Novel Role for Apolipoprotein E in the Central Nervous System

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. 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. apolipoprotein E human apoE isoforms apolipoprotein J Alzheimer's disease ceramide(s) central nervous system cerebrospinal fluid electrospray ionization mass spectrometry galactocerebrosides high density lipoprotein low density lipoprotein acyl chain containing m carbons and n double bonds acyl amide with m carbons and n double bonds choline glycerophospholipids ethanolamine glycerophospholipids phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol(s) phosphatidylinositol(s) phosphatidylserine(s) human recombinant apoE sphingomyelin(s) sulfatide(s) amyloid-β peptide glial fibrillary acidic protein 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 (1Mahley R.W. Science. 1988; 240: 622-630Google Scholar, 2Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Google Scholar). 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) (3Pitas R.E. Boyles J.K. Lee S.H. Hui D. Weisgraber K.H. J. Biol. Chem. 1987; 262: 14352-14360Google Scholar) where apoE is expressed mainly in astrocytes (4Elshourbagy N.A. Liao W.S. Mahley R.W. Taylor J.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 203-207Google Scholar, 5Pitas R.E. Boyles J.K. Lee S.H. Foss D. Mahley R.W. Biochim. Biophys. Acta. 1987; 917: 148-161Google Scholar). In vitro and in vivo data suggest that apoE can play a role in neurite outgrowth and sprouting (4Elshourbagy N.A. Liao W.S. Mahley R.W. Taylor J.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 203-207Google Scholar, 6Ignatius M.J. Shooter E.M. Pitas R.E. Mahley R.W. Science. 1987; 236: 959-962Google Scholar, 7Holtzman D.M. Pitas R.E. Kilbridge J. Nathan B. Mahley R.W. Bu G. Schwartz A.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9480-9484Google Scholar, 8Buttini M. Orth M. Bellosta S. Akeefe H. Pitas R.E. Wyss-Coray T. Mucke L. Mahley R.W. J. Neurosci. 1999; 19: 4867-4880Google Scholar, 9Teter B. Alzheimer's Rep. 2000; 3: 199-212Google Scholar), 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) (10Strittmatter W.J. Roses A.D. Annu. Rev. Neurosci. 1996; 19: 53-77Google Scholar). The effect of apoE in AD is likely to occur at least in part via interactions with the amyloid-β (Aβ) peptide (see Ref. 11Holtzman D.M. J. Mol. Neurosci. 2001; 17: 147-155Google Scholar 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 (1Mahley R.W. Science. 1988; 240: 622-630Google Scholar). 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 (10Strittmatter W.J. Roses A.D. Annu. Rev. Neurosci. 1996; 19: 53-77Google Scholar). Although the role of apoE in the pathogenesis of AD is not entirely clear, studies suggest that apoE influences Aβ deposition and toxicity in the brain (12Wisniewski T. Frangione B. Neurosci. Lett. 1992; 135: 235-238Google Scholar, 13Strittmatter W.J. Weisgraber K.H. Huang D.Y. Dong L. Salvesen G.S. Pericak-Vance M. Schmechel D. Saunders A.M. Goldgaber D. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8098-8102Google Scholar, 14Bales K.R. Verina T. Dodel R.C. Du Y. Altstiel L. Bender M. Hyslop P. Johnstone E.M. Little S.P. Cummins D.J. Piccardo P. Ghetti B. Paul S.M. Nat. Genet. 1997; 17: 263-264Google Scholar, 15Holtzman D.M. Bales K.R. Tenkova T. Fagan A.M. Parsadanian M. Sartorius L.J. Mackey B. Olney J. McKeel D. Wozniak D. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2892-2897Google Scholar). 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. 16Vos J.P. Lopes-Cardozo M. Gadella B.M. Biochim. Biophys. Acta. 1994; 1211: 125-149Google Scholar, 17Merrill Jr., A.H. Schmelz E.-M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol. 1997; 142: 208-225Google Scholar, 18Ishizuka I. Prog. Lipid Res. 1997; 36: 245-319Google Scholar 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 (16Vos J.P. Lopes-Cardozo M. Gadella B.M. Biochim. Biophys. Acta. 1994; 1211: 125-149Google Scholar). 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 (19Kolodny E.H. Fluharty A.L. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York1995: 2693-2739Google Scholar). 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 (20Han X. Holtzman D.M. McKeel Jr., D.W. Kelley J. Morris J.C. J. Neurochem. 2002; 82: 809-818Google Scholar). 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 (21Bosio A. Binczek E. