 |
INTRODUCTION |
Human apolipoprotein E (apoE, protein; APOE,
gene),1 a 34-kDa protein
coded for by a gene on chromosome 19, plays a prominent role in the
transport and metabolism of plasma cholesterol and triacylglycerides
through its ability to interact with the low density lipoprotein (LDL)
receptor and the LDL receptor-related protein (1, 2). It has been
postulated that apoE may also play an important role in the
redistribution of cholesterol and phospholipids within the central
nervous system (CNS) (3) where apoE is expressed mainly in astrocytes
(4, 5). In vitro and in vivo data suggest that
apoE can play a role in neurite outgrowth and sprouting (4, 6-9),
however, whether this is due to effects on cholesterol and lipid
metabolism is unclear. In addition to its role as a plasma lipid
transport protein, apoE participates in pathobiological processes,
including Alzheimer's disease (AD) (10). The effect of apoE in AD is
likely to occur at least in part via interactions with the amyloid-
(A
) peptide (see Ref. 11 for review). To date, the mechanisms of how
apoE is involved in all of these biological processes have not been completely clarified.
There are three common isoforms of human apoE that differ in amino
acids at positions 112 and 158 (1). The most common isoform, apoE3, has
cysteine at position 112 and arginine at 158, whereas apoE2 has
cysteine at both positions, and apoE4 contains arginine at both
positions. The isoforms are encoded by three alleles at the same gene
locus. ApoE4 has been shown to be a risk factor for AD and cerebral
amyloid angiopathy (10). Although the role of apoE in the pathogenesis
of AD is not entirely clear, studies suggest that apoE influences A
deposition and toxicity in the brain (12-15).
Sulfatides (ST) represent a class of sulfated galactocerebrosides that
differ only in the composition of the fatty acid residue that acylates
the amino group of the sphingosine. Sulfatides mediate diverse
biological processes, including the regulation of cell growth, protein
trafficking, signal transduction, cell adhesion, neuronal plasticity,
and cell morphogenesis (see Refs. 16-18 for reviews). Sulfatides are
almost exclusively synthesized by oligodendrocytes in the CNS and are
present predominantly in the myelin sheath surrounding axons and, thus,
are present in both white matter and gray matter (16). Altered levels
of ST in human brain tissues are involved in the pathogenesis of
various human diseases. Accumulation of ST, due to a sulfatidase
deficiency, is responsible for metachromatic leukodystrophy, in which
there is encephalopathy, long tract signs, and degeneration of myelin
in the CNS (19). In contrast, our recent study demonstrated that
substantial ST deficiency occurs at the very earliest stages of AD,
although the one or more causes of this deficiency remain unclear (20).
Mice deficient in ST and galactocerebrosides (GalC), generated by
knocking out a ceramide galactosyltransferase, generally die by 3 months of age and demonstrate several abnormalities, including abnormal
axonal function, dysmyelinosis, and loss of axonal conduction velocity
(21-24).
Numerous studies have demonstrated the role of apoE on the normal
variation of plasma lipids in an isoform-dependent manner (see Refs. 1 and 25 for reviews). Therefore, it has long been
postulated that apoE may also play a role in lipid metabolism in the
brain. However, direct evidence that apoE plays such a role is lacking.
In this study, we investigated whether apoE or apoE isoforms influence
lipid content in the CNS. We found that there were no significant
effects of apoE on the content of multiple phospholipids,
sphingolipids, and cholesterol. In contrast, apoE had a dramatic effect
on the sulfatide content in the CNS. Furthermore, the potential
mechanisms of the relationship between apoE and ST, including ST
in assembly of nascent apoE particles, ST transport and presence within
cerebrospinal fluid (CSF) lipoproteins, and ST hydrolysis mediated by
apoE, were also investigated in the study. Collectively, we have
uncovered a novel role for apoE, modulation of ST content, in the CNS.
This finding may provide some new insights into the role(s) of apoE in
biological and pathological processes in the CNS such as in AD and
following brain injury.
