Novel Role for Apolipoprotein E in the Central Nervous System

MODULATION OF SULFATIDE CONTENT*

Xianlin HanDagger §, Hua ChengDagger , John D. Fryer||, Anne M. Fagan||, and David M. Holtzman||**

From the Dagger  Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Internal Medicine, the  Department of Neurology, the ** Department of Molecular Biology & Pharmacology, and the || Center for the Study of Nervous System Injury, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, December 4, 2002, and in revised form, December 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has long been postulated that apolipoprotein E (apoE) may play a role in lipid metabolism in the brain. However, direct evidence that apoE plays such a role is lacking. We investigated whether apoE isoforms influence lipid content in the brain. We compared the brains of wild-type mice to apoE knockout (-/-) and human apoE3 and apoE4 transgenic mice and compared cerebrospinal fluid (CSF) of humans with different apoE isoforms. We found that there was no effect of apoE on the content of multiple phospholipids, sphingolipids, and cholesterol. There was, however, a marked effect of apoE on the sulfatide (ST) content in both the brain and CSF. The sulfatide mass in hippocampus and cortex of apoE knockout mice was found to be 61 and 114 mol% higher than wild-type mice counterparts at 12 months of age. In contrast, the sulfatide content in brain tissues from human apoE4-expressing mice was ~60% less than those found in wild-type mice of the same age. The ST mass in human CSF was significantly dependent on the APOE genotypes of the subjects. Examination of potential sulfatide carrier(s) in human CSF demonstrated that sulfatides are specifically associated with apoE-containing high density lipoproteins, suggesting that sulfatide levels in the central nervous system (CNS) are likely to be directly modulated by the same metabolic pathways that regulate levels of apoE-containing CNS lipoproteins. This novel role for apoE in the CNS may provide new insights into the connection of apoE with Alzheimer's disease and poor recovery after brain injury.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta (Abeta ) 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 Abeta 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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+/+ (open circle ), and human apoE3 transgenic (triangle ) mice were determined by using negative-ion ESI/MS as described under "Experimental Procedures." Some error bars are within the symbols.

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 black-square denotes the average level of sulfatide mass) and homozygous or heterozygous for apoE4 (open circle ; and  denotes the average level of sulfatide mass) was determined by using negative-ion ESI/MS as described under "Experimental Procedures." *, p < 0.01.

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.

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 epsilon 4 allele of APOE is a strong genetic risk factor for AD and cerebral amyloid angiopathy, whereas the epsilon 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 Abeta 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 Abeta are likely mediated by interactions between apoE-containing HDL (the form of apoE in brain) and Abeta . Given these issues, is there any way that sulfatide association with apoE-containing lipoproteins may be involved in apoE-Abeta interactions?

It has been shown that apoE interaction with Abeta 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 Abeta , we have shown that a portion of Abeta 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.

    ACKNOWLEDGEMENTS

We are grateful to the Washington University Mass Spectrometer Facility Center for use of the electrospray ionization mass spectrometer. We thank the Clinical Core of the Washington University Alzheimer's Disease Research Center for providing subject evaluations and clinical dementia ratings and the Washington University General Clinical Research Center for providing lumbar CSF samples. We also give special thanks to Maia Parsadanian for her skillful assistance in mouse brain dissection.

    FOOTNOTES

* This work was supported by NIA, National Institutes of Health (NIH) Grant P50-AG05681, by NIH Grant AG-13956, by NIH Grant M01 RR00036, and by the Alzheimer's Disease and Related Disorders Program of University of Missouri System.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Division of Bioorganic Chemistry and Molecular Pharmacology, Dept. of Internal Medicine, Washington University School of Medicine, Box 8020, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2690; Fax: 314-362-1402; E-mail: xianlin@pcg.wustl.edu.

Published, JBC Papers in Press, December 24, 2002, DOI 10.1074/jbc.M212340200

    ABBREVIATIONS

The abbreviations used are: apoE, apolipoprotein E; apoE3 or apoE4, human apoE isoforms; apoJ, apolipoprotein J; AD, Alzheimer's disease; Cer, ceramide(s); CNS, central nervous system; CSF, cerebrospinal fluid; ESI/MS, electrospray ionization mass spectrometry; GalC, galactocerebrosides; HDL, high density lipoprotein; LDL, low density lipoprotein; m:n, acyl chain containing m carbons and n double bonds; Nm:n, acyl amide with m carbons and n double bonds; PC, choline glycerophospholipids; PE, ethanolamine glycerophospholipids; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdGro, phosphatidylglycerol(s); PtdIns, phosphatidylinositol(s); PtdSer, phosphatidylserine(s); rhApoE, human recombinant apoE; SM, sphingomyelin(s); ST, sulfatide(s); Abeta , amyloid-beta peptide; GFAP, glial fibrillary acidic protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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