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. Author manuscript; available in PMC: 2025 Apr 3.
Published in final edited form as: Neuron. 2024 Jan 23;112(7):1100–1109.e5. doi: 10.1016/j.neuron.2023.12.018

Apolipoprotein E Secreted by Astrocytes Forms Antiparallel Dimers in Discoidal Lipoproteins

Michael R Strickland 1, Michael Rau 2, Brock Summers 2, Katherine Basore 2, John Wulf II 2, Hong Jiang 1, Yun Chen 1,3, Jason D Ulrich 1,4,5, Gwendalyn J Randolph 3, Rui Zhang 6, James AJ Fitzpatrick 2, Anil G Cashikar 4,7,8, David M Holtzman 1,4,5,9
PMCID: PMC10994765  NIHMSID: NIHMS1960906  PMID: 38266643

Summary:

The Apolipoprotein E gene (APOE) is of great interest due to its role as a risk factor for late onset Alzheimer’s Disease. ApoE is secreted by astrocytes in the central nervous system in HDL-like lipoproteins. Structural models of lipidated ApoE of high resolution could aid in a mechanistic understanding of how ApoE functions in health and disease. Using monoclonal Fab and F(ab’)2 fragments, we characterized the structure of lipidated ApoE on astrocyte-secreted lipoproteins. Our results provide support for the “double-belt” model of ApoE in nascent discoidal HDL-like lipoproteins where two ApoE proteins wrap around the nanodisc in an antiparallel conformation. We further show that lipidated, recombinant ApoE accurately models astrocyte-secreted ApoE lipoproteins. Cryogenic electron microscopy of recombinant lipidated ApoE further supports ApoE adopting antiparallel dimers in nascent discoidal lipoproteins.

Keywords: Apolipoprotein E, ApoE, Lipoprotein, HDL, Glia, Astrocytes, CryoEM, Alzheimer’s Disease

eTOC Blurb:

Apolipoprotein E (ApoE) is the largest genetic risk factor for late onset Alzheimer’s disease. Utilizing cryoEM, Strickland et. al. demonstrate important insights into the structure of lipidated ApoE. ApoE was found to adopt an antiparallel dimer in discoidal lipoproteins secreted by astrocytes and in artificially lipidated recombinant ApoE.

Introduction:

Apolipoprotein E (ApoE) plays an important role in plasma cholesterol and lipid metabolism and is a component of very low density lipoproteins (VLDL), chylomicron remnants, and certain subclasses of high density lipoprotein (HDL) particles 1–3. In the central nervous system (CNS), ApoE is present in HDL-like lipoproteins and is the most abundant lipoprotein4–7. Astrocytes are the main cellular source of ApoE in the CNS6,8–11, and they secrete ApoE-containing lipoproteins that are discoidal and HDL-like in size (~8-20nm in diameter)9,10,12. Astrocyte-secreted lipoproteins are rich in unesterified cholesterol and phospholipids10,12 Once secreted from astrocytes, ApoE-containing lipoproteins undergo a maturation process where they become spherical and can associate with other apolipoproteins9. In addition to astrocytes, microglia strongly upregulate ApoE expression under disease conditions13–15 and secrete ApoE-containing lipoproteins16. ApoE shuttles lipids and cholesterol through binding receptors in the low density lipoprotein receptor (LDLR) superfamily5,6. LDLR and LDLR-related protein 1 (LRP1) regulate ApoE uptake and cholesterol metabolism, with LDLR and LRP1 expressed on glial and neuronal cells17–19.

In humans, the APOE gene has three major isoforms: APOE2, APOE3, and APOE4. APOE3 encodes for the most common ApoE protein isoform and is defined by two single nucleotide polymorphisms leading to a cysteine at position 112 (Cys112) and arginine at position 158 (Arg158). APOE4 differs from APOE3 coding for an arginine at position 112 (Arg112), while APOE2 differs from APOE3, coding for a cysteine at position 158 (Cys158). APOE2 decreases the risk for late onset Alzheimer’s Disease (AD)20–24, but is a risk factor for type III hyperlipidemia25. APOE4 is the strongest risk factor for late onset AD with one copy of the allele increasing disease risk ~4-fold and two copies by ~12-fold3,17,26.