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EXPERIMENTAL PROCEDURES |
Materials--
Synthetic heptadecanoyl ceramide (N17:0
Cer) and phospholipids, including
1,2-dimyristoleoyl-3-phosphatidylcholine (14:1-14:1 PtdCho),
1,2-dipentadecanoyl-3-phosphatidylethanolamine (15:0-15:0 PtdEtn), and
1-myristoyl-2-palmitoyl-3-phosphadylglycerol (14:0-16:0 PtdGro) were
purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Semisynthetic
N-palmitoyl sulfatide (N16:0 ST) and perdeuterated N-stearoyl galactocerebroside (D35-N18:0 GalC) were obtained
from Matreya, Inc. (Pleasant Gap, PA). The Amplex Red cholesterol assay kit was obtained from Molecular Probes, Inc. (Eugene, OR). Human recombinant apoE (rhApoE) isoforms (rhApoE2, rhApoE3, and rhApoE4) were
purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). All rhApoE
isoforms were determined to be ST-free by electrospray ionization mass
spectrometry (ESI/MS). All cell culture supplies were from Invitrogen
(New York, NY) unless specified. Goat anti-apoE and anti-apoJ
antibodies were purchased from BioDesign (Saco, ME). Goat IgG control
antibody was purchased from Sigma (St. Louis, MO). Protein G-Sepharose
was obtained from Pierce (Rockford, IL).
Mouse Brain Tissues--
Wild-type (i.e. apoE+/+),
apoE
/
, and all transgenic mice in these studies were on a C57BL/6
background. Transgenic mice hemizygous (+/
) for a GFAP-apoE3 or
GFAP-apoE4 transgene on a mouse apoE
/
background were produced as
described previously (26). GFAP refers to the glial fibrillary acidic
protein promoter. The GFAP-apoE3 and -apoE4 mice express apoE at the
same level. At the indicated age, mice were anesthetized with
intraperitoneal pentobarbital (150 mg/kg) and were perfused
transcardially with 0.1 M phosphate-buffered saline (pH
7.4) at 4 °C. Brain regions were immediately dissected and frozen in
powdered dry ice before analysis.
Human Cerebrospinal Fluid and Astrocyte-secreted
Lipoproteins--
Human lumbar CSF samples were collected as
previously described (27). Freshly frozen CSF samples at
80 °C
(0.5-1 ml from each subject) were used in the study of ST content.
Lumbar CSF samples were obtained from cognitively normal subjects who
have a clinical dementia rating score of 0, non-demented (28). A clinical dementia rating score is assigned to subjects by experienced clinicians based on extensive clinical and neuropsychological evaluation as described (28). Subjects' genotypes for apoE were kindly
provided by the laboratory of Dr. A. Goate at Washington University
School of Medicine. A total of 16 subjects participated in the study
(apoE3/E3: n = 8; apoE3/E4: n = 6; and
apoE4/E4: n = 2) and had an overall average age of
70.8 ± 4.9 (mean ± S.E.) (71.1 ± 5.7, 71.2 ± 4.2, and 68.5 ± 0.5, respectively).
Astrocyte-secreted apoE3 and apoE4 lipoproteins were purified by using
immunoaffinity columns as described (29). Briefly, primary cultures of
forebrain astrocytes (>95% pure) were prepared from neonatal 1- to
2-day-old wild-type C57BL/6 (apoE+/+), GFAP-apoE3 transgenic, or
GFAP-apoE4 transgenic mice and grown to confluence (10-14 days) in T75
flasks. Cultures were then incubated in 5 ml of serum-free Dulbecco's
modified Eagle's medium/Ham's F-12 (1:1) medium containing N2
supplement (Invitrogen) for an additional 72 h. Conditioned media
were collected and apoE3 or apoE4 lipoproteins were purified from the
corresponding conditioned media with the anti-apoE immunoaffinity column.
