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Developmentally Regulated Ceramide Synthase 6 Increases
Mitochondrial Ca
2ⴙ
Loading Capacity and Promotes
Apoptosis
*
Received for publication, July 14, 2010, and in revised form, December 1, 2010 Published, JBC Papers in Press, December 10, 2010, DOI 10.1074/jbc.M110.164392
Sergei A. Novgorodov
‡§
, Daria A. Chudakova
¶
, Brian W. Wheeler
¶
, Jacek Bielawski
§
, Mark S. Kindy
¶
,
Lina M. Obeid
‡§储
, and Tatyana I. Gudz
¶储1
From the
储
Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29401 and the Departments of
¶
Neuroscience,
‡
Medicine, and
§
Biochemistry and Molecular Biology, Medical University of South Carolina,
Charleston, South Carolina 29425
Ceramides, which are membrane sphingolipids and key me-
diators of cell-stress responses, are generated by a family of
(dihydro) ceramide synthases (Lass1– 6/CerS1– 6). Here, we
report that brain development features significant increases in
sphingomyelin, sphingosine, and most ceramide species. In
contrast, C
16:0
-ceramide was gradually reduced and CerS6 was
down-regulated in mitochondria, thereby implicating CerS6 as
a primary ceramide synthase generating C
16:0
-ceramide. Inves-
tigations into the role of CerS6 in mitochondria revealed that
ceramide synthase down-regulation is associated with dramat-
ically decreased mitochondrial Ca
2ⴙ
-loading capacity, which
could be rescued by addition of ceramide. Selective CerS6
complexing with the inner membrane component of the mito-
chondrial permeability transition pore was detected by immu-
noprecipitation. This suggests that CerS6-generated ceramide
could prevent mitochondrial permeability transition pore open-
ing, leading to increased Ca
2ⴙ
accumulation in the mitochondrial
matrix. We examined the effect of high CerS6 expression on cell
survival in primary oligodendrocyte (OL) precursor cells, which
undergo apoptotic cell death during early postnatal brain devel-
opment. Exposure of OLs to glutamate resulted in apoptosis that
was prevented by inhibitors of de novo ceramide biosynthesis,
myriocin and fumonisin B1. Knockdown of CerS6 with siRNA
reduced glutamate-triggered OL apoptosis, whereas knockdown
of CerS5 had no effect: the pro-apoptotic role of CerS6 was not
stimulus-specific. Knockdown of CerS6 with siRNA improved cell
survival in response to nerve growth factor-induced OL apopto-
sis. Also, blocking mitochondrial Ca
2ⴙ
uptake or decreasing
Ca
2ⴙ
-dependent protease calpain activity with specific inhibitors
prevented OL apoptosis. Finally, knocking down CerS6 decreased
calpain activation. Thus, our data suggest a novel role for CerS6
in the regulation of both mitochondrial Ca
2ⴙ
homeostasis and
calpain, which appears to be important in OL apoptosis during
brain development.
Sphingolipids are essential structural components of cellu-
lar membranes, playing prominent roles in signal transduc-
tion that governs cell proliferation, differentiation, migration,
and apoptosis (1). Most sphingolipids are ubiquitous, but
complex sphingolipids, including sphingomyelin (SM)
2
and
glycosphingolipids, are more abundant in the brain and in
myelin formed by oligodendrocytes (OLs). The building block
of many complex sphingolipids is ceramide, which has nu-
merous cellular signaling functions (2). Ceramides are a fam-
ily of distinct molecular species characterized by various acyl
chains as well as the desaturation and hydroxylation of those
chains. Highly hydrophobic ceramides are generated by mem-
brane-associated enzymes and exert their effects proximal to
the ceramide generation site, or they require specific trans-
porter proteins to reach their targets in other intracellular
compartments (1, 3).
Ceramides are synthesized de novo at the cytosolic side of
the endoplasmic reticulum (4, 5), serving as precursors for the
biosynthesis of glycosphingolipids and SM in the Golgi (6, 7).
Mitochondria are another important intracellular compart-
ment of sphingolipid metabolism (8), and several sphingolip-
id-metabolizing enzymes were found to be associated with
mitochondria, including neutral ceramidase (9), novel neutral
sphingomyelinase (10), and (dihydro) ceramide synthase (EC
2.3.1.24), a key enzyme in de novo ceramide synthesis (11, 12).
Recently, mitochondrial ceramide engagement in apoptosis
has been shown using loss-of-function mutants of ceramide
synthase in the germ cell line of Caenorhabditis elegans (13).
Specifically, ionizing radiation-induced apoptosis of germ
cells was blocked upon inactivation of ceramide synthase, and
apoptosis was restored upon microinjection of long-chain
ceramide. Radiation-induced increases in ceramide localized
to the mitochondria were required for activation of CED-3
caspase and apoptosis.
Each of the 6 mammalian ceramide synthase (CerS, origi-
nally known as Lass) genes appears to regulate synthesis of a
specific subset of ceramides, and each has a unique substrate
specificity for chain-length and/or saturation of fatty acid
*This work was supported, in whole or in part, by National Institutes of
Health Grant P20 RR 17677 for NCCR COBRE in Lipidomics and Pathobiol-
ogy (to T. I. G.), Grant AG16583 (to L. M. O.), and Veterans Affairs Rehabili-
tation Research and Development Merit Awards (to T. I. G. and M. S. K.).
1
To whom correspondence should be addressed: 114 Doughty St., Charleston,
SC 29425. Tel.: 843-792-6439; Fax: 843-876-5099; E-mail: gudz@musc.edu.
2
The abbreviations used are: SM, sphingomyelin; CI, caspase inhibitor; CerS,
ceramide synthase; CLC, Ca
2⫹
-loading capacity; CSR, control siRNA; CSA,
cyclosporin A; FB1, fumonisin B1; Glc-ceramide, glucosyl-ceramide; Lac-
ceramide, lactosyl-ceramide; MPTP, mitochondrial permeability transi-
tion pore; OL, oligodendrocyte; p75
NTR
, p75 neurotrophin receptor; SPH,
sphingosine; S1P, sphingosine-1-phosphate; TrkA, receptor tyrosine ki-
nase; TMPD, N,N,N⬘,N⬘-tetramethyl-p-phenelenediamine; ANT, adenine
nucleotide translocator.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 6, pp. 4644–4658, February 11, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
4644 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 6•FEBRUARY 11, 2011
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acyl-CoA. Overexpression of any CerS protein in mammalian
cells resulted in increases in a specific subset of ceramide spe-
cies. CerS1 has high specificity for C
18:0
-CoA generating C
18:0
-
ceramide (14, 15). CerS2, CerS4, and CerS3 appear to have
broader specificity (16, 17). CerS2 or CerS4 mainly synthe-
sizes C
20:0
-, C
22:0
-, C
24:1
-, C
24:0
-, C
26:1
-, and C
26:0
-ceramide,
but is unable to synthesize C
16:0
-orC
18:0
-ceramide (14, 17).
CerS3 generates C
18:0
-, C
20:0
-, C
22:0
-, and C
24:0
-ceramide (16).
It has been shown that CerS5 generates C
14:0
-, C
16:0
-, C
18:0
-, and
C
18:1
-ceramide (14, 18); and CerS6 produces C
14:0
-, C
16:0
-, and
C
18:0
-ceramide (14).
Our studies described here were designed to ascertain the
functional role of ceramide and CerS6 in mitochondria during
postnatal animal brain development. Herein, we report that,
contrary to most ceramide species, C
16:0
-ceramide was down-
regulated, as was CerS6 expression, in mitochondria. The data
imply that CerS6 could be a primary ceramide synthase, gen-
erating C
16:0
-ceramide in brain mitochondria. Functional
analysis revealed a significant decrease in Ca
2⫹
-loading ca-
pacity in mitochondria from the adult rat brain compared
with the postnatal day 10 (P10) brain, and this decrease oc-
curred with lower CerS6 expression and decreased C
16:0
-cer-
amide. Exogenously added C
16:0
-ceramide completely restored
the Ca
2⫹
-loading capacity of adult mitochondria to that of the
young rat brain. Co-immunoprecipitation studies exposed
selective CerS6 association with adenine nucleotide transloca-
tor (ANT), the mitochondrial permeability transition pore
(MPTP) component in the inner mitochondrial membrane.
This suggests that CerS6 could generate C
16:0
-ceramide in
close proximity of MPTP and prevent pore opening that re-
sults in an increased mitochondrial Ca
2⫹
-buffering capacity.
Gene knockdown experiments revealed a critical role for
CerS6 in promoting OL apoptosis. Thus, knocking down
CerS6 enhanced OL survival in response to glutamate- or
nerve growth factor-induced apoptosis. Investigation of
downstream targets of the CerS6-mediated signaling pathway
revealed an important contribution of mitochondrial Ca
2⫹
and calpain in promoting ceramide-dependent apoptosis in
OLs. Specifically, OL exposure to inhibitors of mitochondrial
Ca
2⫹
uptake or calpain activity enhanced cell survival in re-
sponse to glutamate and NGF. Knocking down CerS6 reduced
calpain activation. These studies identify CerS6 as an impor-
tant regulator of mitochondrial Ca
2⫹
homeostasis and
suggest a pro-apoptotic role in OLs during postnatal brain
development.
EXPERIMENTAL PROCEDURES
Animals and Reagents—Female timed-pregnant Sprague-
Dawley rats (Charles River Laboratories, Wilmington, MA)
were acclimated for 1 week prior to experimentation. Experi-
mental protocols were reviewed and approved by the Institu-
tional Animal Care and Use Committee of Medical University
of South Carolina (MUSC), Charleston SC, and followed the
National Institutes of Health guidelines for experimental ani-
mal use. Cell culture was Dulbecco’s modified Eagle’s medium
(DMEM), fetal bovine serum (FBS), and N2 supplement (from
Invitrogen). Complete Mini Protease Inhibitor Mixture was
from Roche Applied Science. A new generation pan-caspase
inhibitor, Q-VD-OPH, was from BioVision (Mountain View,
CA). Calpain inhibitor PD150606 was supplied by Santa Cruz
Biotechnology (Santa Cruz, CA). Calpeptin was from EMD
Chemicals (Gibbstown, NJ). Nerve growth factor (NGF) was
purchased from Neuromics (Edina, MN). All other chemicals
were purchased from Sigma.
Antibodies—The following antibodies were used: mouse
anti-LASS2/CerS2 (clone 1A6), mouse monoclonal anti-
LASS4/CerS4 (clone7D1), rabbit polyclonal anti-LASS5/
CerS5 (PAB13440), and mouse monoclonal anti-LASS6/
CerS6 (clone 5H7). These antibodies were obtained from
Abnova (Taipei, Taiwan). The rabbit polyclonal anti-LASS1/
CerS1 antibody was from Sigma Genosys (Woodlands, TX).