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13280-13285Google Scholar, 22Coetzee T. Dupree J.L. Popko B. J. Neurosci. Res. 1998; 54: 613-622Google Scholar, 23Bosio A. Büssow H. Adam J. Stoffel W. Cell Tissue Res. 1998; 292: 199-210Google Scholar, 24Coetzee T. Fujita N. Dupree J. Shi R. Blight A. Suzuki K. Popko B. Cell. 1996; 86: 209-219Google Scholar). Numerous studies have demonstrated the role of apoE on the normal variation of plasma lipids in an isoform-dependent manner (see Refs. 1Mahley R.W. Science. 1988; 240: 622-630Google Scholar and 25Plump A.S. Breslow J.L. Annu. Rev. Nutr. 1995; 15: 495-518Google Scholar 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 no effects of apoE on the content of multiple phospholipids, sphingolipids, and cholesterol. In contrast, apoE a effect on the sulfatide content in the CNS. the potential mechanisms of the apoE and ST, including ST in of apoE ST transport and within cerebrospinal fluid (CSF) lipoproteins, and ST by also investigated in the we have a novel role for of ST in the CNS. This may provide new insights into the of apoE in biological and processes in the CNS such as in AD and brain injury. ceramide and phospholipids, including and from sulfatide and from The cholesterol was from Human recombinant apoE isoforms and from isoforms to be by electrospray ionization mass spectrometry cell from and from was from Protein was from and all transgenic mice in these studies on a mice for a or on a as Y. S. Bu G. M.J. Fagan A.M. Holtzman D.M. J. Neurosci. 1998; Scholar). to the glial fibrillary acidic protein The and mice apoE at the same the mice with and with m at and in Human CSF as A.M. Morris J.C. Holtzman D.M. 2000; Scholar). CSF at from in the study of ST CSF from normal have a of L. McKeel D.W. P. M. E.H. Morris J.C. J. M. J. A.M. J.L. M. Saunders A.M. 1998; Scholar). is to by on and as L. McKeel D.W. P. M. E.H. Morris J.C. J. M. J. A.M. J.L. M. Saunders A.M. 1998; Scholar). genotypes for apoE by the of A. at of of in the study n n and n and an age of and apoE3 and apoE4 by as M. J. Fagan A.M. X. Holtzman D.M. Neurochem. 2001; Scholar). of astrocytes from to wild-type or transgenic mice and to in in of containing for an and apoE3 or apoE4 from the with the from brain in the of of of of of and of as for the of phospholipids, phospholipids, lipids and and and as (20Han X. Holtzman D.M. McKeel Jr., D.W. Kelley J. Morris J.C. J. Neurochem. 2002; 82: 809-818Google Scholar, X. Holtzman D.M. McKeel Jr., D.W. J. Neurochem. 2001; X. 2002; Scholar). from human CSF and in the of ST of CSF protein and as for the of of lipids a with an electrospray as (20Han X. Holtzman D.M. McKeel Jr., D.W. Kelley J. Morris J.C. J. Neurochem. 2002; 82: 809-818Google X. Holtzman D.M. McKeel Jr., D.W. J. Neurochem. 2001; Scholar, X. R.W. 1996; Scholar). directly by of the with a for including and after effects X. R.W. 2001; Scholar). the of cholesterol in brain was in of The cholesterol mass of the was by an with an cholesterol of ST by was by of an as M. J. Biol. Chem. 1995; Scholar). of the isoforms was in of containing ST in of was into the for and at of the out and lipids in the by a and W.J. J. Scholar) in the of and for the of and of ST The mass of and was by as (20Han X. Holtzman D.M. McKeel Jr., D.W. Kelley J. Morris J.C. J. Neurochem. 2002; 82: 809-818Google Scholar, X. 2002; Scholar). Human CSF was from a via by a and on There was no CSF was for at to of CSF was to at by to via of and m with of of the in the of ST as an for ST by the of and W.J. J. Scholar), and ST content in was by as was by of human CSF with of and of a protein for 3 at The and three with from both the and by a and and ST content in the lipid was by as Protein was a acid protein as a data from brain to the protein content of the and all data are as the of at least three by an by the where was To the effects of apoE on brain lipid the and mass of including choline glycerophospholipids ethanolamine glycerophospholipids (GalC), ST, and cholesterol in both and brain by or by of from wild-type mice hippocampus at 12 months of age demonstrated multiple to as for a mass of of protein, including of protein of ethanolamine a of an lipid from an hippocampus at 12 months of in which and and of and of protein of and of the from hippocampus the addition of demonstrated multiple to and ST lipid as and the mass of these was mass of of protein of cholesterol in hippocampus was by an with the cholesterol and of lipids in cortex and also and the content in brain of and human apoE3 transgenic mice at 12 months of apoE3 compared with compared with brain lipids by the and W.J. J. Scholar). and directly by as Lipid by mass spectrometry and by of the with after for by a The are expressed