Preparation and Quantitation of Lipids from Mouse Brain Tissues,
Human CSF, and Astrocyte-secreted Lipoproteins--
Lipids from
individual mouse brain tissue were prepared in the presence of
14:1-14:1 PtdCho (20 nmol/mg of protein), 15:0-15:0 PtdEtn (18 nmol/mg of protein), 14:0-16:0 PtdGro (30 nmol/mg of protein),
D35-N18:0 GalC (35 nmol/mg of protein), and N17:0 Cer (5 nmol/mg of
protein) (used as internal standards for the quantification of
choline-containing phospholipids, ethanolamine-containing
phospholipids, anionic lipids (including PtdGro, phosphatidylinositol
(PtdIns), phosphatidylserine (PtdSer), and ST), GalC, and Cer,
respectively) and similarly prepared as described previously (20, 30,
31). Lipids were similarly extracted from human CSF and
astrocyte-secreted lipoproteins in the presence of N16:0 ST (270 pmol/mg of CSF protein and 2 nmol/mg apoE, respectively) (used as
internal standard for the quantification of ST).
ESI/MS analyses of lipids were performed utilizing a Finnigan TSQ-7000
spectrometer equipped with an electrospray ion source as described (20,
30, 32). All molecular species were directly quantitated by comparisons
of the individual ion peak intensities with a properly selected
internal standard (e.g. 14:0-16:0 PtdGro for anionic
lipids, including PtdIns and PtdSer) after correction for
13C isotope effects (33). For the quantification of
cholesterol in brain tissue samples, tissue (10 mg) was homogenized in
0.2 ml of phosphate-buffered saline. The cholesterol mass of the
homogenates was quantitated by using an enzymatic methodology with an
Amplex Red cholesterol assay kit (Molecular Probe, Inc., Eugene, OR).
Hydrolysis Assay of Sulfatide Mediated by Human Recombinant apoE
Isoforms--
Hydrolysis of ST mediated by rhApoEs was performed by
modification of an n-sulfatidase assay as described
previously (34). Briefly, each of the rhApoE isoforms (50 µg) was
incubated in 1 ml (final) of assay buffer (100 mM imidazole
(pH 7.2) containing 2.5 mM MgCl2). N16:0 ST
(0.5 µmol) (dissolved in 50 µl of Me2SO) was injected
into the assay solution. After incubation for 0.5, 1, 2, and 4 h
at 37 °C, aliquots (100 µl) of the assay mixture were pipetted out
and lipids in the mixture were immediately extracted by a modified
Bligh and Dyer technique (35) in the presence of N17:0 Cer (5 nmol) and
D35-N18:0 GalC (5 nmol) (internal standards used for the quantitation
of Cer and GalC, two products of ST hydrolysis). The mass of Cer and
GalC was quantitated by ESI/MS as described previously (20, 31).
Fractionation and Immunoprecipitation Assays of Human
CSF--
Human CSF was obtained from a fasted, healthy subject via
lumbar puncture (L4/L5) by a trained neurologist (D.M.H.) and
immediately put on ice. There was no blood contamination. CSF was then
centrifuged for 5 min (~800 × g) at 4 °C to
pellet any cellular elements. Twenty milliliters of CSF was
concentrated to 1 ml at 4 °C by using Centriplus-10 concentrators
(10-kDa molecular mass, Millipore, Bedford, MA) prior to
fractionation via gel filtration chromatography (BioLogic System,
Bio-Rad, Hercules, CA) using tandem Superose 6 HR 10/30 columns
(Amersham Biosciences, Piscataway, NJ) under conditions of
physiological pH (pH 7.4) and ionic strength (0.15 M NaCl
with 1 mM EDTA). Seventy fractions of 400 µl each were collected. Lipids of the individual fractions were extracted in the
presence of N16:0 ST (70 pmol/fraction, used as an internal standard
for ST quantitation) by using the modified method of Bligh and Dyer
(35), and ST content in each fraction was analyzed by ESI/MS as
described above.
Immunoprecipitation was initiated by incubating 1.7 ml of pooled human
CSF with individual antibody (final concentration of 100 µg/ml) and
100 µl of a 50% protein G-Sepharose slurry for 3 h at room
temperature. The immunoprecipitation complexes were pelleted briefly
and rinsed three times gently with ice-cooled phosphate-buffered
saline. Lipids were extracted from both the final immunoprecipitation
pellets and supernatants by using a modified Bligh and Dyer method, and
ST content in the lipid extracts was analyzed by ESI/MS as described above.
