and anti-S1P1-R antibody.

*Non-specific band. (b–d) Quantitative analyses of immunoblots

probed with anti-SphK1 (b), anti-SphK2 (c), and anti-S1P1-R (d) anti-

bodies. Values [mean ± SEM (n = 3)] are expressed as fold changes

compared with the band intensities of forebrain samples after

normalization with b-actin or b-tubulin.

P1 P4 P7 P10 P13 P16 P19 P25S1P1-RSphK2

Days after birth0 5 10 15 20 25

Pro

tein

exp

ress

ion

(fold

cha

nge)

0

2

4

6

8

10

12

14

16S1P1-RSphK2SphK1

*

*

**

*

* *

*

SphK1*

(a)

(b)

Fig. 1 Developmental profiles of S1P1-R, SphK2, and SphK1 pro-

teins. 50 lg 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. *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 with the content of P1 mice. *Significantly (p < 0.05) dif-

ferent from P4 mice by ANOVA with the Bonferroni’s post hoc test.



We also examined the cellular localization of SphK2 in theP7 brain by immunohistochemistry. Brain sections fromsaline-treated (control) P7 mice were dual immunofluores-cence-labeled with anti-SphK2 antibody and anti-NeuN

antibody (Fig. 3b). The image shows the cingulate cortexregion. In the lower panels, anti-SphK2 antibody was pre-treated with blocking peptides. These results indicate thatSphK2 is localized primarily in neurons. The affinity-purifiedanti-SphK2 antibody (prepared as described in Materials andmethods) also gave similar staining in neurons (data notshown). Immunohistochemistry using anti-SphK1 antibodygave faint staining mainly in astrocytes (data not shown).Also, S1P1-R expression was mostly limited to astrocytes(manuscript in preparation) in agreement with previousstudies on the human brain (Nishimura et al. 2010).

SphK2 NeuN Merged

–Peptide

+Peptide

Fig. 3 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 pre-treated with the blocking peptide.

The bar indicates 10 lm.

1 h 2 h 4 h 8 h 24 h

Ctr Eth Ctr Eth Ctr Eth Ctr Eth Ctr Eth

CC3Ctau

β-actin

Time (hours)

0 5 10 15 20 25

Ratio

(eth

anol/contr

ol)

0

10

20

30

40

CC3

Ctau*

*

* *

(a)

(b)

Fig. 4 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 24 h of

saline (Ctr) or ethanol (Eth) treatment probed with anti-CC3, anti-Ctau,

and anti-b-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 b-actin. Data are

expressed as mean ± SEM, n = 3–5. *Significantly (p < 0.05) different

from the values of the 1 h group by ANOVA with the Bonferroni’s

post hoc test.

Hours0 5 10 15 20 25

ng/m

g w

et w

eigh

t

30

40

50

60

70

80

90

100

ControlEthanol

* *Ceramide

Hours0 5 10 15 20 25

ng/m

g w

et w

eigh

t

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4

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10

ControlEthanol

*S1P

Hours0 5 10 15 20 25

ng/m

g w

et w

eigh

t

0.0

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0.4

0.6

ControlEthanol

**Sphingosine

(a)

(b)

(c)

Fig. 5 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 4–5 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 0 h

with the Bonferroni’s post hoc test.



Ethanol-induced alterations in the levels of ceramide,sphingosine, and S1PP7 mice were exposed to ethanol (5 g/kg) once as describedin Materials and methods. This treatment induced caspase 3

activation in the forebrain 8 h after ethanol injection (Fig. 4).Caspase 3 activation was assessed by CC3 and cleaved tau(Ctau) formation using immunoblotting. Under these ethanoltreatment conditions, time course studies on the effects ofethanol on levels of ceramide, sphingosine, and S1P in thebrain were performed (Fig. 5). Ethanol exposure in P7 micesignificantly increased ceramide levels 8 h after ethanolexposure, and the increase was maintained for at leastanother 16 h (Fig. 5a). The result was similar to the effects oftwo-time ethanol injections (2.5 g/kg each) with a 2-hinterval (Saito et al. 2007a). Sphingosine levels, whichincreased significantly 8 h after ethanol injection, were alsomaintained for another 16 h (Fig. 5b). In contrast, S1Pincreased 2.5 times 4 h after ethanol injection and decreasedto the basal level within the following 4 h (Fig. 5c). Thelevel of sphingomyelin, one of the potential precursors forceramide/sphingosine/S1P, was not altered by ethanol treat-ment (data not shown).

Ethanol transiently increased SphK2 activityEthanol transiently increased SphK2 enzyme activity(p < 0.01 after Bonferroni’s correction) at 2 h (Fig. 6a).