The specificity of each anti-CerS antibody was verified in
knockdown experiments using specific siRNA targeting CerS
in OLs. Anti-

-actin mouse monoclonal (A1978) and rabbit
polyclonal anti-p75
NTR
antibodies were purchased from
Sigma. Rabbit polyclonal antibodies against voltage-depen-
dent anion channel were supplied by EMD Chemicals (Gibbs-
town, NJ). Goat polyclonal anti-myelin basic protein (myelin
marker) antibody, rabbit polyclonal anti-cyclophilin D, anti-
Tom20, and anti-ANT antibodies were obtained from Santa
Cruz (Santa Cruz, CA). The rabbit polyclonal anti-LAMP-2
(lysosomal marker), mouse monoclonal anti-
␣
1 subunit of the
sodium/potassium ATPase (plasma membrane marker), and
the rabbit polyclonal anti-calnexin (ER marker) antibody were
purchased from Abcam (Cambridge, MA). Rat monoclonal
anti-myelin proteolipid protein antibody was generously pro-
vided by Dr. Wendy Macklin (University of Colorado, Denver,
CO). Secondary horseradish peroxidase-conjugated antibod-
ies were supplied by Jackson ImmunoResearch Laboratories
Inc.
Isolation of Rat Brain Mitochondria—All procedures were
performed at 4 °C as described (12). Briefly, tissue was placed
immediately in ice-cold isolation medium containing 230 mM
mannitol, 70 mMsucrose, 10 mMHEPES, and 1 mMEDTA,
pH 7.4. Brain tissue (⬃1 g) was homogenized in 10 ml of iso-
lation medium using a Teflon-glass homogenizer. The homo-
genate was centrifuged at 900 ⫻gfor 10 min. The superna-
tant was then centrifuged at 12,000 ⫻gfor 10 min. The pellet
was re-suspended in the isolation medium and centrifuged
again at 12,000 ⫻gfor 10 min. The pellet was re-suspended
in 2 ml of 15% Percoll-Plus (GE Healthcare) and placed atop
the discontinuous Percoll gradient consisting of a bottom
layer of 4 ml of 40% Percoll and a top layer of 4 ml of 23% Per-
coll. The gradient was spun at 31,000 ⫻gfor 15 min in a SW-
Ti40 rotor in a Beckman LE80K centrifuge. The fraction at
the 23–40% interface, which contained mitochondria, was
washed 3 times with isolation medium by centrifugation at
12,000 ⫻gfor 10 min. Protein concentration was measured
with a bicinchoninic acid assay (Sigma) using bovine serum
albumin as a standard. Typically, the contamination of mito-
chondria with ER was ⬍1% by activity measurements of ER-
specific marker enzyme, NADPH-cytochrome creductase
(12).
Mitochondrial Respiratory Chain Activity—Mitochondrial
respiration was measured by recording oxygen consumption
at 25 °C in a chamber equipped with a Clark-type oxygen elec-
CerS6 Promotes Apoptosis
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trode (Instech Laboratories, Plymouth Meeting, PA) as previ-
ously described (12). Briefly, mitochondria were incubated in
the medium containing 125 mMKCl, 10 mMHEPES, 2 mM
KH
2
PO
4
,5mMMgCl
2
, and 0.5 mg/ml of mitochondrial pro-
tein supplemented with either Complex I substrate (mixture
of5m
Mglutamate and 5 mMmalate) or Complex II substrate
(5 mMsuccinate) in the presence of 1
Mrotenone or Com-
plex IV substrate (1 mMascorbate in the presence of 250
M
TMPD and 1
Mantimycin). A respiratory control ratio was
measured as the oxygen consumption rate in the presence of
the substrate and 100
MADP (State 3) divided by the rate in
the resting state (State 4) in the presence of 2
g/ml of oligo-
mycin. Uncoupler-stimulated (State 3u) respiration was
measured in the presence of 50 nMcarbonyl cyanide
p-trifluoromethoxyphenylhydrazone.
Measurement of Mitochondrial Ca
2⫹
Load—The Ca
2⫹
-
loading capacity (CLC) of mitochondria was monitored using
aCa
2⫹
-selective electrode (Thermo Scientific/Orion, Rock-
ford, IL) in a medium containing 250 mMsucrose, 10 mM
HEPES, and 2 mMKH
2
PO
4
, pH 7.4 (adjusted with Tris base).
Mitochondria were energized by 10 mMsuccinate with 1
M
rotenone and pulsed with 100
MCa
2⫹
every 1.5 min. The
increasing Ca
2⫹
load caused a decline in Ca
2⫹
uptake rates.
Maximal CLC was defined as an amount of Ca
2⫹
(per mg of
protein) required to decrease the Ca
2⫹
uptake rate by ⬎90%.
Simultaneously with CLC measurements, mitochondrial
swelling was monitored using a Brinkman probe colorimeter
as described (19).
Western Blot—Proteins were analyzed by Western blot as
previously described (12, 20). Cells or tissue samples were
lysed in a buffer containing 50 mMTris-HCl, 5 mMEDTA,
150 mMNaCl, 1% Triton X-100, pH 7.4, 1 mMNa
3
VO
4
, and
10 mMNaF, supplemented with a protease inhibitor mixture.
After1honice, cell lysates were centrifuged at 15,000 ⫻gfor
10 min to remove insoluble material. Protein samples were
prepared by boiling lysates in reducing SDS-sample buffer.
Proteins were separated by 8–10% SDS-PAGE, blotted to
PVDF membrane, blocked with 5% nonfat dry milk in TBS-T
buffer (10 mMTris, 150 mMNaCl, and 0.2% Tween 20, pH
8.0) overnight at 4 °C, and subsequently probed with the ap-
propriate primary antibody. Immunoreactive bands were vi-
sualized using a chemiluminescence SuperSignal West Femto
substrate (Thermo Scientific).
Cell Culture—Dissociated rat neonatal cortices were cul-
tured on poly-L-lysine-coated flasks as described (20). Briefly,
the cerebra of rat pups were dissected and minced to generate
a single-cell suspension. Cells were plated into 75-cm
2
flasks
and grown in DMEM with 10% FBS at 37 °C and 5% CO
2
.By
day 10, mixed glial cultures were obtained, consisting of OLs
and microglia growing on an astrocyte monolayer. OLs were
purified from mixed glial cell cultures using a shake-off proce-
dure. Cells were shaken initially for1hat100⫻gto remove
microglia, re-fed, and shaken again for 22–24 h at 37 °C at
200 ⫻g. OLs were collected by centrifugation at 1,200 ⫻gfor
4 min. OLs were used immediately for transfections or further
culturing. Cell culture plates and cell culture dishes were pre-
coated with the 10
g/ml of fibronectin solutions overnight at
37 °C. All cultures contain less than 2% of GFAP
⫹
astrocytes
and non-detectable CD11
⫹
microglia.
siRNA transfection—To down-regulate Lass6/CerS6,
siGenome SMARTpool silencing RNAs were obtained from
Thermo Scientific/Dharmacon (Rockford, IL). The set con-
sists of 4 siRNAs targeting different regions of the gene to
minimize the off-target effects. In addition, silencing RNA
targeting Lass5/CerS5 or Lass6/CerS6 were purchased from
the Qiagen High Performance GenomeWide siRNA bank
(Qiagen, Valencia, CA). The following target sequences were
used: Lass6/CerS6, 5⬘-GAACUGGCGUCCUGACUAG-3⬘,
5⬘-GAACACCGGACUUAACUAU-3⬘,5⬘-GGACAGAG-
GUGCAAGACGC-3⬘,5⬘-CGACACAGGAGUGGACAAA-3⬘;
Lass5/CerS5, 5⬘-TTCGAGCGATTTATTGCTAAA-3⬘; Lass6/
CerS6, 5⬘-GGACAGAGGUGCAAGACGC-3⬘. OLs were
transfected with siRNA using the Nucleofector electropora-
tion system (Amaxa Biosystems, Gaithersburg, MD) accord-
ing to the manufacturer’s instructions with efficiencies of
⬎70% as described (20). Cells (6 ⫻10
6
) were mixed with 100
l of Nucleofector reagent and 0.5
l of siRNA in the cuvette
of the Amaxa electroporation device. AllStars negative con-
trol siRNA (Qiagen, Valencia, CA) was used as a control.
Cell Survival Assay—Cell death was measured using a lac-
tate dehydrogenase-based CytoTox-ONE
TM
Homogeneous
Membrane Integrity Assay (Promega, Madison, WI), accord-
ing to the manufacturer’s recommendations. Cell survival was
expressed as percent of viable cells based on the measure-
ments of lactate dehydrogenase activity associated with the
cells versus the lactate dehydrogenase activity in the medium.
The fluorescence of the sample was measured at 590 nm
emission with 560 nm excitation in a microplate reader
(FLUOstar Optima, BMG LABTECH Inc., Durham, NC).
Caspase Activity Assay—The activities of executioner
caspases 3/7 were determined using Apo-One威Homoge-
neous kit (Promega) according to the manufacturer’s
instructions. Cleavage of non-fluorescent substrate,
Z-DEVD-Rodamine-110 by caspase 3/7 resulted in fluores-
cent rodamine-110. The fluorescence of the sample was
measured at 530 nm emission and 490 nm excitation in the
microplate reader FLUOstar Optima.
Calpain Activity Assay—Calpain activity was measured us-
ing the SensoLyte520 fluorimetric calpain activity assay kit
(AnaSpec, Freemont, CA) according to the manufacturer’s
instructions. The assay employs a novel internally quenched
5-FAM/OXL
TM
520 FRET substrate to increase the sensitivity
of the measurements. Calpain cleaved the FRET substrate
yielding the release of fluorescent 5-FAM, which was moni-
tored at 520 nm emission and 490 excitation in the microplate
reader FLUOstar Optima.
Immunoprecipitation—For immunoprecipitation, cell ly-
sates (500
g) were precleared in buffer A (0.15 MNaCl, 0.5
mMEDTA, 1% Triton X-100, 10 mMNaF, and 1 mMNa
3
VO
4
,
protease inhibitor mixture, 0.05 MTris, pH 7.5, 0.2% BSA) by
incubation with appropriate species-specific, IgG-conjugated
magnetic beads (Dynabeds, Invitrogen/Dynal, Carlsbad, CA)
for 1 h. Antibodies then were added. After incubation at 4 °C
overnight with gentle mixing, antibody-antigen complexes
were captured with Dynabeads and washed two times with
CerS6 Promotes Apoptosis
4646 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 6•FEBRUARY 11, 2011
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buffer A (without BSA), and then washed twice with Tris-
buffered saline, pH 7.5. The immunoprecipitates were eluted
by boiling in SDS-sample buffer. As a control, the same im-
munoprecipitation procedure was performed except for the
primary antibody application.