Miscellaneous--
Protein concentration was determined
utilizing a bicinchoninic acid protein assay kit (Pierce, Rockford, IL)
using bovine serum albumin as a standard. Quantitative data from mouse
brain samples were normalized to the protein content of the tissues, and all data are presented as the means ± S.E. of at least three separate animals. Differences between mean values were determined by an
ANOVA analysis followed by the Dunn's test where p < 0.01 was considered significant.
 |
RESULTS |
Altered Mass of Sulfatides but Not of Other Examined Lipids in the
Brain of ApoE
/
Mice--
To examine the effects of apoE
on mouse brain lipid homeostasis, the molecular species and mass of
lipids, including choline glycerophospholipids (PC), ethanolamine
glycerophospholipids (PE), phosphatidylglycerol (PtdGro),
phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer),
sphingomyelin (SM), galactocerebroside (GalC), ST, and cholesterol in
both apoE+/+ and apoE
/
mouse brain regions were analyzed either by
ESI/MS or by fluorometric assay. Negative-ion ESI/MS analyses of
chloroform extracts from wild-type C57BL/6 mice (apoE+/+) mouse
hippocampus at 12 months of age demonstrated multiple abundant ion
peaks corresponding to PE molecular species as indicated (Fig.
1A). These PE molecular species account for a total mass of 320.0 ± 14.6 nmol/mg of
protein, including 152.7 ± 7.5 nmol/mg of protein of ethanolamine
plasmalogen. Fig. 1B illustrates a positive-ion ESI/MS
spectrum of an identical lipid extract from an apoE+/+ mouse
hippocampus at 12 months of age, in which PC, SM, and GalC molecular
species were identified and quantitated. Total masses of 277.0 ± 3.6, 39.5 ± 3.4, and 106.7 ± 9.7 nmol/mg of protein of PC,
SM, and GalC, respectively, were obtained (Table
I). Negative-ion ESI/MS analyses of the identical chloroform extracts from apoE+/+ mouse hippocampus without the addition of LiOH demonstrated multiple abundant ion peaks corresponding to PtdIns, PtdSer, and ST anionic lipid molecular species
as indicated (Fig. 2A), and
the total mass of these species was tabulated (Table I). A total mass
of 381.4 ± 22.3 nmol/mg of protein of cholesterol in hippocampus
was obtained by using an enzymatic methodology with the Amplex Red
cholesterol assay kit. ESI/MS analyses and fluorometric assays of
lipids in apoE+/+ mouse cortex and cerebellum were also performed and
the results were tabulated (Table I).

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Fig. 1.
Representative ESI mass spectra of lipid
extracts from wild-type mouse hippocampus. Lipids of wild-type
(i.e. apoE+/+) mouse hippocampus at 12 months of age were
extracted by a modified method of Bligh and Dyer as described under
"Experimental Procedures." Negative-ion (A) and
positive-ion (B) ESI/MS of the lipid extracts was performed
in the presence of a trace amount of LiOH under "Experimental
Procedures." All major individual molecular species as indicated were
identified using tandem mass spectrometry. I.S., internal
standard.
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Table I
Lipid content in brain regions of wild-type, apoE / , and human apoE3
transgenic mice at 12 months of age
Mouse brain tissue lipids were extracted by the Bligh and Dyer method
(35). Phospholipids and sphingolipids were analyzed directly by ESI/MS
as described under "Experimental Procedures." Lipid molecular
species were identified by ESI tandem mass spectrometry and quantitated
by comparisons of the individual ion peak intensities with properly
selected internal standards after corrections for 13C isotope
effects. Cholesterol contents were determined by a flurometric
technique. The results are expressed in nmol/mg of protein and
represent means ± S.E. of at least three separate animals.
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Fig. 2.
Representative negative-ion ESI mass spectra
of lipid extracts from wild-type and apoE knockout ( / ) mouse
hippocampi. Lipids of wild-type (i.e. apoE+/+,
panel A) and apoE knockout (i.e. apoE / ,
panel B) mouse hippocampi at 12 months of age were extracted
by a modified method of Bligh and Dyer and negative-ion ESI/MS of the
lipid extracts without the presence of LiOH was performed under
"Experimental Procedures." All major individual molecular species
as indicated were identified using tandem mass spectrometry.
