Lipoproteins, neurobiology, and Alzheimer's disease: structure and function of apolipoprotein E

Lipoproteins, neurobiology, and Alzheimer's disease: structure and function of apolipoprotein E Karl H Weisgraber, Robert E Pitas and Robert W Mahley Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, USA

The structure of apolipoprotein E has provided unique insights into the role of this protein in lipoprotein metabolism and neurobiology. Isoform-specific differences between apolipoprotein E3 and apolipoprotein E4 have been described with respect to neurite extension by cultured neurons, and interactions with proteins characteristic of the lesions of Alzheimer's disease, such as the amyloid [[3 peptide of neuritic plaques and tau, the microtubule-associated protein of neurofibrillary tangles. Apolipoprotein E may play a part in normal neurobiology and in the pathogenesis of Alzheimer's disease.

Current Opinion in Structural Biology 1994, 4:507-515 Introduction

Structure of apoE

The link between lipoproteins and neurobiology has taken on new significance with the recent discovery that apolipoprotein E (apoE) is associated with Alzheimer's disease (AD). It has long been established that apoE, one of the major plasma apolipoproteins, has a prominent role in the transport and metabolism of plasma cholesterol and triglyceride resulting from its ability to interact with lipoprotein receptors [1,2]. It is less well known that lipoproteins containing apoE play a part in peripheral nerve regeneration [3]. The link with AD was established when one of the three common alleles of apoE, the apoE4 allele, was identified as a major risk factor (susceptibility gene), acting in a dose-dependent manner, for Alzheimer's disease [4%5]. This observation has since been confirmed by several groups in a wide variety of populations [6-13], which strongly suggests that the apoE4 protein itself is involved in AD and that the genetic association is linked directly to the apoE4 allele. Identification of apoE4 as a major risk factor has generated widespread interest in its role in this devastating disease, which affects three million people in the United States alone, and has the potential to provide new insight into the cause and treatment of AD, both of which are poorly understood at present. This review will focus on the structure ofapoE, including the recently determined structure of apoE4, and the role of apoE and apoE-containing tipoproteins in nerve growth and repair as they may relate to AD.

Human apoE (a 299-residue protein) contains two structural domains that correlate with defined functions [14,15]. The amino-terminal domain (residues 1-191) contains the lipoprotein receptor-binding region ofapoE [16], and the carboxy-terminal domain (residues 216-299) contains the major lipoprotein-binding determinants of the protein (Fig. 1) [17]. The three common apoE isoforms in humans differ in cysteine and arginine contents at two polymorphic sites, positions 112 and 158: apoE3, the most common isoform, contains cysteine at position 112 and arginine at 158 whereas apoE2 has cysteine at both positions and apoE4 has arginine at both [18,19]. The three-dimensional structure of the amino-terminal domain of apoE3 has been determined by X-ray crystallography and shown to form an extended four-helix bundle (Fig. 1) [20]. The detailed structure of the lipoprotein-binding carboxyLterminal domain is not known and is depicted in Fig. 1 as helical. An interesting feature of the domain structure of apoE is that structural changes in one domain influence the properties of the other [2]. For example, the cysteine-arginine interchange at position 112, which distinguishes apoE3 from apoE4, influences the class of lipoprotein to which the carboxy-terminal domain will bind; apoE3 displays a preference for high density lipoproteins (HDL) and apoE4 for very low density lipoproteins (VLDL) (Fig. 1) [17,21,22]. This difference

Abbreviations AI3 peptide--amyloid beta peptide; AD--Alzheimer's disease;apoA~apolipoprotein A; apoE--apolipoprotein E; CSF--cerebrospinal fluid; HDL---high density lipoproteins; LDL--Iow density lipoproteins; LRP--LDL receptor-related protein; [3-VLDL--beta-migrating very low density lipoproteins;VLDL--very low density lipoproteins. © Current Biology Ltd ISSN 0959-440X

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Amino-terminal domain (residues 1-191 ) Receptor binding function