Analysis of Sphingolipids by Tandem Mass Spectrometry—
Cells or tissues were lysed in a buffer containing 10 mMTris
and 1% Triton X-100, pH 7.4, for analysis by reverse-phase
high pressure liquid chromatography coupled to electrospray
ionization followed by separation by MS. Sphingolipid analy-
sis was performed in the Lipidomics Core Facility at MUSC
using a Thermo Finnigan TSQ 7000 triple quadrupole mass
spectrometer, operating in a multiple reaction monitoring
positive-ionization mode (20, 21). The peaks for the target
analytes and internal standards were collected and processed
with the Xcalibur software system. Calibration curves were
constructed by plotting peak area ratios of synthetic stan-
dards, representing each target analyte, to the corresponding
internal standard. The target analyte peak area ratios from the
samples were similarly normalized to their respective internal
standard and compared with the calibration curves using a
linear regression model. Each sample was normalized to its
respective total protein levels.
Statistical Analysis—All experiments and assays were per-
formed three or more times. Typically, there were at least six
replicates of each treatment in each assay. Data were col-
lected, and the mean value of the treatment groups and the
standard error were calculated. Data were analyzed for statis-
tically significant differences between groups by one-way
analysis of variance with a post hoc Tukey test, which adjusts
for multiple simultaneous comparisons (SAS version 9.1.3).
Statistical significance was ascribed to the data when p⬍
0.05.
RESULTS
C
16:0
-ceramide Is Down-regulated in Mitochondria during
Postnatal Brain Development—Sphingolipids, including
sphingomyelin (SM), sphingosine (SPH), sphingosine
1-phosphate (S1P), and ceramide, were measured in brains
of rats at different postnatal ages: postnatal day 1 (P1)
through P21 and 6-month-old (adult) rats (Fig. 1A). The
data are expressed as fold-increases of P1 brain sphingo-
lipid content (Table 1). Postnatal brain growth was accom-
panied by profound increases in all sphingolipids (Fig. 1A),
which is consistent with the structural role of sphingolipids
in cell membranes.
Reports suggest that some sphingolipids, including cer-
amide, exert pleotropic effects on cell signaling pathways and
can also mediate a variety of cell responses (22). It has been
emphasized that the function of the sphingolipid is deter-
mined by its local concentration, which could vary even be-
tween the two leaflets of the lipid bilayer in the membrane or
FIGURE 1. Sphingolipid changes in brain tissue or brain mitochondria during rat development. Sphingolipids were analyzed in total brain tissue lysate
(A) or isolated brain mitochondria (B) from rats at various ages. Ceramide, SPH, S1P, or SM content was gradually increased in developing rat brain tissue or
mitochondria. The data are expressed as fold-increase in mitochondria content for postnatal day 1 (P1) brains or postnatal day 10 (P10) brains. Data are
mean ⫾S.E., *, p⬍0.05, n⫽16. Each sample was normalized to its respective total protein levels. C, lack of mitochondrial contamination with various cellu-
lar membranes was characterized by Western blot using specific antibodies: anti-
␣
1 subunit Na
⫹
/K
⫹
-ATPase (plasma membrane marker), anti-calnexin (ER
marker), anti-LAMP-2 (lysosomal marker), anti-VDAC (mitochondrial marker), and anti-myelin basic protein (MBP, myelin marker). An equal amount of brain
(Brain) or brain mitochondria (Mito) lysate (10
g) was loaded into the lane.
TABLE 1
Sphingolipid content of rat brain tissue and brain mitochondria
Total ceramide, SPH, S1P, or SM content (pmol/mg of protein) was determined
in the P1 rat brain or mitochondria purified from P10 rat brain. Each sample was
normalized to its respective total protein. Values are mean ⫾S.E., n⫽16.
Sphingolipid Ceramide SPH S1P SM
Brain (P1) 915.4 ⫾24.3 15.9 ⫾0.6 1.3 ⫾0.4 2,313.6 ⫾98.2
Mitochondria (P10) 979.3 ⫾26.8 16.4 ⫾0.8 2.1 ⫾0.5 4,281.4 ⫾121.7
CerS6 Promotes Apoptosis
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between organelles (1, 23). Recently, mitochondria have
emerged as an important intracellular compartment of sphin-
golipid biosynthesis and bioactive sphingolipid-mediated sig-
naling pathways (8). To gain more insight into sphingolipid
function, the content of isolated brain mitochondria was ana-
lyzed (Fig. 1B). The data are presented as fold-increases of
P10 brain mitochondria sphingolipid content (Table 1). Sub-
stantial developmental increases in SM, SPH, or S1P oc-
curred, but there were smaller changes in ceramide content
compared with brain tissue samples. To rule out possible con-
tamination of the mitochondrial preparation with other cellu-
lar membranes as a source of sphingolipids, Western blot was
performed with specific antibodies against calnexin (ER
marker), LAMP-2 (lysosomal marker), Na
⫹
/K
⫹
ATPase
(plasma membrane marker), and myelin basic protein (myelin
marker) (Fig. 1C).
The ceramide species profile of the developing rat brain
revealed increased C
18:0
-, C
18:1
-, C
20:0
-, C
20:1
-, C
22:0
-, C
22:1
-,
C
24:0
-, and C
24:1
-ceramide (Fig. 2A). In mitochondria, there
were substantial increases in very long-chain ceramide spe-
cies, including C
20:0
-, C
22:0
-, C
22:1
-, C
24:0
- and C
24:1
-ceramide.
Although C
18:0
-ceramide or C
20:0
-ceramide appear only to be
moderately increased, there were large mass changes in these
ceramides because of their abundance, whereas C
18:1
- and
C
20:1
-ceramide did not change (Fig. 2B). Data are presented as
fold-increases of P1 brain tissue or P10 brain mitochondria
sphingolipid content (Table 2). Intriguingly, C
16:0
-ceramide
was decreased by 70% in the adult rat brain compared with
the P1 brain (Fig. 3A).
Ceramide serves as a building block for complex sphingo-
lipids, including SM and glycosphingolipids. Two classes of
glycosphingolipids carry galactose or glucose as a first sugar
on the ceramide backbone. Glucosyl-ceramide (Glc-ceramide)
can be further glycosylated to lactosyl-ceramide (Lac-cer-
amide), a precursor for gangliosides, which constitute 10 –12%
of total cell membrane lipids in the animal brain (24). Fig. 3B
illustrates the developmental changes in C
16:0
-ceramide-con-
taining complex sphingolipids in the brain. Glycosphingolipid
content (Glc-ceramide and Lac-ceramide) was increased, but
C
16:0
-SM did not change. Further investigation revealed pro-
found down-regulation of C
16:0
-ceramide in mitochondria,
whereas the content of C
16:0
-SM or C
16:0
-Glc-ceramide was
unchanged during brain development (Fig. 3C). The data sug-
gest that C
16:0
-ceramide could play a specific functional role
in mitochondria.
CerS6 Is Primarily Involved in Producing C
16:0
-ceramide in
Mitochondria—To define the role of ceramide in mitochondria,
we focused on enzymes involved in ceramide biosynthesis. We
measured protein expression of various (dihydro) ceramide syn-
thase (CerS) isoforms in rat brains at different developmental
FIGURE 2. Ceramide species changes in brain tissue or brain mitochondria during rat development. Ceramide species were analyzed in total brain ly-
sate (A) or isolated brain mitochondria (B) from rats at various ages. The data are expressed as fold-increase of ceramide species content of P1 brain tissue or
mitochondria from P10 brain. Data are mean ⫾S.E., *, p⬍0.05, n⫽16. Each sample was normalized to its respective total protein levels.
TABLE 2
Ceramide species content in brain tissue and brain mitochondria
Ceramide species content (pmol/mg of protein) was determined in P1 rat brain or mitochondria purified from P10 rat brain. Each sample was normalized to its
respective total protein levels. Values are mean ⫾S.E., n⫽16.
Ceramide C18:0 C18:1 C20:0 C20:1 C22:0 C22:1 C24:0 C24:1
Brain (P1) 632.9 ⫾13.2 501.4 ⫾10.9 138.1 ⫾7.1 36.9 ⫾2.2 25.1 ⫾2.5 11.8 ⫾1.1 118.8 ⫾7.5 140.5 ⫾6.9
Mitochondria (P10) 328.0 ⫾9.6 241.7 ⫾7.3 135.3 ⫾5.4 23.9 ⫾1.9 3.8 ⫾0.5 2.1 ⫾0.6 15.4 ⫾0.8 16.5 ⫾0.9
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stages (Fig. 4). In agreement with our finding of increases in
C
18:0
-ceramide content (Fig. 2, Aand B), CerS1 protein expres-
sion was gradually increased in brain and mitochondria (Fig. 4, A
and C, respectively). These data are consistent with previous
findings that CerS1 selectively utilizes stearoyl-CoA as acyl do-
nor (14, 15) to generate C
18:0
-ceramide.
The protein expression of CerS2 (Fig. 4A), a ceramide syn-
thase that utilizes very long-chain acyl-CoAs, was up-regu-
FIGURE 3. C
16:0
-containing sphingolipid changes during brain development. A,C
16:0
-ceramide content was down-regulated during brain development.
The data are expressed as fold-decrease of C
16:0
-ceramide content in P1 brain tissue (203.9 ⫾12.5 pmol/mg of protein). B, developmental changes in brain
content of complex sphingolipids containing C
16:0
moeity. The data are expressed as fold-increase of sphingolipid content of P1 brain tissue that contains:
C
16:0
-sphingomyelin (SM), 779.1 ⫾18.9 pmol/mg of protein; C
16:0
-glucosylceramide (Glc-ceramide), 22.9 ⫾1.1 pmol/mg of protein; C
16:0
-lactosylceramide
(Lac-ceramide), 19.3 ⫾0.7 pmol/mg of protein. C.C
16:0
-ceramide content was down-regulated in mitochondria, whereas C
16:0
-sphingomyelin or C
16:0
-glu-
cosyl-ceramide was not changed. The content of sphingolipids in mitochondria from P10 brain: C
16:0
-ceramide, 24.5 ⫾0.9 pmol/mg of protein; C
16:0
-sphin-
gomyelin, 646.4 ⫾17.3 pmol/mg of protein; C
16:0
-glucosyl-ceramide, 20.1 ⫾0.8 pmol/mg of protein. Data are mean ⫾S.E., compared with appropriate
control. *, p⬍0.05, n⫽12. Each sample was normalized to its respective total protein levels.
FIGURE 4. Ceramide synthases are differentially expressed during brain development. Ceramide synthase protein expression was analyzed in brain
tissue (A) or in mitochondria (C) by Western blot using specific antibodies. Membranes were stripped and probed with anti-

-actin antibody (brain) or anti-
voltage-dependent anion channel (VDAC) antibody (mitochondria) to confirm equal loading of samples. Band D, quantification of the ceramide synthases
expression in brain (B) or mitochondria (D). Data are mean ⫾S.E. (from 4 independent experiments).
