Brain Sci. 2013, 3, 670-703; doi:10.3390/brainsci3020670
OPEN ACCESS
brain sciences
ISSN 2076-3425
www.mdpi.com/journal/brainsci/
Review
Involvement of Sphingolipids in Ethanol Neurotoxicity in the
Developing Brain
Mariko Saito 1,2,* and Mitsuo Saito 3
1
2
3
Division of Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, 140 Old
Orangeburg Rd., Orangeburg, NY 10962, USA
Department of Psychiatry, New York University Langone Medical Center, 550 First Ave.,
New York, NY 10016, USA
Division of Analytical Psychopharmacology, Nathan S. Kline Institute for Psychiatric Research,
140 Old Orangeburg Rd., Orangeburg, NY 10962, USA; E-Mail: mitsaito@nki.rfmh.org
* Author to whom correspondence should be addressed; E-Mail: marsaito@nki.rfmh.org;
Tel.: +1-845-398-5537; Fax: +1-845-398-5531.
Received: 21 Febuary 2013; in revised form: 30 March 2013 / Accepted: 12 April 2013 /
Published: 26 April 2013
Abstract: Ethanol-induced neuronal death during a sensitive period of brain development
is considered one of the significant causes of fetal alcohol spectrum disorders (FASD). In
rodent models, ethanol triggers robust apoptotic neurodegeneration during a period of
active synaptogenesis that occurs around the first two postnatal weeks, equivalent to the
third trimester in human fetuses. The ethanol-induced apoptosis is mitochondria-dependent,
involving Bax and caspase-3 activation. Such apoptotic pathways are often mediated by
sphingolipids, a class of bioactive lipids ubiquitously present in eukaryotic cellular
membranes. While the central role of lipids in ethanol liver toxicity is well recognized, the
involvement of sphingolipids in ethanol neurotoxicity is less explored despite mounting
evidence of their importance in neuronal apoptosis. Nevertheless, recent studies indicate
that ethanol-induced neuronal apoptosis in animal models of FASD is mediated or
regulated by cellular sphingolipids, including via the pro-apoptotic action of ceramide and
through the neuroprotective action of GM1 ganglioside. Such sphingolipid involvement in
ethanol neurotoxicity in the developing brain may provide unique targets for therapeutic
applications against FASD. Here we summarize findings describing the involvement of
sphingolipids in ethanol-induced apoptosis and discuss the possibility that the combined
action of various sphingolipids in mitochondria may control neuronal cell fate.
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Keywords: ethanol; sphingolipid; developing brain; apoptosis; neurodegeneration;
mitochondria; ceramide; ganglioside; sphingosine-1-phosphate; fetal alcohol
spectrum disorders
1. Introduction
Sphingolipids, which are a class of bioactive lipids containing sphingoid bases as a basic structure,
are involved in various cellular processes, such as differentiation, proliferation and apoptosis in a wide
variety of cellular systems (reviewed by [1,2]). There are numerous derivatives, including sphingosine,
sphingosine-1-phosphate, ceramide, ceramide-1-phosphate, sphingomyelin, and glycosphingolipids
(reviewed by [3,4]). Apoptosis triggered by various inducers is often mediated or regulated by
sphingolipids. Specifically, ceramide and sphingosine have been recognized as pro-apoptotic mediators,
while sphingosine-1-phosphate (S1P) has been considered anti-apoptotic (reviewed by [2,5–7]). Such
involvement of sphingolipids in cell death and survival has been widely observed in the nervous
system (reviewed by [8–12]), which is highly enriched in sphingolipids (reviewed by [13]). While
ceramides may be necessary to regulate neural cell numbers during brain development [14–17],
dysregulated ceramide formation is involved in neurodegeneration in several neurodegenerative
diseases (reviewed by [11,18–20]), and certain gangliosides (sialic acid-containing glycosphingolipids),
such as GM1, often exert neuroprotection (reviewed by [21–26]).
Ethanol affects lipid metabolism in many cell types, and such alterations are considered a factor
causing or regulating tissue injury. For example, in the liver, alcoholic steatosis is recognized as a
condition leading to steatohepatitis, fibrosis, and cirrhosis (reviewed by [27]). It has been shown that,
along with enhanced lipogenesis induced by ethanol metabolism (reviewed by [28]), many regulators
of lipid metabolism including AMP-activated protein kinase (AMPK) and sterol regulatory
element-binding protein (SREBP)-1 are disturbed by ethanol, causing hepatic steatosis (reviewed
by [29]). The disturbance in lipid metabolism by ethanol is also associated with pro-apoptotic ceramide
elevation [30], (reviewed by [31]).
Prenatal ethanol exposure perturbs brain development in all three trimesters of pregnancy, leading
to long-lasting deficits in cognition and behavior observed in patients with fetal alcohol spectrum
disorders (FASD) (reviewed by [32]). To elucidate mechanisms of ethanol toxicity in the developing
brain, rodent models of FASD have been widely used (reviewed by [33]). As reviewed by Guerri [34],
ethanol exposure in rats during the period between gestational day (GD) 5 and GD11 (roughly
equivalent to the first trimester of human gestation) results in neural tube defects and alterations in
neural precursor cell proliferation. Ethanol exposure during the period between about GD11 and GD18
(roughly equivalent to the second trimester) alters development of radial glia and disturbs proliferation,
generation, and migration of neurons, and ethanol exposure during the period between about GD18
and postnatal days (P) 9 (roughly equivalent to the third trimester) induces severe neuronal loss,
reactive gliosis, and delayed myelination.
While sphingolipids are likely to be involved in these diverse effects of prenatal ethanol, the majority
of in vivo studies so far have focused on testing the involvement of sphingolipids in ethanol-induced
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neuronal death. Ethanol-induced neurodegeneraion is particularly robust during the period of active
synaptogenesis [35,36] (corresponding to the first two postnatal weeks for rodent pups and the third
trimester for human fetuses), and likely contributes to the pathogenesis and outcome of FASD [35,37,38]
(reviewed by [32]). This neuronal death occurs via the Bax-dependent mitochondria-mediated
apoptotic pathway [39–42], and may be related to ethanol-induced changes in lipid metabolism. In
fact, ethanol profoundly alters lipid metabolism in the developing brain and cultured neurons. Studies
show it induces ceramide elevation [43–45], alters fatty acid composition [46,47], changes ganglioside
profiles [45,48,49], and promotes ceramide/sphingosine recycling for ganglioside biosynthesis [50].