Carboxy-terminal domain (residues 216-299) Lipoprotein binding function

isoform

Lipoprotein association

Apo-E4 (Arg 112) . . . . . . . . . . . . . . . . . . . . . . . . . . . Apo-E3 (Cys 112) . . . . . . . . . . . . . . . . . . . . . . . . . . .

in lipoprotein preference has clinical implications, as it is thought that the different lipoprotein preferences are the primary reason apoE4 is associated with elevated concentrations of plasma cholesterol and low density lipoproteins (LDL) and an increased risk for cardiovascular disease [23]. Because of the interest in the differences between apoE3 and apoE4 regarding lipoprotein preference and more recently, in relation to their involvement in AD, the structure of the amino-terminal domain of apoE4 has been determined by X-ray crystallography and compared with that of apoE3 [24"]. As shown in Fig. 2, the amino-terminal domain of apoE4 also adopts the four-helix bundle motif characteristic of the apoE3 fragment. The two structures are virtually superimposable, within experimental error, including all side-chain conformations, with only two exceptions. First, the Arg61 side chain in helix 2 of apoE3 lies over Cys112 in helix 3, whereas substitution with arginine at position 112 in apoE4 forces the Arg61 side chain away from the helical bundle and into the aqueous environment (Fig. 2). Second, Glul09 in apoE3 occupies the position indicated in Fig. 2 and does not form any salt bridges, whereas in apoE4 Glul09 forms a salt bridge with the substituted Arg112. The Arg61 side chain in apoE4 is in an ideal position for interacting with the carboxy-terminal do-

VLDL HDL

Fig. 1. Model of apoE domain structure. The amino-terminal domain (residues 1-191) contains the receptor-binding region of apoE (residues 134-150) and exists as a four-helix bundle. The structure of the carboxy-terminal domain is

not known but is depicted here as helical; it contains the major lipoprotein-binding determinants of apoE. Position 11 2, which distinguishes apoE3 (Cys) from apoE4 (Arg), is indicated along with the lipoprotein preferences of apoE3 (HDL) and apoE4 (VLDL).

main of the apoE molecule. These results suggest that the different properties and characteristics of these two isoforms result from these subtle structural differences. The two simplest possibilities are either that the availability of Glul09 in apoE3 is responsible for the HDL preference of apoE3, or that the repositioning and exposure of Arg61 in apoE4 accounts for its VLDL preference. The involvement of Arg61 in modulating domain interaction appears to be a key factor in determining the isoform-specific lipoprotein preferences of apoE. Site-directed mutagenesis of Glul09 in apoE3 has no effect on lipoprotein preference, i.e. apoE3 retains its preference for HDL. However, when Arg61 in apoE4 is mutated to threonine, the preference shifts from V L D L to HDL [24"]. These results have therefore established the importance of Arg61 in determining the isoformspecific lipoprotein preferences of apoE3 and apoE4. Previously, it had been believed that arginine (or at least a positive charge) at position 112 was responsible for the lipoprotein preferences of apoE4 [17]; however, the present studies have demonstrated that Arg112 is necessary, but not sufficient for VLDL preference. As determined from the three-dimensional structure, the presence of arginine at position 1 t2 displaces the Arg61 side chain from its position between helices two and three and away from the four-helix bundle (Fig. 2).

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Fig. 2. Comparison of the X-ray crystal structures of the amino-terminal domain fragment of apoE3 and apoE4. The ribbon diagram of the four-helix bundle of the apoE3 and apoE4 fragments shows the side-chain differences in the vicinity of the substitution site at position 112. It is interesting to note that of the ten animal species for which the sequence of apoE is known, only human apoE has an arginine at position 61 (or its equivalent); all other species contain threonine at this position. On the other hand, Glul09 is conserved across species. Human apoE is therefore unique with respect to Arg61, and the presence of Arg61 in conjunction with Arg112 makes human apoE4 more unusual still.

been suggested that apoE has a major role in cholesterol homeostasis in the brain [28]. It is important to point out that in CSF, only the apoE-containing HDL possess the ability to bind LDL receptors and to participate in receptor-mediated lipid transport, whereas in plasma, apoB-containing lipoproteins, such as VLDL and LDL, also are involved in transport of cholesterol.