CerS6 Promotes Apoptosis
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lated and this reflected increased levels of very long-chain
ceramides, C
20:0
-, C
20:1
-, C
22:0
-, C
22:1
-, C
24:0
-, and C
24:1
-cer-
amide, during brain development (Fig. 2A). Whereas protein
expression of CerS4, a ceramide synthase with a similar acyl-
CoA specificity, did not differ significantly with developmen-
tal changes (Fig. 4A), we noted differential expression of
CerS2 and CerS4 in mitochondria during brain development
(Fig. 4B). Specifically, CerS2 protein expression was signifi-
cantly down-regulated as CerS4 was up-regulated, suggesting
that CerS4 regulates very long-chain ceramide levels in brain
mitochondria. The data suggest a lack of uniform develop-
mental changes in CerS2 and CerS4 expression in mitochon-
dria compared with cellular levels of these enzymes in the
brain. This finding lends support to the idea that mitochon-
dria represent a separate and unique intracellular compart-
ment involved in sphingolipid metabolism (12, 13, 25).
CerS5 and CerS6 have a similar substrate specificity: both
ceramide synthases can utilize palmitoyl-CoA to produce
C
16:0
-ceramide (14). Fig. 4Aillustrates differential protein
expression of CerS5 and CesR6 in the developing brain. Spe-
cifically, CerS5 expression was gradually up-regulated during
postnatal brain development, whereas CerS6 expression was
significantly down-regulated in the adult rat brain. Consistent
with previous findings (12), CerS5 was not localized to mito-
chondria, whereas CerS6 expression was down-regulated in
the organelle (Fig. 4B). Decreases in CerS6 expression were
associated with reduced C
16:0
-ceramide in mitochondria dur-
ing brain development (Fig. 3C). Altogether, the data suggest
that CerS6 is a primary ceramide synthase that produces
C
16:0
-ceramide in brain mitochondria.
Ceramide Is Involved in Regulation of Mitochondrial Ca
2⫹
Homeostasis—To investigate whether differential CerS6 ex-
pression affected mitochondrial function, respiratory chain
activity was measured. Mitochondrial oxygen consumption
supported by respiratory chain substrates of Complex I, gluta-
mate and malate, or substrates of Complex II, succinate, or of
Complex IV, ascorbate and TMPD, were measured with/
without ADP. Respiration rates in the presence of any tested
substrate alone (state 2) were similar among mitochondria
isolated from P10, P21, and adult (6-month-old) rat brains.
There were no significant differences in state 3 (in the pres-
ence of ADP) respiration rates among brain mitochondria at
various developmental ages, according to measurements of
respiration supported by glutamate and malate (109.3 ⫾6.2
nAO/min/mg of protein), succinate (128.1 ⫾6.9 nAO/
min/mg of protein), or ascorbate and TMPD (150.6 ⫾7.5
nAO/min/mg of protein). Respiratory control ratios were
6.72 ⫾0.31 for mitochondria isolated from P10 or P21 or
adult rat brains. These data indicate a lack of change in oxida-
tive phosphorylation parameters despite differential CerS6
expression in brain mitochondria.
In addition to generating ATP, mitochondria also maintain
low cytosolic Ca
2⫹
levels (26) by sequestering Ca
2⫹
inside the
mitochondrial matrix complexed with phosphate (27, 28).
Energized mitochondria take up Ca
2⫹
via the mitochondrial
calcium uniporter, which has been recently described as a
highly selective, inwardly rectifying channel (29, 30). The mi-
tochondrial calcium uniporter is activated by Ca
2⫹
concentra-
tions greater than 200 nM, and an estimated 10– 40 mito-
chondrial calcium uniporter channels per
m
2
are thought to
be localized in the inner mitochondrial membrane (29).
Excessive accumulation of Ca
2⫹
in the mitochondrial ma-
trix could trigger opening of MPTP at a high conductance
state, which would be accompanied by dissipation of the
transmembrane potential and mitochondrial swelling. In
brain mitochondria, Ca
2⫹
may also activate a limited perme-
ability state of MPTP opening (31) that only depolarizes mito-
chondria without causing swelling (32). This depolarization
dramatically reduces the driving force for Ca
2⫹
influx via mi-
tochondrial calcium uniporter, thus limiting the mitochon-
drial ability to sequester Ca
2⫹
(33).
To determine the effect of differential CerS6 expression on
the ability of mitochondria to regulate Ca
2⫹
, the CLC was
measured in mitochondria from brains of rats at different
postnatal developmental ages (P10, P21, or 6 months old)
(Fig. 5). Pulses of 100
MCa
2⫹
were added to mitochondria
energized by the substrate of Complex II, succinate, whereas
electron transport through Complex I was inhibited by 1
M
rotenone. In line with previous studies (33, 34), sequential
Ca
2⫹
additions caused gradual decreases in the Ca
2⫹
uptake
rates until virtually complete inhibition of Ca
2⫹
uptake was
achieved (Fig. 5A). Notably, mitochondria retained all accu-
mulated Ca
2⫹
and did not swell, which is consistent with the
MPTP opening at a low conductance state (31, 33). The addi-
tion of the pore-forming peptide alamethicin permitted de-
tection of mitochondrial swelling under these conditions.
Quantification of CLC revealed a profoundly reduced ability
of adult rat brain mitochondria to retain Ca
2⫹
(Fig. 5B), com-
pared with P10 or P21 brains.
To investigate whether decreased CLC is dependent on
lower CerS6 expression and C
16:0
-ceramide in mitochondria
from adult brains, mitochondria were supplemented with 1
nmol of C
16:0
-ceramide/mg of protein. Fig. 5Cshows that
C
16:0
-ceramide restored the CLC of adult brain mitochondria
to that of mitochondria from young rat brains. The data sug-
gest an involvement of CerS6 and C
16:0
-ceramide in the regu-
lation of mitochondrial Ca
2⫹
-buffering capacity.
Next, we explored the underlying mechanism of C
16:0
-cer-
amide-mediated increases in CLC of mitochondria from adult
brains. Physiologically relevant concentrations of C
16:0
-cer-
amide have been shown to prevent mitochondrial swelling in
response to high Ca
2⫹
load (35), which is indicative of the
MPTP opening at a high conductance state. However, the
MPTP ceramide-binding site had low specificity toward long-
chain and very long-chain ceramides (35). Consistent with
these findings, addition of C
18:0
-ceramide (Fig. 5C), C
22:0
-
ceramide (Fig. 5C), or C
24:0
-ceramide (not shown) restored
the CLC of adult brain mitochondria to that of mitochondria
from young rat brains. In contrast, sphingosine had no effect.
To examine whether ceramide-dependent modulation of
MPTP opening is responsible for increased mitochondrial
CLC; another inhibitor of MPTP, cyclosporin A (CSA), was
tested (Fig. 5C). CSA is a potent inhibitor of MPTP in heart or
liver mitochondria, but it had a limited ability to prevent the
MPTP opening at a high conductance state in brain mito-
chondria. It was more effective with ADP addition (32). CSA
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was moderately effective in enhancing the CLC of adult brain
mitochondria, but CSA with ADP restored CLC to that of
mitochondria from young rat brains (Fig. 5C). This supports
the concept that Ca
2⫹
could induce MPTP opening at a low
conductance state that results in no swelling and dissipation
of transmembrane potential, the driving force for Ca
2⫹
up-
take (31, 33). Therefore, inhibitors of MPTP opening at a high
conductance state can block MPTP at a low conductance
state resulting in increased mitochondrial CLC.
These data suggest an involvement of CerS6 and C
16:0
-cer-
amide in the regulation of Ca
2⫹
homeostasis in brain mito-
chondria. Thus, lower CerS6 expression and C
16:0
-ceramide
content were associated with reduced mitochondrial CLC in
adult brain mitochondria, whereas exogenous C
16:0
-ceramide
restored CLC to that of young brain mitochondria. Despite
their ability to increase CLC in adult mitochondria, C
18:0
-,
C
22:0
-, and C
24:0
-ceramide seem unlikely candidates for regu-
lating CLC, because there were inverse correlations between
their levels and CLC in mitochondria during brain develop-
ment. In contrast to C
16:0
-ceramide, these three ceramide
species were more abundant in mitochondria from adult
brains with lower CLC compared with mitochondria from
young animal brains (characterized by reduced content of
these ceramides and higher CLC).
Highly hydrophobic ceramide appears to be segregated
within the membrane with its generating enzyme and require
specific transporter proteins to reach other membrane com-
partments (1). This suggests that CerS6 may produce C
16:0
-
ceramide proximal to the MPTP ceramide-binding site in the
inner membrane, whereas CerSs generating C
18:0
-, C
22:0
-, and
C
24:0
-ceramide could be localized to other intra-mitochon-
drial compartments. To examine the intra-mitochondrial lo-
calization of CerS6, co-immunoprecipitations were per-
formed using antibodies against protein components of
MPTP: outer mitochondrial membrane resident protein volt-
age-dependent anion channel, inner mitochondrial mem-
brane resident ANT, and matrix protein cyclophilin D. These
studies reveal a selective CerS6 association with ANT, the
inner membrane component of MPTP (Fig. 6). In contrast,
CerS2 associated with the outer membrane resident protein
Tom20, a receptor of the protein import complex. The data
suggest CerS6/ceramide could regulate the MPTP activity and
mitochondrial Ca
2⫹
homeostasis.
CerS6 and Ceramide Generation Are Required for OL Apo-
ptosis in Response to Glutamate—To further investigate the
role of CerS6 during brain development, our studies were
limited to OLs. OL precursor cells were purified from
mixed glial cultures, plated onto fibronectin-coated plates,
and cultured for 21 days in medium supplemented with
N2, which is known to promote OL differentiation. In cul-
ture, OL precursor cells successfully progressed through
the lineage that was manifested by increased expression of
an OL-specific differentiation marker, myelin proteolipid
protein (Fig. 7A). Similar to the mixed cell population in
the brain (Fig. 4A), CerS6 protein expression was down-
regulated during OL differentiation, whereas CerS5 expres-
FIGURE 5. Mitochondrial CLC is decreased in adult rat brain compared with young rat and could be rescued by ceramide. A, sequential Ca
2⫹
pulses
(100
Meach) caused gradual decreases in the Ca
2⫹
uptake rates (traces a and b). The arrows indicate the addition of mitochondria (MITO) and Ca
2⫹
.No
changes in swelling (traces c and d) at the increasing Ca
2⫹
load were detected. Maximal Ca
2⫹
accumulation was followed by addition of 30
g/ml of pore-
forming peptide alamethicin (ALA). Data are representative of four independent experiments. B, quantitative assessment of CLC (nanomole of Ca
2⫹
/mg of
protein). Data are mean ⫾S.E., *, p⬍0.05, n⫽4. C, CLC was measured in mitochondria purified from an adult (6 month old) rat brain in the presence of
vehicle control (con) or 1 nmol/mg of protein CSA or 1 mMADP with 1 nmol/mg of protein CSA, C
16:0
-ceramide, C
18:0
-ceramide, C
22:0
-ceramide, or SPH. Data
are mean ⫾S.E., *, p⬍0.05, n⫽3.