These effects of ethanol on sphingolipid metabolism and accumulated evidence on the roles of
ceramides in mitochondria-mediated neuronal apoptosis strongly suggest that ethanol-induced
apoptotic neurodegeneration is mediated or regulated by altered sphingolipid metabolism. In this
review, we summarize studies related to this hypothesis, focusing on roles of ceramides, S1P, and
gangliosides in ethanol-induced apoptosis in the developing brain, and discuss the possible functions
of these sphingolipids in mitochondria. We also present the possibility that the developing brain at the
peak of active synaptogenesis may display unique sphingolipid profiles/metabolism, which contribute
to its heightened sensitivity to the apoptotic effects of ethanol. First, we briefly summarize studies
showing mechanisms behind ethanol-induced apoptosis in the developing brain. Then, we describe the
possible involvement of ceramide, S1P, and gangliosides in this apoptotic pathway.
2. Ethanol-Induced Neuronal Apoptosis in the Developing Brain
2.1. Neuronal Apoptosis Triggered by Ethanol during the Period of Synaptogenesis
Previous studies using rodent models for FASD have demonstrated that early postnatal binge
ethanol exposure during the brain growth spurt period, on postnatal days 4–9 (P4–9) for example, causes
high incidence of neuronal loss [51–54], followed by long-lasting behavioral deficits [52,55–57]. The
cell loss appears to be caused by apoptosis, which occurs immediately after ethanol exposure, and is
detected by morphological characteristics of apoptosis (such as cell shrinkage), TUNEL staining,
caspase-3 activation, and involvement of the Bcl-2 family [35,36,58–60]. The developing neurons are
sensitive to the pro-apoptotic effects of ethanol during the brain growth spurt period, which is also the
period of active synaptogenesis, although different neuronal populations display the peak sensitivity at
different time points within this window [35]. Acute ethanol exposure in P7 rodents induces apoptotic
neurodegeneration within one day in many brain regions, including the cortex, thalamus, and caudate
putamen [35,36] in a dose-dependent manner [61], and results in long-lasting behavioral [38,62–65]
and electrophysiological [38,64] deficits observed in adult animals. Acute ethanol also triggers
apoptosis of Purkinje and granule cells in the cerebellum of neonatal rodents, and the peak apoptosis
is found around P4 [40,41,66–70], (reviewed by [71]). Such acute ethanol-induced apoptotic
neurodegeneration during the brain growth spurt is observed not only in rodents but also in fetal
macaque brain at various stages of gestation (G105 to G155) depending on the brain regions [37].
Chronic ethanol exposure during gestation and lactation also induces apoptosis in the early postnatal
rat brain, although cell death is found more in GFAP-positive glial cells [72]. The effects of ethanol on
astrocytes appear to depend on the levels of ethanol, duration, timing of exposure, and the stage of
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glial maturation [73] as shown in the effects of ethanol on neurons. While prenatal ethanol exposure
reduces GFAP expression [73], brief exposure to high levels of ethanol during the brain growth spurt
causes astrogliosis detected by an increase in immunoreactive GFAP [74], along with microglial
activation that appears to facilitate clearance of dead neurons [75]. Ethanol-induced apoptosis is reported
in many types of cultured neurons as well [76–80], (reviewed by [71]). Thus, developing neurons are
particularly sensitive to the pro-apoptotic effects of ethanol during the period of synaptogenesis.
2.2. Mechanisms behind Ethanol-Induced Neuronal Apoptosis
Ethanol exposure in P7 rats or mice induces apoptosis via mitochondria-mediated intrinsic pathway,
involving Bax-induced disruption of mitochondrial membranes, cytochrome c release, and caspase-3
activation in the neonatal brain [39,42]. Although direct targets of ethanol leading to the intrinsic
apoptotic pathway have not been fully elucidated, ethanol’s blocking action at NMDA receptors and its
enhancing action at GABAA receptors may be responsible [35,36]. Ethanol exposure in P4–6 rats
decreases Purkinje cell expression of TrkB and TrkC receptors [81], and apoptotic neurodegeneration
in P7 mice exposed to ethanol is associated with inactivation of Akt [82–84] and extracellular
signal-regulated kinase (ERK) [83,84] and activation of glycogen synthase kinase-β (GSK3β) [75,82].
Lithium, a GSK3β inhibitor [85], attenuates ethanol-induced apoptosis in the P7 brain [82,84,86],
(reviewed by [87]). It is also reported that ethanol-induced apoptosis is accompanied by c-Jun
N-terminal kinase (JNK) activation in neonatal rats [39,88]. Ethanol-induced oxidative stress/free
radical formation is one of the important factors linked to the apoptotic pathway as well. Ethanol
rapidly increases reactive oxygen species (ROS) and the lipid peroxidation product, 4-hydroxynonenal
(HNE), in the neonatal brain [89,90]. Importantly, various anti-oxidant treatment paradigms ameliorate
cell death triggered by ethanol [90–92]. NADPH oxidase (NOX) activation by ethanol in the P7 mouse
brain seems to be a cause of ROS generation [93]. Such oxidative stress induced by ethanol may
trigger the endoplasmic reticulum (ER) stress reported in the P7 brain [94].