Importance of apoE in nerve regeneration Role of apoE in neurobiology Levels of apoE in the brain Early studies of the tissue distribution of apoE m R N A expression have suggested a potential role for apoE in the nervous system. In a number of species, brain tissue has been shown to be second only to the liver (a major organ responsible for lipoprotein synthesis) in apoE m R N A content [25]. Immunochemical localization and metabolic labeling studies have demonstrated that astrocytes, the major cells containing apoE in the brain, actually produce apoE themselves [26,27]. In the peripheral nervous system, however, synthesis of apoE is restricted primarily to nonmyelinating glial cells and resident macrophages [26]. Examination of the lipoproteins in cerebrospinal fluid (CSF) from humans and dogs revealed that both apoE and apoA-I are present in the HDL fraction [28]. In fact, HDL are the only lipoproteins present in CSF, and is capable of binding with high affinity to the LDL receptor. Given that all the components of a lipid transport system, including LDL receptors, are present in the central nervous system, it has

The first indication of the importance of apoE in nerve tissue came when it was discovered that apoE synthesis was dramatically induced following injury of peripheral nerves in a rat model [29-32]: synthesis was increased 250-fold to 350-fold, until apoE constituted 2-5% of the extracellular soluble protein. The induction and accumulation of apoE was also observed in the central nervous system when the optic nerve and spinal cord were injured, but not to the same extent as seen in peripheral injury [3]. Examination of the temporal events following injury to the rat sciatic nerve has led to the development of a model for peripheral nerve regeneration involving apoEcontaining lipoproteins and LDL receptors [3]. Following injury, the nerve distal to the injury site begins to degenerate, liberating large quantities of cholesterol and phospholipid from the degenerating axons (Fig. 3). At the same time, resident macrophages, as well as those entering the injury site, secrete large amounts of apoE in order to scavenge the lipids released by degeneration, and to transport them back to the macrophages for storage. Within a few days following injury, the ax-

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ons begin to regenerate, and express LDL receptors at their growth tips in order to ensure delivery of cholesterol sufficient for continued neurite growth. Associated with this regeneration process is the accumulation of apoE and apoA-I distal to the injury site as the resident macrophages become lipid-laden. The next step in the process, approximately two to three weeks after injury, is the remyelination of the newly developed axons. Once the Schwann cells have depleted their lipid stores, they express LDL receptors, presumably in order to capture the cholesterol that is mobilized from the macrophages and is necessary for continued remyelination. The tissue macrophages become depleted of lipids at this time, and apoE may participate in their redistribution to cells requiring lipids for regeneration. Thus, it appears that cholesterol from the degenerating injured axons is stored locally and then reused in the regeneration and remyelination of the new axons. In this model, apoE-lipoproteins transport cholesterol and other lipids in a local environment from cells where they are stored (macrophages) to cells requiring lipids for repair and membrane biosynthesis, chiefly neurons and Schwann cells (for review see [1]).

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Fig. 3. Role of apoE in peripheral nerve regeneration. Following injury, monocytes/macrophages enter the nerve as degeneration commences. The released lipids, primarily cholesterol, are added to the macarophage stores of lipids and may be delivered via an apoE-mediated uptake. As sprouts appear they express LDL receptors on their growing tips, likely to acquire cholesterol for membrane biosynthesis. As Schwann cells complete the remyelinization of the regenerated axon, they also express LDL receptors, and apoE-containing lipoprotein complexes appear to participate in the redistribution of cholesterol to the Schwann cells. (Reproduced from [1] with permission).

The recent demonstration that peripheral nerve regeneration appears to occur in apoE-deficient mice [33] raises

the hkelihood of redundancy in this critical process and suggests that other apolipoproteins may substitute for apoE. It is known that apoD also accumulates at the injury site and that apoA-I and apoA-IV infiltrate from plasma [34]. However, these apolipoproteins do not possess the ability to interact with the LDL receptor, and therefore would have to deliver lipids by another mechanism, which is likely to be less efficient. A key issue not addressed in the apoE-deficient mouse study was a quantitative comparison of nerve regeneration between the deficient and normal control mice.