Time (hours)0 5 10 15 20 25

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1 h 2 h 4 h 8 h 24 h Ctr Eth Ctr Eth Ctr Eth Ctr Eth Ctr Eth

S1P1-R

b-tubulin

SphK2

b-actin

0

20

40

60

80

100

120

140

Sph

K2 a

ctiv

ity (

% o

f con

trol)

Time (hours)

*

1 2 4 8 24

(a)

(b)

(c)

Fig. 6 The effects of ethanol on SphK2 enzyme activity and protein

levels of SphK2 and S1P1-R. (a) SphK2 activity in the forebrain was

measured at different time points after saline/ethanol treatment. Val-

ues 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. Fifty micrograms of protein

was analyzed by immunoblotting. Representative immunoblots were

probed with anti-S1P1-R, anti-b-tubulin, anti-SphK2, and anti-b-actin

antibodies. (c) Quantitative analysis of immunoblots. Data are ex-

pressed as ratios of band intensities in the ethanol group to those in

the control group after normalizing SphK2 with b-actin and S1P1-R

with b-tubulin. Ethanol treatment induced no significant changes when

analyzed by one-sample t-test with Bonferroni’s correction.

S E S E S E S E S E

Homog P2 Micro Cytosol P1

SphK2

S E S E S E S E S E

Homog P2 Micro Nuc (U) Nuc (P)

SphK2

synaptophysin

PSD95

VDAC

COX IV

Na+,K+-ATPase

β-Glucosidase

Acetyl histone

(a)

(b)

Fig. 7 Subcellular localization of SphK2 in the P7 mouse forebrain.

Brain samples were collected 2 h after either saline or ethanol treat-

ment, and various subcellular fractions were prepared. Thirty micro-

grams 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 anti-

body. (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-b-glucosidase,

and anti-acetyl histone antibodies.



However, SphK1 enzyme activity, which was roughly onefourth of SphK2 enzyme activity, remained unaffected (datanot shown). This transient increase of SphK2 activity afterethanol treatment was not due to an increase in the SphK2level, because the level measured by immunoblot analyses wasunaltered (Fig. 6b and c). The level of S1P1-R was alsounchanged after ethanol treatment (Fig. 6b and c). Immuno-histochemical analyses indicated that ethanol treatment did notchange the cellular distribution of SphK2 (data not shown).

Subcellular localization of SphK2It has been reported that subcellular localization of SphK2 isimportant for exerting its cellular functions (Igarashi et al.2003; Hait et al. 2009; Wattenberg 2010; Strub et al. 2011).P7 mouse brains decapitated 2 h after saline/ethanol injectionwere homogenized and fractionated into P1 (crude nucleus),P2 (mitochondria/lysosome/synaptosome), P3 (microsome),

and soluble (cytosol) fractions, and SphK2 levels in thesefractions were analyzed by immunoblotting (Fig. 7a). Weobserved that SphK2 was highly expressed in the P2 and P3(Micro) fractions while it was not detected in the P1 and verylow in the cytosol fraction. Subcellular distribution of SphK2was not significantly different between saline and ethanol-treated brains. Because the presence of SphK2 in the nucleushas been reported previously (Igarashi et al. 2003; Dinget al. 2007; Sankala et al. 2007; Hait et al. 2009), P1 fractionwas further purified as described in Appendix S1 to increasethe ratio of SphK2 to other proteins, if SphK2 is enriched inthe nucleus. However, SphK2 was not detected in thepurified nuclear fraction [Nuc (P)] (Fig. 7b). The purity ofthe nuclear fraction was confirmed by the abundant presenceof acetyl histone and the absence of other subcellular markerproteins, synaptophysin (synaptic vesicle), postsynapticdensity protein 95 (PSD95, synaptic membrane), voltage-

SphK2Na+,K+-ATPase

Flotillin-1

VDACLAMP1

PSD95

Synaptophysin

BiP78

Homog

Mito

1M

ito (n

s)

Mito

(s)

Synap

1

Synap

2

SphK2Na+,K+-ATPase

Flotillin-1ERp72

Rab5Syntaxin6

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction No

OptiPrep

Fraction number 0 5 10 15 20

% d

istri

butio

n

0

10

20

30

40

SphK2 ERp72 Flotillin-1 Rab5 Na+,K+-ATPase Syntaxin6

(a)

(b)

(c) (d)

Fig. 8 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 fur-

ther subfractionated as described in Mate-

rials 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 lg of protein each) were probed with

anti-SphK2, anti-Na+,K+-ATPase, anti-flotil-

lin-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.