APOE4, in a dose-dependent fashion, is known to contribute to an earlier onset of amyloid-β (Aβ) deposition in humans27,28 and in mouse models of Aβ amyloidosis29–31. APOE3 expressing mice have intermediate levels of fibrillar Aβ pathology in these mouse models and APOE2 expressing mice have the least32,33. Apoe−/− mice have little to no fibrillar Aβ pathology34,35. In the context of tau pathology, ApoE has a dramatic impact on tau-mediated neurodegeneration. APOE4 knock-in mice crossed to PS19 mice (a mouse model of tauopathy) have greater neurodegeneration than APOE3-PS19 and APOE2-PS19 mice36. Apoe−/−-PS19 mice are protected from neurodegeneration36.

The molecular basis underlying the altered risk for AD due to different APOE genotypes is not well understood. Early studies used X-ray crystallography to characterize the structure of the non-lipidated N-terminal domain of ApoE37–39. These studies revealed that the non-lipidated N-terminus of ApoE adopted an extended 4-helix bundle. These structures led to the hypothesis that salt bridge rearrangements present in ApoE2 caused a reduction in the positive charge of the LDLR-binding region in ApoE, reducing the affinity of ApoE2 for its receptor38,40. An important limitation of these studies in interpreting the molecular mechanisms underlying disease risk, is that ApoE is lipidated under physiological conditions3,26. ApoE must be lipidated in order to bind LDLR41. Also, it is known that lipidation of ApoE dramatically alters the conformation of ApoE42–44. Multiple studies have shown that upon lipid binding there is an opening of the 4-helix bundle and an extension of the N-terminal domain of ApoE45–49. This was supported by x-ray crystallography of lipidated ApoE that yielded a low resolution structure suggesting the presence of two ApoE on an ellipsoidal lipoprotein in a “horseshoe” conformation47,50. The lack of information about the lipidated structure of ApoE has limited investigation of the mechanisms underlying the difference in ApoE4 structure, its interaction with other molecules, and contribution to disease pathogenesis.

In this study, we utilized cryogenic electron microscopy (cryoEM) to investigate the structure of lipidated ApoE. CryoEM is an ideal method to study lipidated proteins as this method does not require the crystallization of proteins and is compatible with a wide range of physiological buffers. CryoEM has been widely used to study transmembrane and lipidated proteins in a near native environment51,52. Advancements in direct electron detectors has allowed cryoEM to solve protein structures to atomic resolution, with structures regularly achieving <3Å resolution53–58. Using cryoEM, we have gained new insights into the structure of lipidated ApoE in HDL-like lipoproteins, including those secreted by astrocytes.

Results:

ApoE is an Antiparallel Dimer in Astrocyte Secreted Lipoproteins

Primary astrocyte cultures were obtained from human APOE knock-in mice expressing human APOE2, APOE3, or APOE416. Astrocyte ApoE lipoprotein were immunopurified from astrocyte conditioned media (Figure S1A and S1B). Astrocyte-secreted ApoE lipoproteins form discoidal HDL-like particles and ranged in size from ~8-20 nm (Figure S1B and Figure S3B). Size exclusion chromatography showed a broad peak representing larger ApoE lipoproteins with a second peak representing the majority of ApoE lipoproteins (Figure S1C). Negative stain transmission electron microscopic (TEM) micrographs confirmed the isolation of discoidal ApoE lipoproteins (Figure S1D and S1E). Monoclonal antibody fragments derived from anti-ApoE monoclonal antibodies were used to characterize astrocyte-secreted ApoE lipoproteins. Monoclonal Fab fragments have been previously used to assist with alignment of small particles by cryoEM59. Multiple Fab fragments were generated and tested for binding to lipidated, recombinant ApoE (Figure S2). The combination of antibodies HJ15.1060 and HJ15.3060 were chosen as they gave clear 2D classes (Figure S2A). Epitope mapping showed that HJ15.10 bound ApoE between aa 140-160 and that HJ15.30 bound between aa 40-60 (Figure S2B). TEM imaging of the Fab fragment of HJ15.10 bound to astrocyte ApoE lipoproteins revealed the presence of two ApoE proteins per lipoprotein particle (Figure 1A). The receptor binding domain within ApoE overlaps with the epitope recognized by the HJ15.10 Fab fragment. The 2D classes show that the relative orientation between the two LDLR binding domains ranges from an angle of ~60° to ~170°, with most falling within an angle of 100° (25th percentile) to 170° (75th percentile). To fix the relative orientation of the two ApoE proteins, F(ab’)2 fragments were generated from HJ15.30, which binds the N-terminus of ApoE. Use of the F(ab’)2 fragment of HJ15.30 allowed for the simultaneous binding of the two ApoE proteins on the nanodisc (Figure 1B). The F(ab’)2 fragment contains two Fab domains in an antiparallel orientation with the central heavy chains bound by disulfide bonds at the hinge region61. The binding of the F(ab’)2 fragment supports a model wherein the two ApoE proteins on the nanodisc are also arranged in an antiparallel orientation. Negative stain TEM imaging of astrocyte-secreted ApoE2, ApoE3, and ApoE4 lipoproteins illustrated that astrocyte-secreted ApoE adopts an antiparallel orientation on discoidal lipoproteins (Figure 1B). Thus, adoption of an antiparallel orientation of ApoE on astrocyte-secreted, HDL-like lipoproteins is a general property of ApoE and is not altered by ApoE isoform.

Figure 1: ApoE Forms Antiparallel Dimers in Discoidal Astrocyte Secreted ApoE Lipoproteins.

Figure 1:

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A. Negative stain TEM lowpass filtered images and 2D class averages of anti-ApoE HJ15.10 Fab fragment bound to astrocyte-secreted ApoE lipoprotein. 2D classes show the presence of two ApoE antibody Fab fragments bound to discoidal ApoE lipoprotein.

B. Negative stain TEM lowpass filtered images and 2D class averages of anti-ApoE HJ15.30 F(ab’)2 fragment bound to astrocyte-secreted ApoE lipoprotein. 2D classes show the presence of two ApoE bound to discoidal ApoE lipoprotein. Binding of the F(ab’)2 fragment suggests that ApoE adopts an antiparallel orientation on astrocyte ApoE lipoprotein.

Lipidated, Recombinant ApoE Lipoproteins Resemble Astrocyte Secreted ApoE Lipoproteins

To study the structure of lipidated ApoE in a controlled system, astrocyte lipoproteins were modeled using recombinant ApoE and artificial lipids. This allowed for direct control of the lipid properties and molar ratio of protein to lipid. To model astrocyte ApoE lipoproteins, recombinant ApoE4 (rApoE4) was mixed with different molar ratios of the lipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), based on previous literature62–64. Blue native PAGE analysis of the assembled lipoproteins showed that DMPC at a molar ratio of 1:110 resulted in homogenous, discoidal particles of similar size to astrocyte-secreted lipoproteins (Figure S3A,S3B, and S3C). The molar ratio and lipid type drive lipoprotein particle size. DMPC tends to form lipoproteins ~12.5nm in diameter, DPPC tends to form lipoproteins ~9.7nm in diameter, and POPC tends to form lipoproteins ~20.3nm in diameter (Figure S3A). To assess the homogeneity of artificially lipidated ApoE lipoproteins, samples were assessed by negative stain TEM. To identify ApoE on lipoproteins, a monoclonal Fab fragment (HJ15.10) was incubated with ApoE lipoproteins (Figure S3C). TEM imaging revealed that discoidal particles contained two ApoE proteins per lipoprotein, whereas some large particles, such as ~20.3nm diameter particles formed by POPC, contained two or more ApoE proteins (Figure S3C). This suggests that a minimum of two ApoE cooperate to bind lipid and form discoidal lipoproteins. This result is in agreement with previous publications showing that the addition of ApoE to lipids results in discoidal lipoproteins with at least two ApoE proteins45,62,63. As DMPC at a molar ratio of 1:110 reproducibly formed homogenous lipoproteins similar in size to astrocyte-secreted lipoproteins (~12.5nm, Figure S3B), this condition was chosen for further study by cryoEM.