Asterisks indicate the lower abundant ion peaks of some
sulfatide molecular species present in the lipid extracts. 2OH
N24:1 ST represents the 2-hydroxy N24:1 ST molecular species. Note
the differences in ST.
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Examination of the mass of various lipid molecular species
from hippocampus, cortex, and cerebellum of apoE
/
mice by ESI/MS analyses and fluorometric assays demonstrated no significant
differences in PC, PE, PtdIns, PtdSer, SM, GalC, and cholesterol in
comparison to those obtained from the corresponding regions of apoE+/+
mice (Fig. 2B and Table I). Interestingly, the mass content
of ST in hippocampus, cortex, and cerebellum of apoE
/
mice was
increased by 61, 114, and 7 mol%, respectively, relative to those
found in apoE+/+ mice at the same age, 12 months (Figs. 2B
and 3, Table I). These results indicate that apoE in some way regulates
ST content in the CNS.
Alterations in Sulfatide Content Mediated by apoE: Dependence on
ApoE Isoform and Age--
To examine the effects of human apoE
isoforms on the modulation of ST content in mouse brain, ST molecular
species and mass in lipid extracts of different brain regions from
transgenic mice expressing human apoE3 and apoE4 specifically in brain
astrocytes were compared with apoE
/
mice by ESI/MS analysis (Fig.
3). Importantly, the levels of apoE3 and
apoE4 expression in hemizygous transgenic mice are indistinguishable
from each other and are ~70% of the levels of mouse apoE in
wild-type mice (26, 36). The levels of ST mass in hippocampus and
cortex of apoE3 transgenic mice at 12 months of age were only 53 and 40 mol%, respectively, of those found in apoE
/
mice and were
statistically lower than those found in apoE+/+ mice (Fig. 3). The
levels of ST mass in both cortex and hippocampus of apoE4 transgenic
mice were only 28 and 37 mol%, respectively, of those in the
counterparts of apoE
/
mouse tissues, or 59 and 60 mol%,
respectively, of those found in the corresponding apoE+/+ mouse tissues
at 12 months of age (Fig. 3). The ST mass levels in these brain regions
of apoE4 transgenic mice were significantly lower than those found in
the corresponding apoE3 transgenic mouse tissues (p < 0.01) (Fig. 3), demonstrating isoform-specific regulation of ST mass levels by apoE. ApoE
/
mice had significantly higher ST content in
the thalamus, striatum, and septum compared with that present in
apoE+/+ mice even by 6 months of age (Fig.
4). In contrast, significantly lower ST
content in these brain regions was demonstrated in apoE3 transgenic
mice relative to those in the wild-type (apoE+/+) mice (Fig. 4). The
fact that human apoE transgenic mice, which express apoE only in brain,
have lower levels of sulfatide than wild-type mice demonstrates that
the regulation of brain sulfatide levels is via apoE produced in the
brain.

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Fig. 3.
Comparison of sulfatide mass levels in
hippocampi and cortices of wild-type, apoE / , human apoE3, and apoE4
transgenic mice at 12 months of age. Sulfatide mass of lipid
extracts from hippocampus (A) and cortex (B) of
wild-type, apoE / , apoE3, and apoE4 transgenic mice at 12 months of
age was determined by using negative-ion ESI/MS as described under
"Experimental Procedures." Data represent the means ± S.E. of
at least three separate animals. *, p < 0.01; **,
p < 0.001, relative to those in apoE+/+ mice. There is
also a significant difference between human apoE3 and apoE4 transgenic
mice (p < 0.01) in both hippocampus and cortex.
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Fig. 4.
The absence of apoE results in increase
sulfatide mass levels in several brain regions by 6 months of age.
Total mass of sulfatides in lipid extracts of thalamus, striatum, and
septum from apoE / (unfilled), apoE+/+
(dot-filled), and human apoE3 transgenic
(black-filled) mice at 6 months of age was determined by
using negative-ion ESI/MS as described under "Experimental
Procedures." *, p < 0.01; **, p < 0.001, relative to those in apoE+/+ mice.
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Further experiments were performed to examine the effects of age on
alterations in ST content mediated by the mouse and human apoE.