Twenty-five microliters of each fraction was

immunoblotted and probed with anti-SphK2,

anti-Na+,K+-ATPase, anti-flotillin-1, anti-ER-

p78, anti-Rab 5, and anti-syntaxin 6 anti-

bodies. As no visible bands were detected

with these antibodies in fractions 1 to 4, only

fractions 5–20 are shown here. (c) The fig-

ure illustrates semi-quantitative profiles of

distribution of each marker protein described

in panel (b). (d) Panels show images of the

cingulate cortex region from brain sections

of control P7 mice dual-labeled with anti-S-

phK2 and anti-Na+,K+-ATPase antibodies.

The bar indicates 10 lm.



dependent anion channel (VDAC, mitochondria), ComplexIV (COX IV, mitochondria), Na+,K+-ATPase (plasma mem-brane), and b-glucosidase (lysosome). To better understandthe subcellular localization of SphK2 found in the P2fraction, P2 was further fractionated by different densitygradient centrifugations, followed by western blot analysesof each fraction containing 30 lg of protein (Fig. 8a). Aspredicted from a previous report (Rajapakse et al. 2001),Mito (ns) and Mito (s) fractions were enriched in voltage-dependent anion channel, a mitochondrial marker. Also,Mito (ns) contains LAMP1, a lysosomal/late endosomalmarker. Synap1 and Synap2 were enriched in PSD95 (asynaptic membrane marker) and synaptophysin (a synapticvesicle marker), respectively. These synaptosomal fractionsalso contained Na+,K+-ATPase (a plasma membrane marker)and flotillin-1 (a lipid raft marker). Figure 8a indicates thatSphK2 was absent from mitochondrial/lysosomal fractionsand predominantly localized in synaptosomal vesicle- andsynaptosomal membrane-enriched fractions. The resultsshown in Fig. 7 also indicated that SphK2 was present inthe P3 (microsomal) fraction. To examine the subcellularlocalization of SphK2 in this fraction, components of themicrosomal pellet were separated by iodixanol densitygradient centrifugation and probed with antibodies againstdifferent 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 membraneregions. This notion also agrees with the enrichment ofSphK2 in synaptic membrane and synaptic vesicle fractionsin the P2 pellet (Fig. 8a). Syntaxin6, a Golgi marker, andERp72, an ER marker, showed different distribution patternfrom that of SphK2, although the presence of SphK2 in theER cannot be excluded because of the small difference in thedensities. In Fig. 8d, brain sections from saline-treated(control) P7 mice were dual immunofluorescence-labeledwith anti-SphK2 antibody and anti-Na+,K+-ATPase anti-body. The image shows the cingulate cortex region. TheSphK2 antibody used here was affinity-purified as describedin Materials and methods. The results indicated partial co-localization of SphK2 with Na+,K+-ATPase, which wasconsistent with the subcellular fractionation results.

The effects of DMS on ethanol-induced caspase 3activation and neurodegeneration in the P7 mouse brainTo evaluate the involvement of S1P in the ethanol-inducedapoptotic pathway, DMS, a SphK inhibitor, was adminis-tered into P7 mice via intracerebroventricular injection0.5 h before the ethanol injection, and cleaved caspase 3formation at 8 h after ethanol injection was analyzed bywestern blotting. Figure 9 shows the effects of DMS onCC3 formation induced by the two-time ethanol injections.The results indicated that DMS alone did not affect caspase3 activation, but DMS attenuated ethanol-induced caspase 3activation. Similar effects of DMS were observed using theone-time ethanol injection protocol (data not shown). It hasbeen shown that caspase 3 activation in the P7 mouse brainthat peaks around 8 h after the ethanol injection leads torobust neurodegeneration detected by silver staining (Olneyet al. 2002) and Fluoro-Jade staining (Ieraci and Herrera2006; Saito et al. 2010a). To assess if DMS attenuatesethanol-induced neurodegeneration, the effects of DMS onFluoro-Jade C staining were examined in the cingulate andthe retrosplenial cortex. Figure 10a shows representativeimages of Fluoro-Jade C staining in the cingulate cortexregion from control (Ctr), DMS, ethanol (Eth), etha-nol+DMS (Eth+DMS) mice, and Fig. 10b shows thequantified results calculated from the images and expressedas Fluoro-Jade C-positive cell number per square millime-ter. ANOVA with the Bonferroni’s post hoc test showed thatthe ‘Eth+DMS’ group was significantly different from allother groups, indicating that DMS treatment partiallyblocked ethanol-induced neurodegeneration assessed byFluoro-Jade staining.