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sion was up-regulated (Fig. 7A). During normal brain de-
velopment OL precursor cells are greatly overproduced
and the final OL cell number is adjusted to the number of
axons requiring myelination by increased OL apoptosis
(36). It has been emphasized that neuron-derived factors,
including the neurotransmitter glutamate, could control
OL responses (36, 37).
To ascertain the role of CerS6 in apoptosis, OL precursor
cell response to glutamate was examined. CerS6 expression in
OL precursor cells is depicted in Fig. 7Aat day 1. Glutamate
can damage OLs via excitotoxicity, which is caused by sus-
tained activation of ionotropic glutamate receptors, and by
receptor-independent mechanisms, secondary to glutamate
uptake (38, 39). Glutamate enters the cell via bidirectional
cystine/glutamate antiporter that results in reduced cytosolic
cystine leading to glutathione depletion and cell death (40,
41). Indeed, OL exposure to glutamate reduced cell survival
(Fig. 7C). As expected, blocking glutamate uptake with 200
Mcystine protected OLs against high glutamate concentra-
tions (above 1 mM) indicating the involvement of the cystine/
glutamate antiporter.
To elucidate whether glutamate-induced OL death is medi-
ated by CerS and ceramide, a specific inhibitor of CerS activ-
ity, fumonisin B1 (FB1), and an inhibitor of ceramide biosyn-
thesis, myriocin, were employed. CerS produces ceramide via
two pathways: by de novo ceramide biosynthesis and the recy-
cling or salvage pathway (1, 8, 42). If blocking CerS activity
with FB1 enhanced OL survival, it would indicate CerS activ-
ity is required for glutamate-induced OL death. If blocking
ceramide biosynthesis with myriocin increased OL survival,
the involvement of recycling or the salvage pathway could be
ruled out (42). In fact, both inhibitors protected OL from glu-
tamate toxicity, suggesting that glutamate triggers activation
of CerS via the de novo ceramide biosynthetic pathway lead-
ing to OL death (Fig. 7C). Furthermore, sphingolipid analysis
revealed an about 3-fold increase in ceramide content after
OL treatment with glutamate that was abolished by FB1 or
FIGURE 6. CerS6 is associated with ANT, whereas CerS2 is associated
with protein import complex. Mitochondria were purified from the brain
of an adult rat. Mitochondria lysate (10
g) was loaded into the lane (Input).
Complexing with each CerS was detected by immunoprecipitation using
antibodies against Tom20, a receptor of the outer mitochondrial membrane
protein import complex (A); voltage-dependent anion channel (VDAC),
outer mitochondrial membrane component of MPTP (B); cyclophilin D
(CyD), matrix protein-regulator of MPTP (C); or ANT, the inner mitochondrial
membrane component of MPTP (D). As a control, the same immunoprecipi-
tation procedure was performed except for primary antibody application
(IgG).
FIGURE 7. Glutamate-induced OL death is ceramide-dependent. A, CerS5 and CerS6 protein expression was analyzed in OLs during cell differentiation in
culture by Western blot. Proteolipid protein is a marker for OL differentiation, and

-actin was used for normalization purposes. B, quantification of the cer-
amide synthases expression in cultured OLs during differentiation. C, OLs were exposed to glutamate with/without 20
Mfumonisin B1 (FB1)or1
Mmyri-
ocin (MR)or200
Mcystine, glutamate antiporter inhibitor, and cell death was quantified 24 h later. Data are mean ⫾S.E., *, p⬍0.05, n⫽12. D, OLs were
exposed to 6 mMglutamate (Glut) with/without 20
MFB1 or 1
Mmyriocin and ceramide content was analyzed 24 h later. Data are mean ⫾S.E., *, p⬍
0.05, n⫽12.
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myriocin (Fig. 7D). The data suggest that ceramide is an es-
sential mediator of glutamate-induced OL death.
Glutamate-induced OL death appears to be mediated by
ceramide-dependent apoptotic mechanisms. Fig. 8Ashows
that glutamate-triggered activation of executioner caspases
3/7 peaked at 18 h after treatment with glutamate. A new gen-
eration pan-caspase inhibitor, Q-VD-OPH blocked caspase
activation (Fig. 8A). These studies suggest that glutamate-
induced OL apoptosis is dependent on activation of ceramide
synthase that participates in de novo ceramide biosynthesis.
To identify the ceramide synthase promoting apoptotic
mechanisms in response to glutamate, CerS6 or CerS5 were
knocked down using siRNA. OLs were transfected with 1
nmol of siRNA targeting CerS6 or CerS5 or control siRNA
(CSR), and cultured for 48 h. Western blot analysis indicated
⬃80% knockdown of CerS6 or CerS5 expression (Fig. 8B).
Importantly, there was no interference between effects of
CerS5 and CerS6 siRNA. Thus, siRNA targeting CerS5 did
not affect the protein expression of CerS6, and vice versa. It
should be mentioned that CerS6 siRNA at 10 nmol and higher
could decrease CerS5 protein expression (not shown). Knock-
ing down CerS6 protected OLs from glutamate toxicity,
whereas knocking down CerS5 had no effect on OL survival
(Fig. 8C).
Pan-caspase inhibitor prevented glutamate-induced OL
death, thereby confirming the involvement of apoptotic mecha-
nisms. Furthermore, knockdown of CerS6 impeded the increase
in ceramide after OL exposure to glutamate (Fig. 8D).
Analysis of the ceramide species revealed the most dra-
matic increase in C
16:0
-ceramide (up to 2.3-fold) and smaller
increases in C
18:0
- and C
18:1
-ceramide (up to 1.4- and 1.7-fold,
respectively) after OL treatment with glutamate (Fig. 9).
Knocking down CerS6 attenuated increases in ceramide spe-
cies in response to glutamate. The data are expressed as fold-
increases of OL ceramide content shown in Table 3. The re-
sults of these studies support a critical role for mitochondrial
CerS6-generated C
16:0
-ceramide in glutamate-induced apo-
ptosis and suggest the additional pro-apoptotic input of C
18:0
-
and C
18:1
-ceramide, which could also be generated by CerS6.
To verify these results, OLs were transfected with another
siRNA targeting CerS6 (see “Experimental Procedures”) that
yielded similar OL protection from glutamate toxicity (not
FIGURE 8. CerS6 knockdown decreased ceramide and enhanced cell survival. A, OLs were exposed to glutamate (Glut) with/without 20
Mpan-caspase
inhibitor (CI), and caspase 3/7 activity was measured. Data are mean ⫾S.E., *, p⬍0.05, n⫽12. B, OLs were transfected with 1 nMCerS6-specific siRNA or 1
nMCerS5-specific siRNA or control siRNA (CSR) and cultured for 24 h. CerS5 or CerS6 protein expression was confirmed by Western blot. C, OLs were trans-
fected with 1 nMCerS6-specific siRNA or 1 nMCerS5-specific siRNA or control siRNA (CSR) and cultured for 24 h. Then, cells were treated with glutamate
with/without 20
Mpan-caspase inhibitor (CI). Cell survival was measured 24 h later. Data are mean ⫾S.E., *, p⬍0.05, n⫽12. D, OLs were transfected with
1nMCerS6-specific siRNA or 1 nMCerS5-specific siRNA or control siRNA (CSR) and cultured for 24 h. Then, cells were treated with 6 mMglutamate (Glut), and
ceramide was analyzed 24 h later. Data are mean ⫾S.E., *, p⬍0.05, n⫽18.
FIGURE 9. CerS6 knockdown effect on ceramide species profile in re-
sponse to OL treatment with glutamate. OLs were transfected with 1 nM
CerS6-specific siRNA or control siRNA (CSR) and cultured for 24 h. Then, cells
were treated with 6 mMglutamate, and ceramide species was analyzed 24 h
later. Data are mean ⫾S.E., *, p⬍0.05, n⫽18.
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shown). These data suggest that CerS6 is required for gluta-
mate-induced OL apoptosis.
CerS6 Is Necessary for NGF-induced OL Apoptosis—To de-
termine whether the CerS6 requirement is specific to gluta-
mate-induced OL apoptosis, we utilized another apoptotic
stimulus, NGF. Apoptotic cell death is an essential feature of
normal brain development, and it is controlled in part by a
wide array of neurotrophic factors, including NGF, which
binds and activates both the p75 neurotrophin receptor
(p75
NTR
) and the receptor tyrosine kinase (TrkA) to dictate
specific cell responses. Thus, blocking p75
NTR
function with
antibodies resulted in hypomyelination of peripheral nerves,
whereas blocking TrkA signaling with K252a yielded in-
creased myelination, suggesting that myelination is under
mutual control of p75
NTR
and TrkA signaling (43). Whereas
TrkA receptors have a well defined trophic function, p75
NTR
exerts activities ranging from trophism to apoptosis. OLs have
been shown to undergo p75
NTR
-dependent apoptosis in vitro
(44) mediated by Rac GTPase activity (45), JNK phosphoryla-
tion, and caspase activation (46).
Fig. 10Ashows that treatment of OLs with 1 mMNGF or 20
Mof the specific TrkA inhibitor K252a alone did not affect
cell survival. In contrast, OL exposure to 1 mMNGF in the
presence of 20
MK252a significantly increased OL death
that was completely prevented by anti-p75
NTR
antiserum,
indicating the involvement of p75
NTR
. As expected, the pan-
caspase inhibitor protected OL from NGF-induced toxicity.
To learn whether CerS6 is an important molecular deter-
minant in NGF-induced pro-apoptotic signaling, CerS6 was
knocked down using siRNA. CerS6 knockdown protected
OLs from NGF-induced apoptosis, whereas knocking down
CerS5 had no effect on OL survival (Fig. 10B). Furthermore,
knocking down CerS6 prevented increases in C
16:0
-orC
18:1
-
ceramide in response to NGF ⫹K252a treatment (Fig. 10C).
Data are expressed as fold-increases of OL ceramide species
content (Table 3). Total ceramide did not increase after OLs
exposure to NGF with K252a (not shown). The results of
these studies indicate CerS6 involvement in promoting NGF-
initiated apoptotic signaling in OLs. Collectively, the data sug-
gest CerS6 activation and ceramide generation are important
for OL apoptosis regardless of the apoptotic stimuli.