Also, in cultured neurons, ethanol induces apoptosis via mitochondria-mediated intrinsic pathway,
involving Bax-induced disruption of mitochondrial membranes, cytochrome c release, and caspase-3
activation [41,80,95,96]. Ethanol may trigger apoptosis in cultured rat cerebellar granule neurons
(CGNs) by inhibiting NMDA receptor functions [97] or inhibiting insulin-like growth factor-1 (IGF-1)
receptor functions [98]. The inhibition of NMDA receptors leads to the suppression of brain-derived
neurotrophic factor (BDNF) expression [99,100], and the inhibition of BDNF or IGF-1 function
appears to induce apoptosis through the inhibition of pro-survival PI3K/Akt [101] in CGNs [98,99]
and in cortical neurons [102]. The inhibition of PI3K/Akt pathway triggers caspase-9 and caspase-3
activation, which is an execution phase of apoptosis [103]. The importance of GSK3β activation in
ethanol-induced neuroapoptosis has been also highlighted by studies in vitro; overexpression of
GSK3β sensitizes neurons to ethanol toxicity [104], and lithium attenuates ethanol-induced apoptosis
in cultured neurons [86]. Furthermore, SB216763, a selective GSK3β inhibitor, prevents apoptosis
induced by ethanol in cultured neurons [105]. It is also reported that ethanol-induced apoptosis is
accompanied by JNK activation in SK-N-SH cells [106]. In glial cell cultures, cell death triggered by
ethanol is associated with activation of JNK [107,108], mitogen-activated protein kinase p38 (p38
MAPK) [107,108], and ERK pathways [107–109]. Also, ethanol rapidly increases ROS and 4-HNE, in
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cultured neurons [95,110–114], and various anti-oxidant treatment paradigms ameliorate cell death
triggered by ethanol [95,110,112,113,115]. Further, the neuronal glutathione content appears to
determine selective vulnerability of cultured cortical neurons to ethanol-induced apoptosis [116]. It is
indicated that metabolism of ethanol generates ROS and nitric oxide (NO) by activation of NADPH
oxidase (NOX)/xanthine oxidase and inducible NO synthase in cultured cortical neurons [111].
Thus, ethanol activates a mitochondria-mediated, Bax-dependent apoptotic pathway, involving
ROS formation, inactivation of Akt and ERK, and activation of GSK3β and JNK. Accumulated
evidence suggests that sphingolipids play important roles in such apoptotic pathways. Specifically,
ceramide has been recognized as a pro-apoptotic mediator, while sphingosine-1-phosphate (S1P) and
GM1 ganglioside have been considered anti-apoptotic mediators (reviewed by [2,6,7,21–23,26]). The
relevance of ceramide, S1P, and gangliosides in ethanol-induced apoptosis is described in the
following sections.
3. Ceramide Involvement in Ethanol-Induced Neuronal Apoptosis in the Developing Brain
3.1. Involvement of Ceramide in Apoptosis in the Developing Brain
Ceramides are essential sphingolipid messengers regulating a diverse range of cell-stress responses,
such as apoptosis, cell senescence, and autophagy. Various factors, including the species of ceramides
generated (out of >200 structurally distinct molecules) and its subcellular localization, appear to
determine the ceramide functions (reviewed by [117]). Numerous studies have demonstrated that ceramide
mediates or enhances both extrinsic and intrinsic apoptotic pathways in many cell types (reviewed
by [1–3,118]) including neurons (reviewed by [8–12,119,120]). While such ceramide-mediated
apoptosis can be beneficial during a certain period of neuronal development for regulating neural cell
numbers [14,15,17,121], dysregulated ceramide formation is implicated in neural death in several
neuroinflammatory and neurodegenerative disorders (reviewed by [8,11,12,18–20]). A variety of
studies using cultured neurons and animal models of neurodegenerative diseases support the notion
that ceramide is involved in the apoptotic pathways (reviewed by [8–12,119,120,122]). First, cellular
ceramide elevation, either by adding natural or short-acyl chain analogs of ceramide or by modulating
ceramide metabolizing enzymes, induces apoptosis in cultured neurons (reviewed by [11,119,122]).
Second, many apoptotic inducers elevate endogenous levels of ceramide, and the inhibition of such
ceramide generation by pharmacological or genetic manipulation attenuates cell death (reviewed
by [8,11,12,20,119,120]). As shown in Figure 1, ceramide can be generated by activation of neutral or
acid sphingomyelinase (SMase), by activation of the salvage pathway, which involves ceramide
formation from sphingosine released from the lysosome, or by enhancement of de novo ceramide
synthesis (reviewed by [2,4,11,20,120]). In general, neutral and acidic SMases trigger early and
transient ceramide increase, while de novo ceramide synthesis causes late and sustained ceramide
elevation [123]. Recent studies indicate that molecular species of ceramides thus produced is an
important factor in determining ceramide functions [117]. In untreated cultured neurons, C18 is a
major fatty acid of ceramides [124], and ceramide synthase 1 (CerS1) that catalyzes de novo synthesis
of C18 ceramide is a major and specific CerS in neurons [125] out of six mammalian CerSs
(CerS1–CerS6) (reviewed by [126]). However, the increase in C16 ceramide is associated with
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apoptosis in neurons [124,127,128] as well as in some other cell types [129–131], while increases in
C20 and C24 ceramides in hippocampal tissues from an Alzheimer’s disease (AD) mouse model are
linked to astroglial cell death [132].
The ceramide elevation in the brain or in cultured neurons is followed by activation of pro-apoptotic
signaling pathways, p38 MAPK [133,134], JNK [134,135], and GSK3β [135]. In concert with
activation of these pro-apoptotic pathways, ceramide elevation is associated with inhibition of survival
pathways, PI3K/Akt [135,136] and ERK pathways [133,135]. The inactivation of these pathways may
be caused by direct activation of protein phosphatase 2A (PP2A) by ceramide [137], followed by
dephosphorylation (inactivation) of Akt and ERK ([138], reviewed by [139,140]).
Ceramide is also implicated in mediating oxidative stress via direct effects on mitochondrial ROS
generation or via activation of NOX (reviewed by [141,142]). For example, studies show that ceramide
accumulation by TNFα-stimulated neutral SMase activation results in the formation of ROS by NOX
activation in dorsal root ganglion neurons [143].
Figure 1. Ceramide generating pathways. Three major pathways for ceramide generation
are shown here. Ceramide is synthesized via “de novo synthesis pathway” in endoplasmic
reticulum (ER), which involves several enzymes including serine palmitoyltransferase
(SPT, the initial sphingolipid synthesizing enzyme) and ceramide synthase (CerS).