Role of apoE in nerve growth and neurite extension in vitro

Fetal rabbit dorsal root ganglion cells have been used as a model system to determine the effect ofapoE on neurite branching (the number of branch points along a major extension) and neurite extension (the distance a neurite extends from the cell body) [35]. It has been demonstrated that ~-VLDL (a cholesterol- and apoE-rich lipoprotein) and unesterified cholesterol support membrane synthesis and enhanced neurite branching and extension. Addition of purified rabbit apoE (equivalent to human apoE3) to incubations of cells containing either source of cholesterol reduces the amount of neurite branching but significantly promotes neurite extension. Both LDL receptors and the LDL receptor-related protein (LRP) have been shown to be present in these sprouting neurites [35], suggesting for the first time that the LRP might be involved in nerve growth. These studies therefore demonstrate that apoE, in the presence of a source of cholesterol, could modify the growth patterns of nerve cells in culture and can promote neurite extension. l~ecently, these studies have been extended to compare the isoform-specific effects of human apoE3 and apoE4 on nerve growth in the fetal rabbit dorsal root ganglion cell model [36°]. Cells to which ~-VLDL have been added (Fig. 4b) exhibit an increase in both neurite branching and extension, compared with cells incubated in media alone (Fig. 4a). The addition of human apoE3 with the ~-VLDL results in an increase in neurite extension and a reduction in branching (Fig. 4c) [36°]. These results are very similar to those with rabbit apoE [35]. The provocative result is that addition of human apoE4 reduces both branching and extension (Fig. 4d), suggesting that apoE3 may promote neuronal growth, whereas apoE4 inhibits it. Blocking the interaction of apoE with the receptor, either by incubation with the monoclonal antibody 1D7 (known to prevent apoE-receptor interaction [37]) or by reductive methylation of the protein [38], eliminates the isoform-specific effects [36"]. This suggests the involvement oflipoprotein receptors in binding and/or internalizing the apoE-enriched [~-VLDL particles and that apoE may interact in an isoform-specific manner with one or more cellular proteins, such as the cytoskeletal proteins involved in neurite structure and function.

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Fig.4. Fetal rabbit dorsal root ganglion neurons grown (a) in serum-free medium, (b) with [3-VLDL, (c) with human apoE3 and I3-VLDL, and (d) with human apoE4 and I~-VLDL. The cells were maintained for 24 hr and then nonspecifically stained with 1,1'-dioctodecyl-3,3,3',3'tetramethylindocarbocyanine. The I3-VLDL were used at 401.tg cholesterol m1-1 and the apoE at 30lttgm1-1. In (b), the I3-VLDL enhanced neurite extension and branching, whereas [3-VLDL with apoE3 (c) increased neurite extension and decreased branching. On the other hand, [3-VLDL with apoE4 markedly suppressed neurite extension, in (d). Scale bar: 100 ~m. Role of apoE4 in Alzheimer's disease Alzheimer's disease, an irreversible neurodegenerative disorder characterized by progressive dementia, is difficult to diagnose unambiguously [39]. Definite diagnosis requires supporting pathology that can be obtained only by microscopic examination of the cortex, usually performed at autopsy. Confirmatory neuropathology requires the presence of both neuritic amyloid plaques and neurofibrillary tangles in the brain. Both structures are characteristic of AD; however, the relationship of plaques and tangles to each other, and to the pathogenesis of AD, is unknown and quite controversial. It is clear, however, that AD is a heterogeneous disorder, with a number of genes and chromosomal loci affecting its cause and progression [40]. Neuritic plaques consist ofextracellular deposits ofamyloid (Fig. 5). The major core component of these deposits and the associated vascular deposits of amyloid (cerebral vessel angiopathy) is the amytoid beta (At3) peptide, which has also been detected in plasma and CSF [41]. Ranging in length from 39 to 42 residues, the A~ peptide is derived by an incompletely understood prote-