CerS6 Promotes OL Apoptosis by Increasing Ca
2⫹
Influx in
Mitochondria and Calpain Activation—To identify down-
stream targets of CerS6-mediated pro-apoptotic signaling, we
investigated the possibility that CerS6-dependent disturbance
of mitochondrial Ca
2⫹
homeostasis could be crucial for OL
apoptosis. Having shown the ceramide-induced increases in
mitochondrial CLC in isolated brain mitochondria, we fo-
FIGURE 10. CerS6 knockdown protected OLs against NGF-induced apoptosis. A, OLs were exposed to 1 mMNGF with/without TrkA receptor inhibitor,
50
MK252a, or 1
g/ml of anti-NGF receptor antibody, and cell death was measured 24 h later. B, OLs were transfected with 1 nMCerS6-specific siRNA or 1
nMCerS5-specific siRNA or control siRNA (CSR) and cultured for 24 h. Then, cells were treated with 1 mMNGF, 50
MK252a, and cell survival was measured
24 h later. Data are mean ⫾S.E., *, p⬍0.05, n⫽12. C, OLs were transfected with 1 nMCerS6-specific siRNA control siRNA (CSR) and cultured for 24 h. Then,
cells were treated with 1 mMNGF, 50
MK252a, and ceramide species content was measured 24 h later. Data are mean ⫾S.E., *, p⬍0.05, n⫽12.
TABLE 3
Ceramide species content of OLs transfected with CSR or CerS6 siRNA
Ceramide species were determined in OL cultures transfected with 1 nmol of siRNA targeting CerS6 or 1 nmol of control siRNA and cultured for 24 h. Each sample was
normalized to its respective total protein levels. Values are mean ⫾S.E., n⫽18.
Ceramide C16:0 C18:0 C18:1 C20:0 C24:0 C24:1
CSR 89.7 ⫾6.8 179.2 ⫾10.6 5.9 ⫾0.8 18.1 ⫾1.3 192.0 ⫾11.3 221.1 ⫾8.4
CerS6 siRNA 80.6 ⫾5.2 177.8 ⫾9.7 4.9 ⫾1.1 20.9 ⫾1.1 205.9 ⫾10.8 218.3 ⫾9.7
CerS6 Promotes Apoptosis
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cused our studies on the role of mitochondrial Ca
2⫹
accumu-
lation in promoting OL apoptosis using a specific inhibitor of
the mitochondrial uniporter channel, RU360 (IC
50
⫽2nM)
(29). Blocking mitochondrial Ca
2⫹
uptake with 1
MRU360
significantly enhanced OL survival in response to glutamate
or NGF (Fig. 11, Aor B, respectively). This suggests that glu-
tamate or NGF triggers activation of CerS6 and generation of
ceramide in OL mitochondria leading to MPTP closure, in-
creased Ca
2⫹
influx via the uniporter channel, and reduced
cell survival.
Increased Ca
2⫹
accumulation in OL mitochondria could
result in activation of a Ca
2⫹
-dependent cysteine protease
calpain (EC 3.4.22.17). Calpains are a 15-member family of
non-lysosomal enzymes that degrade diverse proteins via lim-
ited proteolysis. Although calpains are mainly cytosolic en-
zymes, recent studies have shown that calpains 1, 2, and 10
exist in mitochondria and participate in the cleavage of aspar-
tate aminotransferase, apoptosis-inducing factor, respiratory
chain Complex I subunits, and the MPTP (47–49). Calpains
exhibit different Ca
2⫹
sensitivities in vitro,K
d
⫽25
MCa
2⫹
for calpain 1, and K
d
⫽750
MCa
2⫹
for calpain 2 (50).
Mounting evidence supports mitochondrial calpain involve-
ment in both caspase-dependent and -independent pathways
of apoptotic cell death (51). Specifically, calpain has been
shown to truncate apoptosis-inducing factor, a caspase-inde-
pendent death effector, and to induce its release from the mi-
tochondria (48, 51).
To elucidate whether calpain activation is required for OL
apoptosis, structurally unrelated cell-permeable specific in-
hibitors of calpain activity were used. Peptide derivative (ben-
ziloxycarbonyl-Leu-nLeu-H) calpeptin (52) competes for the
active site of calpain, whereas epoxysuccinyl peptide (E64d)
covalently and irreversibly binds to a critical sulfhydryl group
in the active site of the enzyme (53). In contrast, an
␣
-mer-
captoacrylic acid derivative, PD150606, is a selective nonpep-
tide uncompetitive inhibitor of calpain activity (K
i
⫽0.21
M
for calpain 1, and 0.37
Mfor calpain 2) (54). OLs were ex-
posed to glutamate with/without 1
Mcalpeptin or 1
M
PD150606, and cell death was assessed 24 h later. Both inhibi-
tors of calpain activity enhanced OL survival in response to
glutamate (Fig. 11A). These data agree with previous reports
of calpain involvement in pro-apoptotic signaling triggered by
glutamate in motor neurons (55). Similarly, OLs were pro-
tected by 1
Mcalpeptin or 1
ME64c against NGF receptor-
induced cell death (Fig. 11B). Whereas E64c, a cell-imperme-
able analog of E64d, had no effect. Study results suggest that
glutamate- or NGF receptor-triggered pro-apoptotic signaling
leads to a disturbance of mitochondrial Ca
2⫹
homeostasis and
activation of calpain in OLs.
To learn whether calpain is a downstream target in the
CerS6-mediated pro-apoptotic signaling pathway, calpain
activity was measured in OLs with down-regulated CerS6. Fig.
11Cshows that glutamate increased (⬃2.6 fold) calpain activ-
ity in OLs transfected with non-targeting siRNA (CSR). Glu-
tamate-induced calpain activation was partly attenuated by
knocking down CerS6. The data indicate CerS6 involvement
in regulation of calpain activity in OLs. Altogether, the results
of these studies suggest that CerS6-ceramide-mediated signal-
ing increases mitochondrial Ca
2⫹
load and calpain activity to
promote apoptosis in OLs.
DISCUSSION
The present studies are unique in establishing a novel pro-
apoptotic signaling pathway mediated by CerS6 in OLs. We
have shown that an apoptotic stimulus triggers activation of
CerS6 and generation of ceramide, thereby disturbing mito-
chondrial Ca
2⫹
homeostasis and calpain activation, which
results in OL death. Furthermore, CerS6 is down-regulated
during postnatal brain development and appears to generate
C
16:0
-ceramide in brain mitochondria. This is the first dem-
onstration of an essential role of CerS6 in the neural cell
apoptosis.
Apoptosis is important during brain development, elimi-
nating excess cells and ensuring the establishment of a proper
synaptic connection network. In contrast to OLs, neuronal
apoptosis has been extensively studied, and two waves of neu-
ronal cell death have been described. The first wave consists
of a large number of dividing neurons being eliminated during
a peak of neurogenesis at mid-embryogenesis due to competi-
tion for a limited supply of neurotrophic factors and intracel-
lular processes (56). The second wave consists of differenti-
ated neurons dying while migrating toward their target
location or while connecting to target cells during the early
postnatal period (57).
FIGURE 11. Glutamate- or NGF-induced OL apoptosis involves disturb-
ance of mitochondrial Ca
2ⴙ
and calpain activation. A, OLs were exposed
to glutamate with/without the mitochondrial Ca
2⫹
-uniporter inhibitor, 1
MRU360, or calpain inhibitors, 1
Mcalpeptin or 1
MPD150606. Cell
death was assessed 24 h later. Data are mean ⫾S.E., *, p⬍0.05, n⫽16.
B, OLs were exposed to 1 mMNGF plus 20
MK252a with/without 1
M
RU360 or 1
Mcalpeptin or 1
ME64d or 1
ME64c, and cell death was
measured 24 h later. Data are mean ⫾S.E., *, p⬍0.05, n⫽12. C, OLs were
transfected with 1 nMCerS6-specific siRNA or control siRNA (CSR) and cul-
tured for 24 h. Then, cells were treated with glutamate for 24 h and calpain
activity was measured. Data are mean ⫾S.E., *, p⬍0.05, n⫽6.
CerS6 Promotes Apoptosis
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To provide electrical insulation and maximize their con-
duction velocity, the axonal tracts in the central nervous sys-
tem are myelinated by OLs in early postnatal life. The myelin
biogenesis is coordinated by neuronal signals that control OL
proliferation, differentiation, and survival (36, 58). OLs are
greatly overproduced and the cell number is adjusted to the
number and length of axons requiring myelination (36). Only
the OLs that manage to ensheath the axon survive, whereas
those that fail degenerate (36). Given the significance of neu-
ron-derived factors in regulation of OL survival, the charac-
terization of a ceramide-mediated apoptotic pathway trig-
gered by glutamate and nerve growth factor is a valuable
contribution to our understanding of sphingolipid signaling in
OL apoptosis during brain development.
Our studies identify CerS6 as a novel determinant of the
pro-apoptotic signaling cascade in OLs. Here, we show that
OL exposure to high concentrations of glutamate activates de
novo ceramide biosynthesis, leading to cell death. Blocking
ceramide production with a specific inhibitor of de novo cer-
amide biosynthesis or an inhibitor of ceramide synthase en-
hanced OL survival (Fig. 7, Dand C). Gene knockdown exper-
iments suggested involvement of CerS6, but not CerS5, in
glutamate-induced OL death (Fig. 8, B–D). Further investiga-
tions revealed that CerS6-mediated OL death involves apo-
ptotic mechanisms through activation of functionally similar
executioner caspases 3 and 7 (Fig. 8A). Based on the knock-
out mice studies, caspases 3 and 7 have been implicated as key
mediators of apoptotic events downstream of mitochondria
(59). Caspase activation requires the release of pro-apoptotic
proteins from mitochondria due to mitochondrial dysfunc-
tion and loss of integrity. Indeed, glutamate-induced OL apo-
ptosis appears to involve a disturbance in mitochondrial Ca
2⫹
homeostasis and activation of the Ca
2⫹
-dependent protease,
calpain (Fig. 11, Aand C). Knocking down CerS6 reduced cal-
pain activation in response to glutamate, suggesting that cal-
pain is a downstream target of CerS6.
Furthermore, these studies show that a CerS6/ceramide-
mediated pro-apoptotic signaling pathway is essential for
p75
NTR
-induced OL apoptosis (Fig. 10). Whereas p75
NTR
-
induced responses in OLs are certainly understudied, cer-
amide participation in p75
NTR
-initiated signaling activities,
ranging from growth and differentiation to apoptosis, is well
established in neurons (60, 61). Thus, stimulation of p75
NTR
activates neutral and/or acid sphingomyelinases in the vicinity
of the receptor in the plasma membrane that results in SM
hydrolysis and ceramide generation. It has been emphasized
that transient ceramide production upon sphingomyelinase acti-
vation takes place within 1–5 min and mainly serves the mem-
brane structure, thereby facilitating the clustering of the death
receptors localized in lipid rafts and promoting apoptosis (62,
63). Our studies point to an important pro-apoptotic role of cer-
amide generated by CerS6 in mitochondria, and they agree with
the concept that endogenous ceramide production should be
considered in its topological context (1, 63).