Overall, the ST content in most brain regions of wild-type (apoE+/+)
mice was relatively unchanged after 6 months of age (Fig.
5). However, the levels of ST mass in
apoE
/
mice at 6 and 12 months of age were substantially increased
relative to those in apoE+/+ mice of advanced age (Fig. 5). In
contrast, the ST content in all examined regions of apoE3 transgenic
mice were substantially lower compared those found in apoE+/+ mice at
ages of 12 months and older, although the ST content in these two types of mice were almost identical at 6 months of age (Fig. 5). It was also
found that alterations in ST mass mediated by apoE was smaller in
cerebellum than that found in cortex or hippocampus at the early ages
(Fig. 5).

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Fig. 5.
Age-dependent regulation of
sulfatide mass levels in hippocampus, cortex, and cerebellum in
wild-type, apoE / , and human apoE3 transgenic mice. Sulfatide
mass levels of lipid extracts from hippocampus (A), cortex
(B), and cerebellum (C) of apoE / ( ),
apoE+/+ ( ), and human apoE3 transgenic ( ) mice were determined by
using negative-ion ESI/MS as described under "Experimental
Procedures." Some error bars are within the
symbols.
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Sulfatide Content in Cultured Astrocyte-secreted ApoE Particles and
Human CSF--
To investigate the possible mechanism(s) of
apoE-mediated alterations in ST content in the CNS, two experiments
were performed. First, purified, astrocyte-secreted lipoproteins
containing either apoE3 or apoE4 were prepared from cultured astrocytes
as described under "Experimental Procedures." The ST content in
these particles was quantitated by ESI/MS. It was found that the ST
content in these particles was negligible, although apoE4 particles
contained more ST mass than that found in apoE3 particles (731 ± 45 versus 562 ± 22 pmol/mg apoE, respectively).
Second, CSF (which contains CNS-produced apoE-containing lipoprotein
particles) samples were collected from age-matched cognitively normal
human subjects. The ST content in the CSF samples was analyzed by
ESI/MS. The ST mass (nmol/mg apoE) in apoE3/E3 homozygous subjects was
46.4 ± 3.3, whereas the level was significantly higher in
subjects with one or two alleles of apoE4 (53.7 ± 4.6, p < 0.01) (Fig. 6). It
should be noted that the ST content in CSF samples from subjects who
were either heterozygous or homozygous for APOE4 (3/4 or
4/4) were indistinguishable in this study. Therefore, it can be
concluded that human CSF contains a significant amount of ST and the
mass level of ST in human CSF is apoE
isoform-dependent.

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Fig. 6.
Relationship between sulfatide mass of human
cerebrospinal fluid and the APOE genotypes of the
subjects. Cerebrospinal fluid samples were collected from
age-matched (70.8 ± 4.9 years) cognitively normal human subjects
and lipids were extracted by a modified method of Bligh and Dyer as
described under "Experimental Procedures." The mass of ST in lipid
extracts of human cerebrospinal fluid in the subjects homozygous for
apoE3 ( ; and denotes the average level of sulfatide mass) and
homozygous or heterozygous for apoE4 ( ; and denotes the average
level of sulfatide mass) was determined by using negative-ion ESI/MS as
described under "Experimental Procedures." *, p < 0.01.
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Identification of the ST Carrier(s) in Human CSF--
Examination
of the mass levels of ST in both cultured astrocyte-secreted apoE
particles and human CSF (see last subsection) suggests two
possibilities. First, ST molecules are acquired and directly associate
with apoE particles in the CNS after apoE particles are secreted from
astrocytes. Alternatively, ST molecules may be influenced by apoE in an
indirect fashion but not directly associate and interact with
apoE-containing lipoproteins. Therefore, to understand the one or more
mechanisms of apoE-mediated alterations in ST content in the CNS, it is
necessary to identify the carrier or carriers of ST molecules. The
location of ST molecules was first investigated by analyzing ST content
in human CSF fractions after gel filtration chromatography. It was
found that sulfatides co-localized exactly with HDL-like lipoproteins
present in human CSF (Fig.