Discussion

Our studies showed that ethanol treatment transientlyincreased SphK2 activity and S1P content in the P7 mouse

Ctr DMS Eth Eth+DMS

Rat

io (t

reat

men

t/con

trol)

0

1

2

3

4

5

*

Ctr DMS Eth Eth+DMS

CC3

β-actin

(a)

(b)

Fig. 9 Effects of DMS, a SphK inhibitor, on ethanol-induced caspase

3 activation. 0.5 h before saline/ethanol injection, DMS (3 lg in 1.5 lL

DMSO) or vehicle was administered to P7 mice via intracerebroven-

tricular injection. Saline or ethanol was injected twice with a 2-h

interval, and forebrains were taken 8 h 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 immu-

noblot probed with anti-CC3 antibody and anti-b-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 b-actin. *Significantly different from all other groups

by ANOVA with the Bonferroni’s post hoc test.



brain prior to peak caspase 3 activation, and that pre-treatmentwith DMS (an inhibitor of SphK) attenuated the caspase 3activation and the subsequent neurodegeneration. As far as weknow, this is the first report describing the effects of ethanol onthe endogenous S1P metabolism in the brain. Under thepresent conditions, ethanol increased ceramide, sphingosine,and S1P in the P7 mouse brain (Fig. 5) along with inducingrobust caspase 3 activation (Fig. 4). While the time course ofsphingosine elevation was similar to that of ceramide, theelevation of S1P occurred transiently 4 h after ethanolexposure. The similar transient activation of SphK2 by ethanol(Fig. 6a) shortly before the elevation of S1P levels stronglysuggests that ethanol-induced SphK2 enzyme activationmediates S1P elevation. As protein levels of SphK2 remainedunaltered (Fig. 6b and c), post-translational modifications,such as phosphorylation described previously (Ding et al.2007; Hait et al. 2007), may cause ethanol-induced SphK2activation. The contribution of SphK1 to ethanol-inducedelevation of S1P is expected to beminimal, because the SphK1level was low in the brain (Figs 1 and 2), and the SphK1enzyme activity, which was one fourth of the SphK2 activity,did not show any change by ethanol treatment. Our observa-tion that SphK2 is the major SphK isoform in the mouse brain(Figs 1 and 2) agrees with previous studies (Blondeau et al.2007; Pfeilschifter et al. 2011). The immunohistochemicaldata (Fig. 3) suggest that SphK2 is localizedmainly in neurons

as indicated in a previous study (Blondeau et al. 2007), whileSphK1 seemsmainly expressed in astrocytes (data not shown).The localization of SphK1 in astrocytes and microglia has alsobeen reported by others (Lee et al. 2010; Nayak et al. 2010;Fischer et al. 2011). These results indicate that ethanol triggersS1P elevation via activation of SphK2 in neurons in the P7mouse brain, although we cannot exclude the possibility thatS1P 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 rear-rangements, angiogenesis, and immunity, and the level ofS1P is mainly controlled by the SphK activity (Spiegel andMilstien 2003). In contrast to the proliferative and anti-apoptotic effects of S1P produced by SphK1, S1P generatedby SphK2 has been implicated to cause apoptosis and othercellular functions through intracellular targets in severalcultured cells (Igarashi et al. 2003; Maceyka et al. 2005;Okada et al. 2009). Our subcellular fractionation studiesshowed that SphK2 was localized mainly in the P2 and P3fractions but not in the nuclear fraction, and the localizationwas unchanged by ethanol treatment (Fig. 7). Furtherfractionation of P2 and P3 indicated that SphK2 was enrichedin synaptic vesicles (identified by synaptophysin), synapticmembranes (identified by PSD95), and the plasma membrane(identified by Na+,K+-ATPase and flotillin) fractions (Fig. 8),

Ctr DMS Eth Eth+DMS

FJ-p

ositi

ve c

ells

/mm

2

0

200

400

600

800

1000Cingulate Cx

*

#

Ctr DMS Eth Eth+DMS

FJ-p

ositi

ve c

ells

/mm

2

0

200

400

600

800

1000

1200Retrosplenial Cx

*

#

(a)

(b)

Fig. 10 Effects of DMS, a SphK inhibitor,

on ethanol-induced neurodegeneration.