Ceramide can be generated by activation of neutral (n) or acid (a) SMases (“SMase
pathway”), often found in the plasma membrane. In the “salvage pathway”, ceramide is
synthesized by CerS from sphingosine released from the lysosome. Although not shown
here, ceramide generation may also occur in the mitochondria, where ceramide generating
enzymes, such as CerS, have been found. These pathways are activated by a variety of
apoptotic inducers in various cell types including neurons as described in the text. (CDase,
ceramidase; SphK, sphingosine kinase; SPPase, S1P phosphatase; GCS, glucosylceramide
synthase; SMS, sphingomyelin synthase; GlcCer, glucosylceramide.)
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Thus, while ceramide is involved in many apoptotic pathways, mechanisms of ceramide formation,
sites of the ceramide action, ceramide species generated, and the downstream pathways may differ
depending on the type of apoptotic inducers and cellular conditions. However, it is generally recognized
that mitochondria are key sites of the ceramide action in a variety of apoptotic pathways [144].
Specifically, the role of mitochondrial ceramide (or ceramide metabolites) in Bax/Bak-dependent
mitochondrial outer-membrane permeabilization (MOMP) and the following release of mitochondrial
proteins (including cytochrome c) have been reported not only in mammalian cells [144–148], but also
in yeast cells [146,149]. The brain mitochondria contain SM and ceramide [150], and the related
enzymes, such as ceramide synthases (CerS1, CerS2, CerS4 and CerS6) [15,128], SMases [151], and
neutral ceramidase [152], providing potentially important intracellular compartment for ceramide
metabolism [153]. The elevation of ceramide content and CerS activity in mitochondrial ceramide-rich
microdomain appears to be necessary for Bax insertion, MOMP, and the following release of
pro-apoptotic factors in radiation-induced apoptosis [148]. Ceramides in mitochondria are also
implicated in regulation of Ca2+ levels. Studies indicate that ceramide is responsible for increased Ca2+
levels in mitochondria prior to calpain-mediated apoptosis in retinal photoreceptor cells [154] and in
primary oligodendrocyte precursors [15]. CerS6/ceramide in mitochondrial inner membrane is
suggested to regulate mitochondrial Ca2+ homeostasis by inhibiting mitochondrial permeability
transition pore (MPTP) opening [15]. Because excessive accumulation of Ca2+ in mitochondrial matrix
can trigger MPTP opening at a high conductance state and lead to cell death by necrosis, MPTP may
regulate necrosis [153] in contrast to MOMP, which releases small pro-apoptotic molecules, leading to
apoptosis. It is indicated that MPTP-dependent, apoptosis-independent process is critical for brain
injury in the adult, whereas Bax-dependent apoptotic mechanisms prevail in the immature brain [155].
This notion may be related to studies indicating that mitochondrial Ca2+-loading capacity and the
threshold of MPTP opening are higher in immature brain [153], probably due to the elevated
expression of C16-ceramide (and CerS6) in mitochondria [15]. These studies suggest that ceramides
regulate apoptosis by affecting both MOMP and MPTP, although further investigation is necessary to
reveal precise roles of mitochondrial ceramides in neuronal apoptosis in the developing brain.
3.2. Ceramide in Ethanol-Induced Apoptosis in the Developing Brain
As described above, ethanol-induced apoptosis in the developing brain shows similar characteristics
to those observed in the ceramide-mediated apoptotic pathway. Both ethanol and ceramide activate
mitochondria-mediated apoptotic pathways involving Bax-induced disruption of mitochondrial
membranes, ROS formation, inactivation of Akt and ERK, and activation of GSK3β and JNK,
suggesting that ceramide is involved in ethanol-induced apoptosis.
In cultured neurons, our studies [45] have shown that ethanol-induced cell death in CGNs and
SK-N-SH human neuroblastoma cells are associated with significant accumulation of ceramide.
Further, ethanol-induced cell death in these neurons is attenuated by myriocin, an inhibitor of serine
palmitoyltransferase (SPT) (the first rate limiting enzyme for sphingolipid synthesis, as shown in
Figure 1), implying that de novo ceramide synthesis is important for this ethanol-induced cell death.
Concomitant increases in levels of triglycerides (TG) and GM2 ganglioside [45] indicate that ethanol
may enhance lipid synthesis or inhibit fatty acid β-oxidation, leading to ceramide accumulation, as
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reported in the liver [30], although our studies have also shown that glucosylceramide (GlcCer)
decreases by ethanol treatment in these neurons. Given that GlcCer synthase expression protects
against ceramide-induced stress in keratinocytes [156] and elevation of GlcCer is detected in
multidrug-resistant cancer cells [157], GlcCer itself or the ceramide/GlcCer ratio may be important in
apoptotic/survival pathways.
Increases in ceramide, TG, and GM2 are also observed in vivo in the brain 4 to 24 h after P7 mice
are acutely exposed to ethanol (2.5 g/kg, s.c., twice with a 2 h interval as described in [36]) [43,44,49].
Ceramide elevation is specifically prominent in brain regions where strong neurodegeneration
occurs [43]. Such ceramide elevation as well as apoptotic neurodegeneration assessed by cleaved
(activated) caspase-3 immunostaining and Fluoro-Jade staining is attenuated by SPT inhibitors
(myriocin and L-cycloserine) [43]. Concomitantly, SPT immunostaining is enhanced in cleaved
caspase-3 positive neurons, and the SPT activity increases in ethanol-treated forebrain samples [43].