in of protein and represent of at least three compared with in a new mass of lipid from wild-type and apoE knockout (−/−) of wild-type and apoE knockout at 12 months of age by a of and and of the lipid the of was as mass the of sulfatide present in the lipid ST the ST the in brain lipids by the and W.J. J. Scholar). and directly by as Lipid by mass spectrometry and by of the with after for by a The are expressed in of protein and represent of at least three Examination of the mass of various lipid from and of mice by and demonstrated no in and cholesterol in to those from the of mice and the mass content of ST in and of mice was by and to those found in mice at the same 12 months that apoE in ST content in the CNS. To the effects of human apoE isoforms on the of ST content in ST and mass in lipid of different brain from transgenic mice human apoE3 and apoE4 specifically in brain astrocytes compared with mice by the levels of apoE3 and apoE4 in transgenic mice are from and are of the levels of apoE in wild-type mice Y. S. Bu G. M.J. Fagan A.M. Holtzman D.M. J. Neurosci. 1998; Scholar, D.M. Bales K.R. S. P. Parsadanian M. Fagan A.M. Y. Paul S.M. J. 1999; Scholar). The levels of ST mass in hippocampus and cortex of apoE3 transgenic mice at 12 months of age only and of those found in mice and than those found in mice The levels of ST mass in both cortex and hippocampus of apoE4 transgenic mice only and of those in the counterparts of or and of those found in the tissues at 12 months of age The ST mass levels in these brain of apoE4 transgenic mice significantly than those found in the apoE3 transgenic tissues regulation of ST mass levels by mice significantly higher ST content in the and compared with that present in mice by months of age In contrast, significantly ST content in these brain was demonstrated in apoE3 transgenic mice to those in the wild-type mice The that human apoE transgenic which apoE only in have levels of sulfatide than wild-type mice that the regulation of brain sulfatide levels is via apoE in the brain. to the effects of age on in ST content by the and human the ST content in most brain of wild-type mice was after months of age However, the levels of ST mass in mice at and 12 months of age to those in mice of age In contrast, the ST content in all of apoE3 transgenic mice compared those found in mice at of 12 months and although the ST content in these of mice almost at months of age It was also found that in ST mass by apoE was in than that found in cortex or hippocampus at the To the of in ST content in the containing apoE3 or apoE4 from astrocytes as The ST content in these was by It was found that the ST content in these was although apoE4 more ST mass than that found in apoE3 CSF contains apoE-containing lipoprotein from normal human subjects. The ST content in the CSF was by The ST mass in was whereas the was significantly higher in with one or alleles of apoE4 It be that the ST content in CSF from or for or in this Therefore, it can be that human CSF contains a of ST and the mass of ST in human CSF is apoE Examination of the mass levels of ST in both apoE and human CSF (see ST are and directly with apoE in the CNS after apoE are from ST may be by apoE in an not directly and interact with apoE-containing lipoproteins. Therefore, to the one or more mechanisms of in ST content in the it is to the or of ST The of ST was investigated by ST content in human CSF after It was found that sulfatides with present in human CSF The most in the CNS and as as several are present in human CSF. Therefore, to the carrier(s) of ST in human CSF by (see the of and by of ST content in both and It was found that most not all ST with whereas ST not specifically with demonstrated that ST specifically associated with apoE-containing in CSF and that apoE mainly ST in the CNS after from we have demonstrated that human CSF apoE-containing ST, it is most likely that the effects of apoE on ST are via its with apoE-containing CNS and However, it was that one for ST content in the CNS occur through the of ST and in brain from and human apoE transgenic mice by There no in content and content not these was from the lipid of the sulfatide after of of isoforms with these to suggest that in the ST content of different apoE mice are not by ST The of apoE in metabolism of phospholipids and cholesterol in the plasma is (1Mahley R.W. Science. 