Mitochondria are being appreciated as vital intracellular
compartments for ceramide metabolism. Mitochondria have
been shown to contain a variety of sphingolipids, including
SM and ceramide (64, 65). Although several enzyme activities
involved in ceramide metabolism have been shown in mito-
chondria, the nature of ceramide biosynthesis enzymes in this
organelle is still a matter of debate (17).
Ceramide synthase activity was first detected (66, 67) and
partially purified from a bovine brain mitochondria-enriched
fraction (68). Mitochondrial enzymes had ⬃2-fold higher spe-
cific ceramide synthase activity than the ceramide synthase
from the ER. The mitochondrial enzyme had a pH optimum
⬃7.5 and maximal catalytic efficiency with C
16:0
-orC
18:0
-
acyl-CoA (68). Purification of ceramide synthase from bovine
liver mitochondria yielded two major protein bands: 62 and
72 kDa (69). Detailed analysis of ceramide synthase activity in
highly purified mitochondria by Bionda et al. (11) essentially
confirmed previous findings. Thus, ceramide synthase activity
was shown in rat liver mitochondria and in the subcompart-
ment of the ER that is closely associated with mitochondria.
Further submitochondrial investigation of ceramide synthase
activity revealed enzyme localization to both outer and inner
mitochondrial membranes (11).
Our studies describing CerS1, CerS2, CerS4, and CerS6, in
purified brain mitochondria support the idea that several cer-
amide synthesizing enzymes could be localized to the mito-
chondria and/or to ER fragments tethered to the outer mito-
chondrial membrane (25, 70). The results of our studies
suggest that CerS6 could be localized to the inner mitochon-
drial membrane proximal to the MPTP, whereas CerS1,
CerS2, and CerS4 are likely to be found in the outer mito-
chondrial membrane. The additional source of ceramide in
mitochondria is a reverse reaction of a neutral ceramidase, e.g.
formation of ceramide as a result of condensation of palmitate
and sphingosine (71). On the basis of molecular cloning and
confocal microscopy data, this activity was ascribed to mito-
chondria (9), and it was demonstrated in purified mitochon-
dria (11). A recent report suggests that ceramide could be
generated by novel mitochondrial neutral sphingomyelinase
hydrolyzing SM (10). Continued research efforts are required
to better understand the mechanisms of mitochondrial cer-
amide generation and utilization along with its influence on
mitochondrial functions.
Our studies provide further support for the concept of dis-
tinct roles of ceramide species in cell metabolism. As ex-
pected, sphingolipids and most ceramide species were in-
creased in mitochondria during postnatal brain growth and
development (Figs. 1 and 2). In contrast, C
16:0
-ceramide was
severely reduced concomitantly with the down-regulation of
CerS6 expression (Figs. 3 and 4). Although overexpressed in
mammalian cells, CerS6 could generate C
14:0
-, C
16:0
-, and
C
18:0
-ceramide (14); in brain tissue, CerS6 appears to specifi-
cally regulate C
16:0
-ceramide content in brain mitochondria
during organ development. Increasing evidence suggests that
the fatty acid chain of ceramide is an important characteristic
of the biological effect mediated by the individual ceramide
species. Generation of C
18:0
-ceramide, and not C
16:0
-cer-
amide, has been shown to repress the human telomerase re-
verse transcriptase promoter in lung carcinoma cells (72).
Activation of acid sphingomyelinase in the salvage pathway
brought about a selective accumulation of C
16:0
-ceramide (20,
73) due to the involvement of CerS5 (73). Another study re-
CerS6 Promotes Apoptosis
4656 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 6•FEBRUARY 11, 2011
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vealed a specific role for dihydro-C
16:0
-ceramide in the adap-
tive cardiac tissue response to hypoxia (74). Although certain
ceramide species could have different effects on biophysical
properties of the membrane lipid bilayer (75), it remains un-
clear how ceramides containing different fatty acids exert
their effects upon cell physiology. These studies suggest that
regulated expression of specific CerS in intracellular compart-
ments in conjunction with availability of certain fatty acyl-
CoA species could be an important mechanism for control-
ling the fatty acid composition of ceramides and their
biological effects on cell metabolism.
In the present study, we investigated the effect of differen-
tial CerS6 protein expression on mitochondrial functions in
brain mitochondria from young (high CerS6 expression) or
adult (low CerS6 expression) rat brains (Fig. 5). Assessment of
respiratory chain enzyme activities revealed no changes in
oxidative phosphorylation parameters between brain mito-
chondria from animals at different ages. Consistent with pre-
vious reports (76), mitochondria from young animal brains
were characterized by higher CLC compared with brain mito-
chondria from adult rats (Fig. 5). Remarkably, long-chain or
very long-chain ceramide addition to brain mitochondria
from adult rats enhanced their ability to retain Ca
2⫹
such that
the mitochondria were similar to mitochondria from young
animal brains. Ceramide-mediated blockade of MPTP open-
ing seems to be the underlying mechanism of the increased
CLC. Further studies reveal selective association of CerS6
with ANT the inner mitochondrial membrane component of
MPTP thereby linking CerS6 to the regulation of MPTP activ-
ity (Fig. 6). These studies suggest a novel role for CerS6/cer-
amide in governing Ca
2⫹
homeostasis in brain mitochondria.
The results from our study implicate CerS6 as an upstream
regulator of calpain activity in the cellular response to apopto-
tic stimuli in OLs (Fig. 11). Calpains are part of a broad family
of intracellular cysteine proteases that are independent from
caspases. Typically, calpains function as key regulators in cy-
toskeletal remodeling through their substrates, including the
microtubule-associated proteins neurofilament, Tau, and ac-
tin (77). Conversely, calpain activation was found to increase
as intracellular Ca
2⫹
increased during oxidative stress, leading
to induction of apoptotic pathways (55, 78).
Whereas calpains are mainly cytosolic proteins, numerous
reports exist of mitochondrial calpain-like activity (51). Cyto-
solic contamination of mitochondrial preparations has been a
concern, and a few studies report the presence of calpain in
the inner membrane fraction (47). Mitochondrial calpains are
thought to facilitate apoptosis-inducing factor release from
the intra-membrane space, inducing caspase-independent
apoptosis, but direct experimental evidence has been elusive
(79). Relevant to this study is the ability of mitochondrial cal-
pain to modulate the activity of the MPTP, and the subse-
quent release of proteins initiating the caspase-dependent
apoptotic cascade. Overexpression of calpain 10 induced mi-
tochondrial fragmentation and swelling, consistent with the
MPTP opening at a high conductance state and this altered
mitochondrial morphology was blocked by MPTP inhibitors
in kidney cells (49).
Conceivably, an apoptotic stimulus triggers a cytosolic
Ca
2⫹
influx into the mitochondria in OLs and an activation of
mitochondrial CerS6 that then elevates ceramide. Ceramide
blocks the MPTP opening at a low conductance state, leading
to increased Ca
2⫹
in the mitochondrial matrix. Rising mito-
chondrial Ca
2⫹
activates calpain 10, which could cleave pro-
tein components of the MPTP resulting in the MPTP opening
at a high conductance state, swelling, and rupture of the outer
mitochondrial membrane and release of cytochrome cto ini-
tiate caspase activation. Noteworthy, all 8 calpain 10 splice
variants seem to possess a mitochondrial targeting sequence
localized to the NH
2
-terminal 15 amino acids (49). Mitochon-
drial calpain expression appears to be tissue-specific. Also, it
has been shown that smooth muscle and rat liver mitochon-
dria do not contain calpain 10, whereas kidney mitochondria
express only calpain 10 and not calpains 1 or 2 (51). Identifi-
cation of the calpain isoform activated by the CerS6/cer-
amide-dependent pro-apoptotic pathway in OL mitochondria
would be helpful, and these studies are currently underway in
our laboratory.
In summary, this study provides experimental evidence that
apoptotic stimuli trigger activation of CerS6 and accumula-
tion of ceramide that results in an increased Ca
2⫹
in mito-
chondrial matrix and activation of calpain in OLs, and the
data shed more light on the compartmentalization of sphin-
golipid metabolism and function in brain.
Acknowledgments—We are very grateful to Drs. Narayan Bhat and
Edward Goetzl for stimulating discussions regarding the manu-
script. We thank Dr. Wendy Macklin for generously providing anti-
proteolipid protein antibody. We thank Dr. Jennifer G. Schnellmann
for help with preparation of the manuscript.
REFERENCES
1. Hannun, Y. A., and Obeid, L. M. (2008) Nat. Rev. Mol. Cell Biol. 9,
139–150
2. Kolesnick, R. N., Gon˜i, F. M., and Alonso, A. (2000) J. Cell. Physiol. 184,
285–300
3. Futerman, A. H., and Riezman, H. (2005) Trends Cell Biol. 15, 312–318
4. Merrill, A. H., Jr. (2002) J. Biol. Chem. 277, 25843–25846
5. Mandon, E. C., Ehses, I., Rother, J., van Echten, G., and Sandhoff, K.
(1992) J. Biol. Chem. 267, 11144–11148
6. Kolter, T., Proia, R. L., and Sandhoff, K. (2002) J. Biol. Chem. 277,
25859–25862
7. Futerman, A. H., Stieger, B., Hubbard, A. L., and Pagano, R. E. (1990)
J. Biol. Chem. 265, 8650–8657
8. Novgorodov, S. A., and Gudz, T. I. (2009) J. Cardiovasc. Pharmacol. 53,
198–208
9. El Bawab, S., Roddy, P., Qian, T., Bielawska, A., Lemasters, J. J., and Han-
nun, Y. A. (2000) J. Biol. Chem. 275, 21508–21513
10. Wu, B. X., Rajagopalan, V., Roddy, P. L., Clarke, C. J., and Hannun, Y. A.
(2010) J. Biol. Chem. 285, 17993–18002
11. Bionda, C., Portoukalian, J., Schmitt, D., Rodriguez-Lafrasse, C., and
Ardail, D. (2004) Biochem. J. 382, 527–533
12. Yu, J., Novgorodov, S. A., Chudakova, D., Zhu, H., Bielawska, A., Bielaw-
ski, J., Obeid, L. M., Kindy, M. S., and Gudz, T. I. (2007) J. Biol. Chem.