0.5 h before saline/ethanol injection, DMS

(3 lg in 1.5 lL DMSO) or vehicle was

administered to P7 mice via intracerebro-

ventricular injection. Saline or ethanol was

injected twice with a 2-h interval, and the

mice were perfusion-fixed 19 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 repre-

sentative image here shows the cingulate

cortex region. The bar indicates 50 lm. (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.



although we cannot rule out the possibility that SphK2 wasalso localized in endosomes (identified by Rab5), endoplas-mic reticulum (identified by ERp72), and other organelleswhich have similar densities. To the best of our knowledge,this is the first in depth subcellular fractionation studyreporting the localization of SphK2 in the brain. It appearsthat the subcellular localization of SphK2 in the P7 brain isdifferent from that reported mainly in cultured cells, whereSphK2 is detected in the nucleus (Igarashi et al. 2003; Dinget al. 2007; Sankala 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 inthe plasma membrane has been reported in some cell types(Maceyka et al. 2005), apoptotic functions of SphK2 aregenerally associated with the presence of SphK2 in thenucleus (Igarashi et al. 2003; Okada et al. 2005) and ER(Maceyka et al. 2005; Hagen et al. 2009). Interestingly, ourresults indicated that DMS, a SphK inhibitor, significantlyattenuated ethanol-induced caspase 3 activation (Fig. 9) andneurodegeneration (Fig. 10), although further studies arenecessary because DMS is not a specific inhibitor for SphK2(French et al. 2010). Nonetheless, our results suggest thatSphK2 primarily localized in or near the plasma membranein neurons is activated by ethanol, produces S1P, and inducesor enhances apoptotic action of ethanol. Because ourprevious studies have shown that inhibitors of serinepalmitoyl transferase, a rate limiting enzyme for sphingolipidsynthesis, attenuate ethanol-induced apoptosis (Saito et al.2010a), de novo ceramide synthesis may be involved in theS1P formation catalyzed by SphK2. Also, the partialblocking of ethanol-induced neurodegeneration by DMS(Fig. 10) suggests that ceramide/sphingosine may enhanceneurodegeneration independent of S1P action. It has beenshown that S1P metabolism is affected under pathologicaland stressful conditions in the nervous system. For example,increases in the expression levels of SphK1 have been shownin kainic acid-treated hippocampal astrocytes (Lee et al.2010) and lipopolysaccaride-treated cultured microglia (Na-yak et al. 2010), whereas increases in SphK2 expression oractivity have been reported in cerebral microvessels underhypoxic pre-conditioning (Wacker et al. 2009), in theischemic brain (Blondeau et al. 2007), and in the brains ofpatients with Alzheimer’s disease (Takasugi et al. 2011).While S1P elevation produced by SphK2 is consideredapoptotic in the cerebellar granule neurons derived from S1Plyase-deficient mice (Hagen et al. 2009) as well as in othernon-neural cells (Liu et al. 2003; Maceyka et al. 2005;Okada et al. 2005), SphK2 activation is found neuroprotec-tive in some animal models of brain ischemia (Wacker et al.2009; Hasegawa et al. 2010; Pfeilschifter et al. 2011; Yunget al. 2012). Whether SphK2 activation leads to neuropro-tection or not may depend on the subcellular targets of S1Pproduced. The efficacy of S1P receptor agonists in neuro-protection in some of these studies (Wacker et al. 2009;

Hasegawa et al. 2010; Pfeilschifter et al. 2011) suggests thatS1P produced by SphK2 may activate S1P receptors, leadingto neuroprotection, or S1P in mitochondria may exertcytoprotection as indicated in a myocardial injury model(Gomez et al. 2011). S1P increased by ethanol treatment inthis study may have different targets, inducing or enhancingneuroapoptosis. Our observation that S1P1-R, a major S1Preceptor in the brain (Brinkmann 2007), was mainly localizedin astrocytes as reported previously (Nishimura et al. 2010)suggests that S1P produced by SphK2 in neurons may exertits function independent of S1P1-R. S1P may interact withother S1P receptor isoforms or may have other targets, suchas Na+,K+-ATPase as recently reported (Dakroub andKreydiyyeh 2012).

In conclusion, we demonstrated that ethanol transientlyelevated SphK2 activity and S1P levels in the P7 mousebrain, which may be related to ethanol-induced apoptoticneurodegeneration.

Acknowledgements

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

Supporting information

Additional supporting information may be found in the onlineversion of this article:

Appendix S1. Supplementary Materials and Methods.Table S1. List of primary antibodies used in immunoblotting (IB)

and immunohistochemistry (IHC).Figure S1. Developmental changes in levels of ceramide,

sphingosine, and S1P.As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such materials arepeer-reviewed and may be re-organized for online delivery, but arenot copy-edited or typeset. Technical support issues arising fromsupporting information (other than missing files) should beaddressed to the authors.

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

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