1988; 240: 622-630Google Scholar, 25Plump A.S. Breslow J.L. Annu. Rev. Nutr. 1995; 15: 495-518Google Scholar). To whether apoE influences the mass of brain we the of lipids including multiple phospholipids, sphingolipids, and cholesterol in different brain of and human apoE transgenic We found that there are no effects of apoE on the content of all lipids with the of ST which in both the apoE knockout and human apoE transgenic this in ST content in the CNS is by as as by apoE in the the human apoE transgenic mice only apoE in brain and have plasma cholesterol Y. S. Bu G. M.J. Fagan A.M. Holtzman D.M. J. Neurosci. 1998; Scholar). The from the of the ST content in apoE that apoE can a of ST directly from that However, most brain ST is by oligodendrocytes and in myelin in the it is likely that the of ST in apoE in the CNS is from myelin after apoE from its expressed The effect of and human apoE to be in ST levels in brain at a than that present in the of This effect is due to apoE-containing of Although of ST directly by apoE was not in this study, ST in vivo can not be completely we that ST directly with apoE-containing in it is likely that transport of ST by apoE in brain fluid and CSF and of apoE through LDL receptor or LDL receptor such as LDL receptor-related M. J. Annu. Rev. 1994; Scholar) are the pathways by which apoE ST metabolism in the CNS. The of APOE is a risk factor for AD and cerebral amyloid whereas the is Although there are several mechanisms as to these there is very evidence to suggest that the apoE and Aβ play an important role in the effect of apoE on AD (see Refs. 11Holtzman D.M. J. Mol. Neurosci. 2001; 17: 147-155Google Scholar and T. J. Frangione B. 1997; Scholar for reviews). in transgenic that and the of apoE in to no amyloid K.R. Verina T. Dodel R.C. Du Y. Altstiel L. Bender M. Hyslop P. Johnstone E.M. Little S.P. Cummins D.J. Piccardo P. Ghetti B. Paul S.M. Nat. Genet. 1997; 17: 263-264Google Scholar, D.M. Fagan A.M. Mackey B. Tenkova T. Sartorius L. Paul S.M. Bales K. K.H. 2000; Scholar), and the of human apoE in an deposition of amyloid within with the effect apoE4 D.M. Bales K.R. Tenkova T. Fagan A.M. Parsadanian M. Sartorius L.J. Mackey B. Olney J. McKeel D. Wozniak D. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2892-2897Google Scholar, A.M. M. Parsadanian M. Bales K.R. Paul S.M. Holtzman D.M. 2002; Scholar). effects of apoE on Aβ are likely by interactions apoE-containing of apoE in and these is there that sulfatide with apoE-containing may be involved in It has been shown that apoE with Aβ is by the of lipids M.J. A.M. G.S. J. Biol. Chem. 1994; Scholar, M.J. G.S. J. Biol. Chem. 1995; Scholar, T. M. E. R. A. B. B. M.J. A. Frangione B. J. J. 2000; Scholar). However, A.M. Morris J.C. Holtzman D.M. 2000; Scholar) and the data have shown that there is not a in the of most phospholipids and cholesterol present in brain or CSF in the or of apoE or apoE isoforms. In to we a in ST levels in human CSF lipoproteins. Although we have not whether sulfatide levels in apoE influence interactions with we have shown that a of Aβ with apoE-containing shown to also ST in human CSF A.M. Morris J.C. Holtzman D.M. 2000; Scholar). In addition to the effect of apoE and apoE on risk for AD, there is substantial evidence that apoE4 is also a risk factor for poor CNS such as multiple and (see Ref. 9Teter B. Alzheimer's Rep. 2000; 3: 199-212Google Scholar for recent review). it is that apoE plays a role in these processes unclear. It is that the effect is via an in as has been shown in in vitro and in vivo (see Ref. 9Teter B. Alzheimer's Rep. 2000; 3: 199-212Google Scholar for recent review). It is also in the of multiple and that apoE is CNS processes such as as has been in D. S. J. 1997; Scholar). studies have shown that sulfatides are for a of receptor on and G. J. K.A. A. J. Biol. 1992; Scholar, Y. Y. T. T. Suzuki T. T. K. M. A. K. I. M. Biophys. Res. 1993; Scholar, J. D. G. Jr., W. A. 1994; Scholar, J. G. K. Suzuki Y. 1995; Scholar, M. Suzuki M. H. M. A. M. 1995; Scholar, C.R. 2001; Scholar, M. P. 2001; Scholar). Although the the role of apoE in and sulfatide as a for is it has been shown that sulfatides are A. A. J. R. P. B. M. Jr., G. J. D. A. J. Chem. 1997; Scholar). This potential likely a role of ST in apoE as an In although apoE in the brain not play a role in brain and cholesterol our data that apoE plays an important role in brain sulfatide via apoE-containing CNS as an ST that its on the apoE and ST may provide important to the pathogenesis of the effects of apoE on both AD and different CNS We are to the for of the electrospray ionization mass We the of the Alzheimer's for and and the for CSF We also to Parsadanian for in brain