These results suggest the importance of de novo ceramide synthesis in ethanol-induced apoptosis,
which agrees with our studies using cultured neurons [45]. However, the contribution of SMase
activation by ethanol cannot be excluded, because small but significant increases in both neutral and
acid-SMase activity are observed when forebrain slices from P7 mice are treated with ethanol
in situ [158]. The neuronal localization of SPT [43] and our preliminary data indicating strong
ceramide staining in cleaved caspase-3 positive neurons [159] suggest that increased ceramide is
localized mainly in neurons. The inhibition of AMPK and activation of acetyl-CoA carboxylase
(a lipogenic enzyme) found in the P7 brain exposed to ethanol [44] suggest that ethanol-enhanced
lipogenesis may be linked to the ceramide elevation as indicated in other organs [160,161]. However,
it has been also proposed in the liver that the effects of ethanol on AMPK inactivation
(dephosphorylation) may be mediated by the direct activation of protein phosphatase 2A (PP2A) by
ceramide [137] produced by activated acid SMase [162,163]. It is possible that ethanol induces both
transient SMase activation and long-lasting enhancement of de novo ceramide synthesis. Figure 2
illustrates the possible involvement of ceramide in the ethanol-induced apoptotic pathway in the P7
mouse brain. Because apoptotic neurons produced by P7 ethanol exposure are cleared promptly by
activated microglia, which return to the shape of resting microglia within 48 h [75], it is expected that
lipid alterations, including ceramide elevation, are short-lived. However, neonatal ethanol exposure has
been shown to induce persistent neocortical astrogliosis [164,165] and increased cytokine (such as
TNF-α) formation [165] in adolescent rats, suggesting that neonatal ethanol exposure induces
prolonged neuroinflammation. It has been reported that administration of ethanol to pregnant mice on
GD15–16 induces elevation of ceramide and sphingosine in the brain of juvenile progeny mice [166].
Whether the elevation of these lipids in the juvenile brain is associated with cell death and/or
neuroinflammation, or whether P7 ethanol exposure induces long-term ceramide elevation remains to
be explored.
Ceramide has been also linked to ethanol-induced apoptosis in neural crest-derived cells both
in vivo and in vitro [167]. In this case, ceramide appears to increase by enhanced SM hydrolysis or
impaired conversion of ceramide to SM [167]. In cultured astrocytes, ethanol-induced cell death is
associated with ceramide elevation via activation of neutral and acid SMases, along with activation of
JNK, p38, and ERK [107]. Ethanol-induced oxidative stress may activate SMases because changes in
intracellular redox seem to regulate neutral SMases (reviewed by [141]). It is also suggested that
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ethanol induces astroglial apoptosis by disrupting phospholipase D signaling, thereby reducing
phosphatidic acid and increasing ceramide formation [168].
Ceramide, thus elevated by ethanol treatment, may function in mitochondria as suggested in other
ceramide-mediated apoptotic pathways. Our preliminary studies [169] indicate that ceramide increases
in the mitochondrial fraction isolated from the P7 mouse brain exposed to ethanol, but not in the
synaptic plasma membrane or microsomal fractions, where ceramides are mainly localized. A recent
study [170] indicates that the interaction of Bax with proteins associated with MPTP is crucial for the
initiation/progression of apoptotic cascade in the cerebellum of P4 rats exposed to ethanol. As
described in the previous section, ceramide in mitochondria is implicated in the regulation of apoptosis
by affecting functions of MOMP and MPTP in several apoptotic models [145–149,153]. Whether
ceramide elevated in mitochondria during ethanol-induced apoptosis influences the functions of
MOMP and MPTP remains to be explored.
Thus, apoptosis in the P7 brain exposed to ethanol is associated with changes in lipid metabolism
including ceramide elevation. In contrast, ethanol exposure in the P19 mouse brain under the same
condition barely induces lipid changes [44] or apoptosis [35,44]. Causes of this heightened sensitivity
of the early postnatal rodent brain have not been fully elucidated. However, several factors, which may
collectively confer this sensitivity, have been proposed. First, blockade of NMDA receptors induces
neuronal death during an early postnatal period [35,171–174], indicating an important neurotrophic
role of NMDA receptor activation during this period in controlling natural programmed cell death
(reviewed by [175]) as well as apoptosis induced by ethanol, which is an inhibitor of NMDA receptor
functions (reviewed by [176,177]). Secondly, because of the need of the natural programmed cell
death, early postnatal neurons are primed to undergo apoptosis [175] by expressing higher levels of
apoptotic effectors, such as caspase-3, APAF-1, and Bax [178,179]. Studies show that ethanol exposure
in P7 rat brains induces changes in neurotrophic factors, apoptosis-related proteins, antioxidant
enzymes, and ROS, which favor both cell death and survival, while ethanol exposure in P21 brains
elicits changes, which mostly promote cell survival [59]. In addition, basal levels of nerve growth factor
and BDNF are higher in rat brains at P21 than at P7 [59]. It should be also noted that lipid metabolism
characteristic to the neonatal brain may confer the additional sensitivity to ethanol-induced apoptosis
in these brains. For example, higher expression of CerS6 in neonatal brain [15] may be correlated with
the susceptibility of these brains to ethanol-induced apoptosis, because C16 ceramide (a product of
CerS6) accumulated in mitochondria inhibits necrotic cell death and promotes apoptosis [15]. Our
previous studies indicate that S1P, which is generally neuroprotective, increases during brain
development [180]. However, ceramide, TG, lipogenic enzymes (fatty acid synthase and acetyl-CoA
carboxylase), and putative lipid metabolism regulators (SREBP-1 and AMPK) are highly expressed in
neurons at the early postnatal period and decline thereafter [181]. These results suggest that lipogenesis
is more active and ceramide is more readily synthesized in the neonatal brain than in the mature brain.
It has been indicated that ceramide produced by chronic ethanol treatment in adult liver is transferred
across the blood–brain barrier and causes neurodegeneration [182]. However, during the early
postnatal period, ceramide is likely to be synthesized in the brain, although contribution of ceramide
generated in the liver cannot be excluded.
Thus, the involvement of ceramide in ethanol-induced apoptosis is indicated in the neonatal brain.
Likewise, elevated ceramide has been linked to apoptosis of neural cells in neurodegenerative diseases
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and neurological injuries, such as Alzheimer’s disease, HIV-associated dementia, multiple sclerosis,
and ischemia/reperfusion injury [8,11,12,18–20]. Although mechanisms of ceramide elevation are
different among these disorders, ceramide-related activation of mitochondrial cell death pathway as
well as JNK activation has been often observed [8,11,12,18–20]. However, neuronal cell death
processes in these disorders appear to be more complicated than those in ethanol-induced neuronal
death in the neonatal brain, because in many cases apoptosis is not the major cell death mode, and the
impact of neuroinflammation on cell death is more significant in these disorders [8,11,12,18–20].