7A). The two most abundant
apolipoproteins produced in the CNS (apoE and apoJ) as well as several
other apolipoproteins are present in human CSF. Therefore, experiments
were performed to identify the specific carrier(s) of ST in human CSF
by immunoprecipitation (see the inset of Fig. 7B
for Western blots) and followed by ESI/MS quantitation of ST content in
both supernatants and pellets. It was found that most (over 80 mol%)
if not all ST molecules were immunoprecipitated with anti-apoE
antibodies, whereas ST molecules were not specifically
immunoprecipitated with anti-apoJ antibodies (Fig. 7, B and
C). These results clearly demonstrated that ST molecules
were specifically associated with apoE-containing HDL-like lipoproteins
in CSF and suggested that apoE particles mainly acquired ST in the CNS
after secretion from astrocytes.

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Fig. 7.
Identification of the sulfatide carrier in
human cerebrospinal fluid. A, quantitation of sulfatide
content in human cerebrospinal fluid fractions after gel filtration
chromatography. Human cerebrospinal fluid was fractionated by employing
tandem Superose 6 columns as described under "Experimental
Procedures." Seventy fractions of 400 µl each were collected.
Lipids of the each individual fraction were extracted in the presence
of N-palmitoyl sulfatide (70 pmol/fraction, used as an
internal standard for sulfatide quantitation) by using a modified Bligh
and Dyer method. Sulfatide content in each fraction was quantitated by
ESI/MS as described under "Experimental Procedures." The
x axis indicates fraction number and known elution profile
of CSF lipoproteins as reported previously (27). Quantitation of
sulfatide content in the immunoprecipitated lipoprotein particles
(B) and supernatants before or after immunoprecipitation
(C) was initiated by incubating 1.7 ml of pooled human
cerebrospinal fluids with individual antibodies as indicated as
described under "Experimental Procedures." Lipids of each
immunoprecipitation pellet were extracted in the presence of
N-palmitoyl sulfatide (70 pmol). Sulfatide content in the
lipid extracts was quantitated by ESI/MS as described under
"Experimental Procedures." Data represent means ± S.E. of at
least three separate experiments. The inset in B
shows a typical Western blot using anti-apoE and anti-apoJ antibodies
to analyze apoE and apoJ content in CSF samples prior to
(Pre-IP) immunoprecipitation or following
(Post-IP). Following immunoprecipitation of CSF, the pellet
was also analyzed for apoE and apoJ.
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|
Examination of the Hydrolysis of Sulfatide Mediated by
ApoEs--
Because we have demonstrated that human CSF apoE-containing
lipoproteins contain ST, it is most likely that the effects of apoE on
ST are via its association with apoE-containing CNS lipoproteins and
subsequent apoE-regulated metabolism. However, it was possible that one
determinant for apoE-mediated ST content differences in the CNS might
occur through apoE-catalyzed hydrolysis. Accordingly, the hydrolytic
products of ST (i.e. GalC and Cer) in brain tissue samples
from wild-type, apoE
/
, and human apoE transgenic mice were
determined by ESI/MS. There were no significant differences in GalC
content (Table I) and Cer content (data not shown) between these mice.
Additionally, neither GalC nor Cer was detected from the lipid extracts
of the sulfatide vesicles after incubation of each type of rhApoE
isoforms with these vesicles up to 4 h. These results suggest that
differences in the ST content of different apoE mice are not caused by
apoE-mediated ST hydrolysis.
 |
DISCUSSION |
The involvement of apoE in metabolism of phospholipids and
cholesterol in the plasma is well known (1, 25). To investigate whether
apoE influences the mass distribution of brain lipids, we
systematically quantified the distribution of lipids including multiple
phospholipids, sphingolipids, and cholesterol in different brain
regions of wild-type, apoE
/
, and human apoE transgenic mice. We
found that there are no significant effects of apoE on the content of
all examined lipids with the exception of ST levels, which were
dramatically altered in both the apoE knockout and human apoE
transgenic mice. Furthermore, this apoE-mediated alteration in ST
content in the CNS is regulated by aging as well as by apoE produced in
the brain, because the human apoE transgenic mice only express apoE in
brain and still have markedly elevated plasma cholesterol (26). The
results from the examination of the ST content in astrocyte-secreted
apoE particles indicate that apoE can acquire a small amount of ST
directly from cells that secrete apoE. However, because most brain ST
is produced by oligodendrocytes and located in myelin in the CNS, it is
likely that the majority of ST in apoE particles in the CNS
is acquired from myelin after apoE secretion from its expressed cells.