olytic process from the amyloid precursor protein [42]. Present on the surface of a variety of cells, the amyloid precursor protein is processed to the A~ peptide at low rates in both healthy and AD subjects. The first suggestion that apoE might be associated with AD arose when apoE was detected by immunochemical localization in amyloid deposits in neuritic plaques and cerebral vessel angiopathy [4",43,44]. In contrast to amyloid deposits, neurofibrillary tangles appear to be primarily intracellular (Fig. 5). These tangles are characterized by paired helical filaments [45], the major component of which is an extensively phosphorylated ('hyperphosphorylated') form of the tau protein. Tau is a member of the microtubule-associated family of proteins and is known to facilitate microtubule assembly and stability. It consists of several isoforms that result fi~om differential splicing; tau contains three or four repeating sequences, depending on the isoform, that constitute the microtubule-binding region. These repeats are homologous to those found in other microtubule-binding proteins. Phosphorylation of a limited number of sites on tau reduces its binding affinity for microtubules, resulting in their destabihzation.

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Fig. 5. Model of a nervecell from an AD patient.The intracellular locationsof the neurofibrillafytangles(composedof paired-helicalfilaments of hyperphosphorylatedtau) and the extracellular location of the amyloid plaques(composedof the A~ peptide) are compared. ApoE has been identified in both locations, Isoform-specific interactions of apoE3 and apoE4 with AI3 peptide in vitro The interactions of apoE3 and apoE4 with the A[~ peptide and tau have been examined in an effort to identify isoform-specific effects that could provide biochemical correlations with the genetic evidence linking apoE4 to Alzheimer's disease. It was first demonstrated that apoE binds A~ peptide with high attinity [4",44]. This led to the observation that when apoE4 and apoE3 are incubated with the A[~ peptide, and then heated in a sodium dodecyl sulfate (SDS) buffer and electrophoresed, an SDS-stable complex is formed [46°]. The most interesting observations have been that apoE4 is more effective than apoE3 in the formation of the complex, and that the pH range of the interaction is different for the two isoforms [46°]. This isoform-specific interaction with the A~ peptide provides the first functional correlation with the genetic evidence. It has also been demonstrated that residues 244-272 in the carboxyterminal domain of apoE are required for A[3 peptide binding [46°]. Thus, the interaction of apoE with the peptide appears to be another example of domain interaction, similar to the isoform-specific lipoprotein preferences discussed above, in which an amino acid substitution in the amino-terminal domain, distinguishing apoE3 from apoE4, influences the binding properties of the carboxy-tenninal domain.

strands [47]. Inclusion of apoE in the A~ peptide incubation mixture results in the production of monofibrils -7 nm in diameter. The A~ peptide forms a denser, more extensive matrix with apoE4 than with apoE3 [47]. Furthermore, the monofilaments appear earlier with apoE4 than with apoE3. In addition, incubations with apoE4 result exclusively in monofibrils, whereas those with apoE3 result in some double- and triple-stranded ribbons, characteristic of the A[~-only incubations. With both isoforms, immunogold labeling revealed apoE to be distributed along the entire length of the monofilaments [47]. These results demonstrate that both isoforms interact with the A~ peptide to form novel monofibrillar structures, with apoE4 more effective. It is reasonable to speculate, therefore, that A[3 peptide produced by neurons or other cells may form monofibrils more readily in the presence of apoE4. ApoE is abundant in the brain, being synthesized and secreted by astrocytes and some microglia. Fibrils in the amyloid plaques have similar dimensions to monofilaments formed in vitro, suggesting that these monofilaments may represent a relevant in vitro model of the amyloid component of plaques.