Ethanol-induced apoptosis in the neonatal brain provides a suitable model to study the mechanisms of
sphingolipid involvement in neurodegeneration. Further studies, such as cellular and subcellular
localization of increased ceramides, identification of molecular species of these ceramides, and the
effects of ethanol on mice and neurons expressing genetically modified enzymes related to ceramide
metabolism may offer better understanding of roles of ceramide in ethanol neurotoxicity. Furthermore,
ceramide metabolites, such as S1P and gangliosides, may also be involved in ethanol-induced
apoptosis, because these lipids are often recognized as apoptosis regulators, and are known to modify
ceramide functions as well (reviewed by [7,21–23,26,183–185]).
4. S1P in Ethanol-Induced Apoptosis in the Developing Brain
It is generally postulated that ceramide induces apoptosis (reviewed by [118]) while S1P promotes
cell survival (reviewed by [7]), and the balance between these bioactive lipids, termed “sphingolipid
rheostat”, determines the cell fate [7]. 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. The pro-survival effects of S1P produced by SphK1 are
usually mediated by the interaction of S1P with five G-protein-coupled cell surface receptors termed
S1P receptor 1–5 (reviewed by [7,186]), and the S1P binding to S1P receptors is associated with
activation of pro-survival ERK [187], PI3K/Akt [187–190], and BclXL [189]. However, the
pro-apoptotic action of S1P has been reported occasionally [191–195]. Particularly, S1P produced by
SphK2 in the nucleus [191] or ER [192,194] is considered pro-apoptotic.
In the nervous system, S1P plays critical roles in neurogenesis, neurite formation,
neuroprotection ([196,197], reviewed by [183]), astrocyte proliferation [198–202], and microglial
activation [203]. Most of the SphK activity in the brain appears to be attributed to that of SphK2,
which is mainly localized in neurons, while SphK1 is localized primarily in astrocytes [180,204]. It is
generally assumed that SphK1 activation exerts a pro-survival influence [205,206], whereas SphK2
activation enhances apoptosis [191]. In fact, S1P produced by SphK2 has been implicated in causing
apoptosis through intracellular targets in CGNs derived from S1P lyase-deficient mice [192]. However,
in some animal models of brain ischemia, SphK2 activation is considered neuroprotective [207–210].
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 [207–209]
suggests that S1P produced by SphK2 may activate S1P receptors, leading to neuroprotection, or S1P
in mitochondria [211] may exert cytoprotection as indicated in a myocardial injury model [212]. In
contrast, S1P produced in other sites, such as nucleus and the ER, may have other targets and enhance
apoptosis [191,192,194].
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Figure 2. Sphingolipid involvement in ethanol-induced apoptosis in the P7 mouse brain.
This figure, which summarizes possible involvement of ceramide, sphingosine-1-phosphate
(S1P), GM2 and GM1 in ethanol-induced apoptosis, is based on previous studies by us and
others. Ceramide generated via enhanced de novo synthesis and SM hydrolysis inactivates
PI3K/Akt pathway and activates GSK3β. Ceramide may also directly affect mitochondrial
membrane permeability. Increases in GM2 and S1P may enhance apoptosis while
exogenous GM1 shows neuroprotection. ROS generation and AMPK inhibition triggered
by ethanol are likely to be linked to the altered sphingolipid metabolism during apoptosis.
The coordinate action of sphingolipids in mitochondria may be crucial for the regulation of
the ethanol-induced, mitochondria-mediated apoptotic pathway.
Our studies have demonstrated that ethanol exposure in P7 mice (5.0 g/kg, once) induces, within
2–4 h, transient activation of SphK2 and a similar transient increase in S1P in the brain. Because an
inhibitor of SphKs, dimethylsphingosine, attenuates ethanol-induced apoptosis, S1P may enhance
apoptosis in this system [180] (Figure 2). However, it has been shown that exogenous addition of S1P
protects rat liver sinusoidal endothelial cells [213] and corneal epithelial cells [214] from
ethanol-induced apoptosis. In 3T3 fibroblasts, ethanol enhances the stimulatory effects of S1P on both
DNA synthesis and cell proliferation [215]. Our preliminary results [216] also indicate that SEW2871,
an agonist for S1P receptor 1, which is a major receptor isoform in the brain [217] and is expressed
mainly in astrocytes [180], attenuates ethanol-induced apoptosis in the P7 brain. S1P produced by
SphK2 activation by ethanol may have a target different from S1P receptor 1, although our studies
suggest that SphK2 is primarily localized in the plasma membrane/synaptic membrane of neurons in
Brain Sci. 2013, 3
681
P7 mice, and not in the nucleus or the ER that are implicated as the sites for apoptotic action of S1P in
the previous studies [191,192,194].
Recent studies have begun to uncover S1P functions in mitochondria. S1P reduces the membrane
depolarization and the elevation in Ca2+ in mitochondria during oxygen-glucose deprivation [196]. S1P
produced by SphK2 regulates complex IV assembly and respiration via interaction with mitochondrial
prohibitin-2 [211], and the mitochondrial S1P is required for the downstream protective modulation of
MPTP [212]. Although it is generally recognized that the balance between ceramide and S1P
determines cell fate [7], mechanisms by which these sphingolipids reciprocally regulate apoptosis are
not fully understood. Considering the roles of S1P and ceramide in mitochondria, elucidation of the
coordinated action of these sphingolipids in mitochondria is important for understanding apoptotic
pathways, including those triggered by ethanol.