The major effect of mouse and human apoE appears to be in keeping ST
levels in brain at a lower level than that present in the absence of
apoE. This effect is probably due to apoE-containing lipoproteins
facilitating cellular clearance of ST. Although hydrolysis of ST
directly catalyzed by apoE was not observed in this study,
apoE-associated ST hydrolysis in vivo can not be completely
excluded. Because we showed that ST directly associates with
apoE-containing lipoproteins in CSF, it is likely that transport of ST
by apoE particles in brain interstitial fluid and CSF and endocytotic
recycling of apoE particles through LDL receptor or LDL receptor family
members such as LDL receptor-related proteins (37) are the major
pathways by which apoE mediates ST metabolism in the CNS.
The
4 allele of APOE is a strong genetic risk factor for
AD and cerebral amyloid angiopathy, whereas the
2 allele is
protective. Although there are several possible mechanisms as to why
these associations exist, there is very strong evidence to suggest that the interaction between apoE and A
play an important role in the
effect of apoE on AD (see Refs. 11 and 38 for reviews). For example, in
transgenic animal models that develop AD-like amyloidosis and neuritic
plaques, the lack of apoE results in little to no fibrillar amyloid
formation nor neuritic dystrophy (14, 39), and the expression of human
apoE results in an isoform-specific deposition of amyloid within
neuritic plaques with the effect order being apoE4 > E3 > E2 (15, 40). These effects of apoE on A
are likely mediated by
interactions between apoE-containing HDL (the form of apoE in brain)
and A
. Given these issues, is there any way that sulfatide
association with apoE-containing lipoproteins may be involved in
apoE-A
interactions?
It has been shown that apoE interaction with A
is markedly
influenced by the presence of lipids (41-43). However, previous work
(27) and the data presented here have shown that there is not a
quantitative difference in the amount of most phospholipids and
cholesterol present in brain or CSF lipoproteins in the presence or
absence of apoE or apoE isoforms. In contrast to this, we observed a
significant isoform-specific difference in ST levels (E4 > E3) in
human CSF lipoproteins. Although we have not yet explored whether sulfatide levels in apoE particles influence interactions with A
, we
have shown that a portion of A
co-elutes with apoE-containing HDL
particles shown here to also contain ST in human CSF (27).
In addition to the effect of apoE and apoE isoform on risk for AD,
there is substantial evidence that apoE4 is also a risk factor for poor
outcome following certain CNS injuries such as head trauma, multiple
sclerosis, and possibly stroke (see Ref. 9 for recent review). How it
is that apoE plays a role in these processes remains unclear. It is
possible that the effect is via an isoform-specific difference in
regeneration as has been shown in some in vitro and in
vivo models (see Ref. 9 for recent review). It is also possible,
particularly in the case of multiple sclerosis and possibly stroke,
that apoE is somehow regulating CNS inflammatory processes such as
cytokine production as has been seen in some models (e.g.
Ref. 44).
Interestingly, numerous studies have shown that sulfatides are ligands
for a family of receptor proteins, called selectins, located on
activated vascular endothelial cells, leukocytes, and activated
platelets (45-51). Although the relationship between the role of apoE
in immunoregulation and sulfatide as a ligand for selectins is unclear,
it has been shown that sulfatides are potent anti-inflammatory agents
(52). This potential relationship likely indicates a possible role of
ST in apoE particles as an anti-inflammatory immunomodulatory molecule.
In sum, although apoE in the brain does not play a major role in
regulating brain phospholipid and cholesterol levels, our data show
that apoE plays an important role in regulating brain sulfatide levels,
via apoE-containing CNS lipoproteins serving as an extracellular ST
carrier that regulates its metabolism. Further work on understanding
the relationship between apoE and ST may provide important clues to the
pathogenesis of the effects of apoE on both AD and following different
CNS injuries.