282, 25940–25949
13. Deng, X., Yin, X., Allan, R., Lu, D. D., Maurer, C. W., Haimovitz-Fried-
man, A., Fuks, Z., Shaham, S., and Kolesnick, R. (2008) Science 322,
110–115
14. Mizutani, Y., Kihara, A., and Igarashi, Y. (2005) Biochem. J. 390,
CerS6 Promotes Apoptosis
FEBRUARY 11, 2011•VOLUME 286•NUMBER 6 JOURNAL OF BIOLOGICAL CHEMISTRY 4657
at MUSC Library, on July 22, 2011www.jbc.orgDownloaded from
263–271
15. Spassieva, S., Seo, J. G., Jiang, J. C., Bielawski, J., Alvarez-Vasquez, F.,
Jazwinski, S. M., Hannun, Y. A., and Obeid, L. M. (2006) J. Biol. Chem.
281, 33931–33938
16. Mizutani, Y., Kihara, A., and Igarashi, Y. (2006) Biochem. J. 398,
531–538
17. Laviad, E. L., Albee, L., Pankova-Kholmyansky, I., Epstein, S., Park, H.,
Merrill, A. H., Jr., and Futerman, A. H. (2008) J. Biol. Chem. 283,
5677–5684
18. Lahiri, S., and Futerman, A. H. (2005) J. Biol. Chem. 280, 33735–33738
19. Novgorodov, S. A., Gudz, T. I., Milgrom, Y. M., and Brierley, G. P.
(1992) J. Biol. Chem. 267, 16274–16282
20. Chudakova, D. A., Zeidan, Y. H., Wheeler, B. W., Yu, J., Novgorodov,
S. A., Kindy, M. S., Hannun, Y. A., and Gudz, T. I. (2008) J. Biol. Chem.
283, 28806–28816
21. Bielawski, J., Szulc, Z. M., Hannun, Y. A., and Bielawska, A. (2006) Meth-
ods 39, 82–91
22. Hannun, Y. A., and Obeid, L. M. (2002) J. Biol. Chem. 277, 25847–25850
23. van Meer, G. (2005) EMBO J. 24, 3159 –3165
24. Degroote, S., Wolthoorn, J., and van Meer, G. (2004) Semin. Cell Dev.
Biol. 15, 375–387
25. Futerman, A. H. (2006) Biochim. Biophys. Acta 1758, 1885–1892
26. Nicholls, D. G. (1985) Prog. Brain Res. 63, 97–106
27. Chalmers, S., and Nicholls, D. G. (2003) J. Biol. Chem. 278,
19062–19070
28. Kristian, T., Pivovarova, N. B., Fiskum, G., and Andrews, S. B. (2007)
J. Neurochem. 102, 1346–1356
29. Kirichok, Y., Krapivinsky, G., and Clapham, D. E. (2004) Nature 427,
360–364
30. Hoppe, U. C. (2010) FEBS Lett. 584, 1975–1981
31. Novgorodov, S. A., and Gudz, T. I. (1996) J. Bioenerg. Biomembr. 28,
139–146
32. Brustovetsky, N., and Dubinsky, J. M. (2000) J. Neurosci. 20, 8229 – 8237
33. Brustovetsky, N., and Dubinsky, J. M. (2000) J. Neurosci. 20, 103–113
34. Kushnareva, Y. E., Wiley, S. E., Ward, M. W., Andreyev, A. Y., and Mur-
phy, A. N. (2005) J. Biol. Chem. 280, 28894–28902
35. Novgorodov, S. A., Gudz, T. I., and Obeid, L. M. (2008) J. Biol. Chem.
283, 24707–24717
36. Barres, B. A., Hart, I. K., Coles, H. S., Burne, J. F., Voyvodic, J. T., Rich-
ardson, W. D., and Raff, M. C. (1992) Cell 70, 31–46
37. Gudz, T. I., Komuro, H., and Macklin, W. B. (2006) J. Neuros ci. 26,
2458–2466
38. Matute, C., Domercq, M., and Sa´nchez-Go´mez, M. V. (2006) Glia 53,
212–224
39. Goldberg, M. P., and Ransom, B. R. (2003) Stroke 34, 330 –332
40. Wang, L., Hinoi, E., Takemori, A., Nakamichi, N., and Yoneda, Y. (2006)
J. Biol. Chem. 281, 24553–24565
41. Iemata, M., Takarada, T., Hinoi, E., Taniura, H., and Yoneda, Y. (2007)
J. Cell. Physiol. 213, 721–729
42. Kitatani, K., Idkowiak-Baldys, J., and Hannun, Y. A. (2008) Cell Signal.
20, 1010–1018
43. Cosgaya, J. M., Chan, J. R., and Shooter, E. M. (2002) Science 298,
1245–1248
44. Casaccia-Bonnefil, P., Carter, B. D., Dobrowsky, R. T., and Chao, M. V.
(1996) Nature 383, 716–719
45. Harrington, A. W., Kim, J. Y., and Yoon, S. O. (2002) J. Neurosci. 22,
156–166
46. Gu, C., Casaccia-Bonnefil, P., Srinivasan, A., and Chao, M. V. (1999)
J. Neurosci. 19, 3043–3049
47. Kar, P., Chakraborti, T., Samanta, K., and Chakraborti, S. (2008) Arch.
Biochem. Biophys. 470, 176–186
48. Ozaki, T., Yamashita, T., and Ishiguro, S. (2009) Biochim. Biophys. Acta
1793, 1848–1859
49. Arrington, D. D., Van Vleet, T. R., and Schnellmann, R. G. (2006) Am. J.
Physiol. Cell Physiol. 291, C1159–1171
50. Elce, J. S., Hegadorn, C., and Arthur, J. S. (1997) J. Biol. Chem. 272,
11268–11275
51. Kar, P., Samanta, K., Shaikh, S., Chowdhury, A., Chakraborti, T., and
Chakraborti, S. (2010) Arch. Biochem. Biophys. 495, 1–7
52. Tsujinaka, T., Kajiwara, Y., Kambayashi, J., Sakon, M., Higuchi, N.,
Tanaka, T., and Mori, T. (1988) Biochem. Biophys. Res. Commun. 153,
1201–1208
53. Lampi, K. J., Kadoya, K., Azuma, M., David, L. L., and Shearer, T. R.
(1992) Toxicol. Appl. Pharmacol. 117, 53–57
54. Wang, K. K., Nath, R., Posner, A., Raser, K. J., Buroker-Kilgore, M., Haji-
mohammadreza, I., Probert, A. W., Jr., Marcoux, F. W., Ye, Q., Takano,
E., Hatanaka, M., Maki, M., Caner, H., Collins, J. L., Fergus, A., Lee, K. S.,
Lunney, E. A., Hays, S. J., and Yuen, P. (1996) Proc. Natl. Acad. Sci.
U.S.A. 93, 6687–6692
55. Das, A., Sribnick, E. A., Wingrave, J. M., Del Re, A. M., Woodward, J. J.,
Appel, S. H., Banik, N. L., and Ray, S. K. (2005) J. Neurosci. Res. 81,
551–562
56. Meier, P., Finch, A., and Evan, G. (2000) Nature 407, 796 – 801
57. de la Rosa, E. J., and de Pablo, F. (2000) Trends Neurosci. 23, 454 – 458
58. Barres, B. A., and Raff, M. C. (1999) J. Cell Biol. 147, 1123–1128
59. Lakhani, S. A., Masud, A., Kuida, K., Porter, G. A., Jr., Booth, C. J., Me-
hal, W. Z., Inayat, I., and Flavell, R. A. (2006) Science 311, 847–851
60. Brann, A. B., Scott, R., Neuberger, Y., Abulafia, D., Boldin, S., Fainzilber,
M., and Futerman, A. H. (1999) J. Neurosci. 19, 8199–8206
61. Blo¨chl, A., and Blo¨chl, R. (2007) J. Neurochem. 102, 289 –305
62. Grassme´, H., Cremesti, A., Kolesnick, R., and Gulbins, E. (2003) Onco-
gene 22, 5457–5470
63. van Blitterswijk, W. J., van der Luit, A. H., Veldman, R. J., Verheij, M.,
and Borst, J. (2003) Biochem. J. 369, 199–211
64. Ardail, D., Popa, I., Alcantara, K., Pons, A., Zanetta, J. P., Louisot, P.,
Thomas, L., and Portoukalian, J. (2001) FEBS Lett. 488, 160–164
65. Tserng, K. Y., and Griffin, R. (2003) Anal. Biochem. 323, 84 –93
66. Morell, P., and Radin, N. S. (1970) J. Biol. Chem. 245, 342–350
67. Ullman, M. D., and Radin, N. S. (1972) Arch. Biochem. Biophys. 152,
767–777
68. Shimeno, H., Soeda, S., Yasukouchi, M., Okamura, N., and Nagamatsu,
A. (1995) Biol. Pharm. Bull. 18, 1335–1339
69. Shimeno, H., Soeda, S., Sakamoto, M., Kouchi, T., Kowakame, T., and
Kihara, T. (1998) Lipids 33, 601–605
70. Csorda´s, G., and Hajno´czky, G. (2009) Biochim. Biophys. Acta 1787,
1352–1362
71. El Bawab, S., Birbes, H., Roddy, P., Szulc, Z. M., Bielawska, A., and Han-
nun, Y. A. (2001) J. Biol. Chem. 276, 16758–16766
72. Wooten-Blanks, L. G., Song, P., Senkal, C. E., and Ogretmen, B. (2007)
FASEB J. 21, 3386–3397
73. Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., Taha, T. A., Jenkins, R. W.,
Senkal, C. E., Ogretmen, B., Obeid, L. M., and Hannun, Y. A. (2006)
J. Biol. Chem. 281, 36793–36802
74. Noureddine, L., Azzam, R., Nemer, G., Bielawski, J., Nasser, M., Bitar, F.,
and Dbaibo, G. S. (2008) Prostaglandins Other Lipid Mediat. 86, 49–55
75. Sot, J., Aranda, F. J., Collado, M. I., Gon˜i, F. M., and Alonso, A. (2005)
Biophys. J. 88, 3368–3380
76. Wang, X., Carlsson, Y., Basso, E., Zhu, C., Rousset, C. I., Rasola, A., Jo-
hansson, B. R., Blomgren, K., Mallard, C., Bernardi, P., Forte, M. A., and
Hagberg, H. (2009) J. Neurosci. 29, 2588–2596
77. Potter, D. A., Tirnauer, J. S., Janssen, R., Croall, D. E., Hughes, C. N.,
Fiacco, K. A., Mier, J. W., Maki, M., and Herman, I. M. (1998) J. Cell
Biol. 141, 647–662
78. Ishihara, I., Minami, Y., Nishizaki, T., Matsuoka, T., and Yamamura, H.
(2000) Neurosci. Lett. 279, 97–100
79. Joshi, A., Bondada, V., and Geddes, J. W. (2009) Exp. Neurol. 218,
221–227
CerS6 Promotes Apoptosis
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