A recent study indicates that ethanol induces not only apoptosis but also autophagy in the P7 brain,
and the enhanced autophagy formation is proposed to be the cells’ neuroprotective reaction [218],
while ethanol suppresses autophagy in embryonic cerebral cortical progenitors, which are resistant to
ethanol-induced apoptosis [219]. Both acute and chronic ethanol administration enhance autophagy
formation in the liver [220–224] via ethanol oxidation [220–222,224] and through inactivation of Akt
and the downstream mTOR signaling that controls autophagy formation [220,221]. Specifically,
ethanol appears to enhance autophagy for removing damaged mitochondria and accumulated lipid
droplets [221]. Recent findings indicate that sphingolipids are also involved in these autophagic
processes (reviewed by [144,225,226]). The de novo synthesis of ceramide is reported to be essential
for the induction of autophagy ([227–229], reviewed by [230]). Ceramide may induce autophagy
through inactivation of Akt/mTOR signaling or up-regulation of beclin 1, which is required for the
autophagosome formation (reviewed by [144,225,226]). It has been also reported that ceramide binds
to LC3B-II and anchors LC3B-II-positive autophagolysosomes to mitochondrial membranes to
induce mitophagy [231]. On the other hand, S1P has been reported to counteract amino acid
deprivation-induced autophagy and cell death by suppressing mTOR inactivation through binding to
S1P receptor 3 [232]. However, S1P produced by overexpression of SphK1 in MCF cells stimulates
autophagy and attenuates apoptosis during nutrient starvation by increasing the formation of LC3
positive autophagosomes and the rate of proteolysis [233]. It is suggested that intrinsic regulation of
autophagy by S1P is different from its extrinsic action via S1P receptors [232]. The S1P-induced
autophagy is characterized by the inhibition of mTOR signaling without affecting Akt signaling and by
the lack of robust accumulation of autophagy, and is considered cytoprotective [233]. Because the
crosstalk between apoptosis and autophagy through sphingolipids (specifically ceramide and S1P)
appears to be critical to determine cell fate [144,225,226], future studies of the possible involvement of
sphingolipids in autophagy formation during ethanol-induced apoptosis are important to clarify the
dynamic roles of sphingolipids in this apoptotic pathway.
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682
5. Gangliosides in Ethanol-Induced Neuronal Apoptosis in the Developing Brain
5.1. Pro- and Anti-Apoptotic Effects of Gangliosides in the Brain
Gangliosides (sialic acid-containing glycosphingolipids) are particularly abundant in the nervous
system and exert many biological functions as antigens, mediators of cell adhesion, and modulators of
signal transduction (reviewed by [234–236]). Gangliosides are also known to be involved in apoptotic
pathways. Specifically, GD3 ganglioside is reported to be pro-apoptotic in neurons [237–241], as
indicated earlier in myeloid and lymphoid tumor cells ([242], reviewed by [243,244]). Also, elevated
GD3 expression has been found in brain tissue with various neurodegenerative disorders ([245–247],
reviewed by [248]). Although the majority of gangliosides are found in glycosphingolipid-enriched
microdomains (lipid rafts) in the plasma membrane [235], GD3 accumulates within mitochondria of
cells undergoing apoptosis [249,250], and direct interaction of GD3 with mitochondria induces
cytochrome c release and caspase-3 activation [251]. There is also a report indicating nuclear
localization of GD3 during apoptosis [238,252], which may affect histone H1 modification [252]. In
addition to GD3, the involvement of GM3 in apoptotic death of dividing astrocyte precursors has been
reported [253].
While some gangliosides mediate apoptosis in certain cell types, other gangliosides, specifically
GM1, show anti-apoptotic/neuroprotective effects against cell damage caused by various types of
stress and injury (reviewed by [21–26]). In serum-deprived PC-12 cells, exogenously added GM1
exerts anti-apoptotic effects through the augmented phosphorylation of NGF receptors [254], possibly
by interacting with high-affinity Trk-type receptors for NGF [255]. GM1 increases phosphorylation of
Trks (TrkA > TrkC > TrkB) and Erks in slices of striatum, hippocampus and frontal cortex of rat
brain [256]. Thus, GM1 mimics or potentiates certain actions of neurotrophic factors (reviewed
by [21,23]. It has been also reported that gangliosides activate Trk receptors by increasing the release
of neurotrophins, such as neurotrophin-3 [257] and BDNF [258]. Activation of Trk receptors by GM1
leads to the stimulation of the PI3K/Akt pathway in the brain [259]. Although GM1 is mainly localized
in lipid rafts in the plasma membrane, which contain many signaling molecules [235] including Trk
receptors [260,261], it is also found in the nucleus. GM1 in the nucleus regulates nuclear and cellular
calcium levels through sodium-calcium exchanger in the nuclear envelope and maintains neuronal
viability [22]. Not only exogenously added gangliosides but also endogenous gangliosides increased
by an enhancer of ganglioside biosynthesis confer neuroprotection in cortical neurons [262]. The
neuroprotective function of endogenous GM1 has been also indicated in studies using animals or cells
with totally or partially lacking GM1. Mice lacking B4galnt1 for GM2/GD2 synthase, which depletes
GM2, GD2 and all the gangliotetraose-series gangliosides including GM1, are susceptible to
kainate-induced seizures and neuronal apoptosis, and administration of membrane permeable derivative
of GM1, LIGA20, attenuates the susceptibility of these mice [263]. Also, B4galnt1 knockout mice and
their heterozygotes manifest Parkinson’s disease (PD)-like symptoms along with the loss of
dopaminergic neurons, and such abnormalities are attenuated by administration of LIGA20 [264,265].
Also, decreased levels of GM1 found in cells from Huntington’s disease (HD) patients or the animal
models contribute to HD cell susceptibility to apoptosis, which is restored by GM1 administration
probably through Akt activation [266]. It has also been proposed that changes in the lipid rafts induced
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683
by ganglioside (including GM1) deficiency may cause neurodegeneration [24,236]. These studies
indicate that endogenous GM1 in the lipid rafts and nucleus may contribute to neuronal cell survival.
The neuroprotective effects of GM1 thus observed provide foundation for clinical applications in
neurological disorders, such as AD [267], Parkinson’s disease [268–270], spinal cord injury [271], and
stroke [272].