With longer incubation times, the SDS-stable complexes of both apoE3 and apoE4 form very high molecular weight complexes that precipitate from solution. These aggregates have been examined by negative staining electron microscopy and compared with aggregates of the A~ peptide, which, when incubated alone, had been shown to form twisted ribbons containing two or more

The interaction of apoE with the tau protein also is isoform-specific. ApoE3 forms an SDS-stable complex with tau, whereas apoE4 forms little, if any, complex [48"]. The molecular weight of the apoE3-tau complex is consistent with a 1:I adduct. Phosphorylation of tau with a crude brain extract eliminates its ability to form a complex with apoE3 [48°]. The amino-ter-

isoform-specific interaction of apoE3 and apoE4 with tau in vitro

Apolipoprotein E Weisgraber, Pitas and Mahley minal domain of apoE (residues 1-191) binds to tau [48"[, whereas with the AI3 peptide the carboxy-terminal domain of apoE appears to contain the binding site. What is the potential significance of apoE3 binding to tau? It could stabilize microtubules and the cytoskeleton, and perhaps help maintain the structure and function of neurons. The binding of apoE3 to tau could inhibit phosphorylation of tau and thus retard the paired-helical filament formation that appears to be involved in the development of neurofibrillary tangles. These possibilities have led to the hypothesis that apoE4 is associated with AD, because it does not have the protective features of apoE3 [48"]; that is, the protection of tau from hyperphosphorylation, and the stabilization of its interactions with microtubules (thought to arise from apoE3) would be lost with apoE4. It is important to note that in order for apoE3 to modulate tau metabolism, it must act within the cytoplasm. Although conclusive evidence that apoE actually exists in the cytoplasm of nerve cells is lacking, imnmnochemical studies suggest that apoE occurs in the cytoplasm of hepatocytes [49] and in the cytoplasm of muscle cells from people with inclusionbody myositis [50]. Interestingly, this neuromuscular disorder has several features in common with AD, including the presence of amyloid deposits and paired-helical filaments, and apoE localization. However, it remains to be proven whether apoE exists in the cytoplasm of nerves, and if it does, how it comes to be there. Presumably, apoE entering neurons via the LDL receptor or LRP would have to escape the endosomal pathway in order to be available for interaction with cytoskeletal elements within the cell.

Conclusion Apolipoprotein E, a critical protein involved in plasma lipoprotein metabolism and cholesterol transport, appears to play an important part in nerve regeneration and neurite extension. Specifically, apoE3, the most common isoform of apoE, supports neurite extension in the presence of a source of cholesterol, whereas apoE4 does not. This suggests that, depending on the isoform, apoE modulates the cytoskeletal activity of neurons either to facilitate or to retard neurite extension. Clearly, there are also isoform-specific differences between apoE3 and apoE4 that may help to clarify the association of apoE4 with AD. The A[~ peptide forms a complex with apoE4 more readily and effectively than with apoE3, as assayed on SDS gels and as observed by negative staining electron microscopy. ApoE is a major component of the anwloid plaques found in the brains of Alzheimer's patients. It may be involved in the pathogenesis of AD by affecting cytoskeletal elements of neurons. ApoE3 readily interacts with tau, a microtubule-associated protein that stabi-

lizes cytoskeletal compounds of a cell, including microtubules, and may prevent hyperphosphorylation of tau. Hyperphosphorylated tau constitutes the neurofibrillary tangles seen in the neurons of Alzheimer's patients. ApoE4 does not bind tau; therefore, the presence of apoE4 could lead to microtubule instability and tangle formation. These are intriguing and thought-provoking observations relating apoE to normal nervous system physiology and to the pathogenesis of AD. However, additional studies are required to identify the precise mechanism(s) whereby this protein is involved in normal and abnormal pathophysiology within the nervous system.

Acknowledgements This work is supported in part by the National Heart, Lung, and Blood Institute Program Project Grant HL 41636, and by the L K Whittier Foundation. The authors thank K Humphrey for manuscript preparation, A Corder and L Jach for graphics, and L DeSimone and D Levy for editorial assistance.

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2.

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3.

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4. •

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Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, Pericak-Vance MA: Gene Dose of Apolipoprotein E Type 4 Allele and the Risk of Alzheimer's Disease in Late Onset Families. Science 1993, 261:921-923.