5.2. Gangliosides in Ethanol-Induced Apoptosis in the Developing Brain
It has been shown that ethanol alters brain ganglioside metabolism. In adult rats, chronic
administration of ethanol reduces GM1 and GD1a in the synaptosomal fraction and GD1a in the
microsomal fraction with decreases in UDP-Gal: GlcCer galactosyltransferase and UDP-Gal: GM2
galactosyltransferase activities [273]. In addition, ratios of the long chain base C20 sphingosine/C18
sphingosine increase in GM1 of synaptosomes and microsomes and in GD1a of myelin [274], which
may reduce membrane fluidity and affect the lipid-protein interactions in the lipid rafts [275]. Also,
GD3 and GD1a decrease by augmented activity of sialidase [276], which appears to be the plasma
membrane sialidase [277], and by reduction of CMP-NeuAc: GM3 alpha2,8-sialyltransferase gene
expression [278]. Prenatal ethanol exposure on GD7 and GD8 and/or GD13 and GD14 increases GM1
and decreases polysialogangliosides in the fetal brain on GD20 [279]. However, chronic ethanol
exposure during the gestation and lactation period increases ganglioside concentration in the offspring
brain analyzed on P21 [280], while ethanol exposure during the gestation period does not significantly
affect synaptic and axolemmal gangliosides in the offspring (P17 to P34) [281].
In cultured CGNs, ethanol increases sialidase activity and changes ganglioside profiles [48], and
increases sphingosine recycling for ganglioside biosynthesis [50]. Importantly, ethanol exposure
decreases the membrane GM1 content in the mouse neural crest cells, and a significant correlation has
been found between the GM1 content and the viability of these cells [282].
In agreement with the general neuroprotective effects of GM1 described in the previous section,
GM1 induces neuroprotection against ethanol toxicity. When added to culture media, GM1 diminishes
ethanol-induced neural crest cell death and the membrane fluidity elevation [283], and provides
protection against ethanol neurotoxicity in rat hippocampal neurons and in chick dorsal root ganglion
neurons [284]. Our laboratory has shown that ethanol-induced apoptosis in rat CGNs is attenuated by
pretreatment with GM1, GD1a, GD1b, GT1b, or LIGA20 [285]. LIGA20 was the most effective,
followed by GD1b and GT1b, while asialo GM1 was ineffective. When administered in vivo, GM1
diminishes the teratogenic/toxic effects of prenatal ethanol exposure (reviewed by [286]). GM1
pretreatment blocks changes in ganglioside profiles, phospholipase A2 activation, and fatty acid ethyl
ester production in the brain induced by fetal ethanol exposure in rodents, and minimizes the alteration
in brain maturation and associated behavioral dysfunction [279,287,288]. Also, pre-administration of
GM1 or LIGA20 attenuates ethanol-induced apoptosis in the P7 mouse brain [289]. These results
indicate that gangliosides, specifically GM1, significantly attenuate brain abnormalities induced by
prenatal or neonatal ethanol exposure, although roles of endogenous GM1 in the process of ethanol
neurotoxicity in the brain have not been elucidated yet.
In contrast, certain gangliosides, such as GD3, may function as an apoptotic inducer during
ethanol-induced apoptosis. We have analyzed the time course of changes in ganglioside profiles in the
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684
brain of mouse pups exposed to ethanol (5.0 g/kg, s.c., one time injection) on P7 and found significant
increases in GM2, but not in other major gangliosides including GD3. The small increase in GM2
observed 2 h after ethanol exposure is followed by a marked increase around 24 h [49]. GM2 may be
associated with apoptosis, because GM2 is accumulated in cleaved caspase-3 positive neurons and
increases in mitochondria in the P7 brain exposed to ethanol [49]. Furthermore, GM2, as well as GD3,
induces cytochrome c release from mitochondria isolated from P7 brains. Interestingly, the addition of
GM1 attenuates GM2-induced cytochrome c release from isolated mitochondria [49] indicating that
the balance between GM1 and GM2 in mitochondria may affect membrane permeability. Thus, as
illustrated in Figure 2, ethanol-induced apoptosis, which is promoted by ceramide/S1P, is attenuated
by GM1 ganglioside. In addition, in vitro experiments suggest that GM2 may be pro-apoptotic.
Although subcellular localization of these spingolipids remains to be clarified, it is tempting to speculate
that the coordinate action of these sphingolipids in mitochondria may regulate ethanol-induced
apoptosis in the developing brain.
6. Conclusion
Ethanol induces acute apoptotic neurodegeneration in the rodent brain during the brain growth spurt
period, corresponding to the third trimester of human fetuses. This apoptosis occurs via the
Bax-dependent mitochondria-mediated pathway involving many factors, including ROS formation,
inactivation of Akt and ERK, and activation of GSK3β. Although published studies are still scant,
available data indicate that not only proteins but also sphingolipids are involved in the ethanol-induced
apoptotic neurodegeneration in the developing brain. Ethanol alters sphingolipid metabolism
profoundly in the developing brain as well as in cultured neurons. Particularly, elevation of de novo
ceramide synthesis and S1P formation appear to mediate or enhance apoptosis. Ethanol-induced
increase in GM2 ganglioside may also promote apoptosis, while exogenously added GM1ganglioside
exerts the anti-apoptotic effect. Studies also indicate that ceramide and GM2 increase in mitochondria
in the brain exposed to ethanol. The coordinated action of sphingolipids in mitochondria may be
crucial for the regulation of this mitochondria-mediated apoptotic pathway. Some of these roles of
sphingolipids, such as the pro-apoptotic action of ceramide and the neuroprotective action of GM1, are
widely observed in neuronal apoptosis triggered by diverse apoptotic inducers. However, some of the
effects of other sphingolipids may be specific to this ethanol-induced apoptosis, because of the unique
and profound effects of ethanol on lipid metabolism in the developing brain. Obviously, further studies
are needed to better understand the sphingolipid involvement in developmental ethanol neurotoxicity.
These include; subcellular localization and trafficking of sphingolipids and related enzymes involved
in ethanol-induced apoptosis, molecular species of ceramide and other sphingolipids altered by ethanol
exposure, the relationship between glial activation and sphingolipids, the long-lasting effects of
developmental ethanol exposure on sphingolipid metabolism, the involvement of sphingolipids in
ethanol-induced autophagy, and the comparison of ethanol-induced apoptosis/autophagy between
wild-type mice and the mice with disrupted genes for sphingolipid metabolism. These studies may help
identify unique targets for therapeutic applications against FASD.
Brain Sci. 2013, 3
685
Acknowledgments
The authors were supported by AA015355 from the NIAAA.
Conflict of Interest
The authors declare no conflict of interest.
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