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18.

Weisgraber KH, Rail SC Jr, MoNey RW: Human E Apoprotein Heterogeneity. Cysteine-Arginine Interchanges in the Amino Acid Sequence of the Apo-E Isoforms. J Biol Chem 1981, 256:9077-9083.

Boyles JK, Notterpek LM, Anderson LJ: Accumulation of Apolipoproteins in the Regenerating and Remyelinating Mammalian Peripheral Nerve. Identification of Apolipoprotein D, Apolipoprotein A-IV, Apolipoproteln E, and Apolipoprotein A-I. J Biol Chem 1990, 265:17805-17815.

35.

19.

Rail SC Jr, Weisgraber KH, Mahley RW: Human Apolipoprotein E. The Complete Amino Acid Sequence. J Biol Chem 1982, 257:4171-4178.

Handelmann GE, Boyles JK, Weisgraber KH, Mahley RW, Pitas RE: Effects of Apolipoprotein E, 13-Very Low Density Lipoproteins, and Cholesterol on the Extension of Neurites by Rabbit Dorsal Root Ganglion Neurons in Vitro. J Lipid Res 1992, 33:1677-1688.

20.

Wilson C, Wardell MR, Weisgraber KH, Mahley RW, Agard DA: Three-Dimensional Structure of the LDL Receptor-Binding Domain of Human Apolipoprotein E. Science 1991, 252:1817-1822.

21.

Gregg RE, Zech LA, Schaefer FJ, Stark D, Wilson D, Brewer HB Jr: Abnormal in Vivo Metabolism of Apolipoprotein E4 in Humans. J Clin Invest 1986, 78:815-821.

22.

23.

Steinmetz A, Barbaras R, Ghalim N, Clayey V, Fruchart FC, Ailhaud G: Human Apolipoprotein A-W Binds to Apollpoprotein A-I/A-II Receptor Sites and Promotes Cholesterol Efflux from Adipose Cells. J Biol Chem 1990, 265:7859-7863.

36.

Nathan BP, Bellosta S, Sanan DA, Weisgraber KH, MoNey RW, Pitas RE: Differential Effects of Apolipoprotelns E3 and E4 on Neuronal Growth in Vitro. Science 1994, 264:850-852. Establishesthat human apoE3 and apoE4 possess isoform-specific effects on neuronal growth. Evidence indicates that these isoform-specific effects involve binding and/or internalization of apoE-containing lipoproteins, suggesting an interaction with one or more cellular proteins. 37.

Weisgraber KH, Innerarity TL, Harder KJ, Mahley RW, Milne RW, Marcel YL, Sparrow JT: The Receptor-Binding Domain of Human Apolipoprotein E. Monoclonal Antibody Inhibition of Binding. J Biol Chem 1983, 258:12348-12354.

38.

Weisgraber KH, Innerarity TL, Mahley RW: Role of the Lysine Residues of Plasma Lipoproteins in High Affinity Binding to Cell Surface Receptors on Human Fibroblasts. J Biol Chem 1978, 253:9053-9062.

39.

McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM: Clinical Diagnosis of Alzheimer's Disease: Report of the NINCDS-ADRDA Work Group under the Auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 1984, 34:939-944.

40.

RosesAD: The Alzheimer Diseases. Curt Neurol 1994, 40:in press.

41.

Seubert P, Vigo-Pelfrey C, Esch F, Lee M, Dovey H, Davis D, Sinha S, Schlossmacher M, Whaley J, Swindlehurst C, et al. : Isolation and Quantification of Soluble Alzheimer's ~-Peptide from Biological Fluids. Nature 1992, 359:325-327.

Davignon J, Gregg RE, Sing CF: Apolipoprotein E Polymorphism and Atherosclerosis. Arteriosclerosis 1988, 8:1-21.

24 •

Dong L-M, Wilson C, Wardell MR, Simmons T, Mahley RW, Weisgraber KH, Agard DA: Human Apolipoprotein E: The Role of Arginine-61 in Mediating the Lipoprotein Preferences of the E3 and E4 Isoforms. J Biol Chem 1994, in press. identifies that an important structural difference between human apoE3 and apoE4 is a conformation of the arginine-61 side chain. This difference is determined to be a major feature of the apoE domain interaction with respect to apoE3 and apoE4 lipoprotein preferences. 25.

Elshourbagy NA, Liao WS, Mahley RW, Taylor JM: Apolipoprotein E mRNA Is Abundant in the Brain and Adrenals, as Well as in the Liver, and Is Present in Other Peripheral Tis-

A p o l i p o p r o t e i n E Weisgraber, Pitas and M a h l e y 42.

HaassC, Selkoe DJ: Cellular Processingof ~Amyloid Precursor Protein and the Genesis of Amyloid [3-Peptide. Cell 1993, 75:1039-1042.

43.

Namba Y, Tomonaga M, Kawasaki H, Otomo E, Ikeda K: Apolipoprotein E Immunoreactivity in Cerebral Amyloid Deposits and Neurofibrillary Tangles in Alzheimer's Disease and Kuru Plaque Amyloid in Creutzfeldt-Jakob Disease. Brain Res 1991, 541:163-166.

44.

Wisniewski T, Frangione B: Apolipoprolein E: A Pathological Chaperone Protein in Patients with Cerebral and Systemic Amyloid. Neurosci Lett 1992, 135:235-238.

45.

Crowther RA: Tau Protein and Paired Helical Filaments of Alzheimer's Disease. Curt Opin Struct giol 1993, 3:202-206.

46. "

Strittmatter WJ, Weisgraber KH, Huang DY, Dong L-M, Salvesen GS, Pericak-Vance M, Schmechel D, Saunders AM, Goldgaber D, Roses AD: Binding of Human Apolipoprotein E to Synthetic Amyloid [3 Peptlde: Isoform-specific Effects and Implications for Late-onset Alzheimer Disease. Proc Nat/ Acad Sci USA 1993, 90:8098-8102. Report demonstrating an isoform-specific binding of apoE to the A[3 peptide. ApoE4 was demonstrated to bind more effectively to the peptide than apoE3. This was the first demonstration of a biochemical correlation with the genetic evidence that the apoE4 allele is a major risk factor for AD. 47.

Sanan DA, Weisgraber KH, Mahley RW, Huang D, Saunders A, Schmechel D, Wisniewski T, Frangione B, Roses AD, Strittmatter WJ: Apolipoprolein E Associates with [3 Amyloid Peptide of Alzheimer's Disease to Form Novel Monofibrils: Isoform

ApoE4 Associates More Efficiently Than ApoE3. J Clin Invest 1994, in press. 48. •

Strittmatter W/, Weisgraber KH, Goedert M, Saunders AM, Huang D, Corder EH, Dong L-M, lakes R, Alberts MI, Gilbert JR, et al.: Hypothesis: Microtubule Instability and Paired Helical Filament Formation in the Alzheimer Disease Brain Are Related to Apolipoprotein E Genotype. E×p Heurol 1994, 125:163-171. Based on an isoform-specific binding of apoE3 to tau, a major component of neurofibrillary tangles, it was suggested that apoE3 may be protective against Alzheimer's disease by modulating tau hyperphosphorylation. 49.

Hamilton RL, Wong lS, Guo LSS, Krisans S, Havel RJ: Apolipoprotein E Localization in Rat Hepatocytes by Immunogold Labeling of Cryothln Sections. J Lipid Res 1990, 31:1589-1603.

50.

AskanasV, Mirabella M, Engel WK, Alvarez RB, Weisgraber KH: Apolipoprotein E Immunoreactive Deposits in Inclusion-Body Muscle Diseases. Lancet 1994, 343:364-365.

KH Weisgraber and RE Pitas, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, California 941419100, USA, and Department of Pathology, University of California, San Francisco, California 94143, U S A. R.W Mahley, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, California 94141-9100, U SA, and Departments of Medicine and Pathology, University of California, San Francisco, California 94143, U S A.

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