Cryogenic Electron Microscopy of Recombinant ApoE Lipoproteins

Lipidated rApoE4 samples bound by HJ15.10 were imaged by cryoEM. 2D classes of the particles revealed two ApoE proteins bound to the nanodisc (Figure 2A). Side views of the nanodisc show the HJ15.10 Fab fragment bound to the “top” and “bottom” of the nanodisc. This result suggests that one ApoE protein is on the “top” of the nanodisc while the other ApoE protein is on the “bottom” of the nanodisc. This orientation supports a model of ApoE where ApoE exists as an extended α-helical chain with the hydrophobic face of the α-helix interacting with the nonpolar fatty acid chains of the lipids and the polar face being solvent exposed65. However, resolution was limited due to the relative orientation of the two ApoE proteins to each other. To limit the relative orientation between the two ApoE proteins, F(ab’)2 fragments were generated from HJ15.30. The binding of the F(ab’)2 to the two proteins on the nanodisc suggest that ApoE is in an antiparallel orientation (Figure 2B). The resolution was resolved to 8.73Å (Figure S5A) which allowed us to see two protein densities in contact with the F(ab’)2 fragment and circling the nanodisc (Figure 3A, cyan arrows).

Figure 2: Lipidated Recombinant ApoE Forms Antiparallel Dimers in Discoidal Lipoproteins.

Figure 2:

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A. 2D classes of rApoE4-DMPC lipoprotein bound by anti-ApoE HJ15.10 Fab fragment. Micrographs represent cryoEM images taken on Titan Krios operating at 300kV at 59,000x magnification with volta phase plate (1.081Å/pixel). Side views show lipid bilayer consistent with ApoE forming discoidal nanodiscs. Side views also show monoclonal Fab fragment binding to the “top” and “bottom” of the nanodisc.

B. 2D classes of rApoE4-DMPC lipoprotein bound by anti-ApoE HJ15.30 F(ab’)2 fragment. Micrographs represent cryoEM images taken on Titan Krios operating at 300kV at 75,000x magnification with volta phase plate (0.928Å/pixel). Binding of the F(ab’)2 fragment suggests that ApoE adopts an antiparallel orientation consistent with the “double belt” model.

C. 2D classes of gradient fixed rApoE4-DMPC lipoprotein bound by anti-ApoE HJ15.30 F(ab’)2 fragment. Micrographs represent cryoEM images taken on Glacios operating at 200kV at 150,000x magnification (0.928Å/pixel).

D. Representative 2D classes from negative stain TEM imaging of rApoE2-DMPC, rApoE3-DMPC, rApoE4-DMPC bound by anti-ApoE HJ15.30 F(ab’)2 fragment. These data show that ApoE adopts an antiparallel conformation on discoidal HDL-like lipoprotein regardless of ApoE isoform.

E. 2D classes from negative stain TEM micrographs of rApoE4-DMPC bound simultaneously by anti-ApoE antibody HJI 5.10 monoclonal Fab fragment and anti-ApoE antibody HJ15.30 monoclonal F(ab’)2 fragment.

Figure 3: CryoEM Density Maps of Lipidated ApoE.

Figure 3:

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A. 3D cryoEM density map of lipidated rApoE4-DMPC bound by anti-ApoE HJ15.30 F(ab’)2 fragment. CryoEM density map shows binding of the F(ab’)2 fragment to the “top” and “bottom” of the nanodisc. Strong electron density is observed on the side of the nanodisc consistent with two α-helical chains wrapping around the nanodisc (cyan arrows). Strong electron density is also observed opposite of the F(ab’)2 binding site where the lipoprotein is slightly “pointed” (fuchsia arrow). Micrographs used were collected on a Titan Krios operating at 300kV at 75,000x magnification (0.928Å/pixel).

B. 3D cryoEM density map of lipidated rApoE4-DMPC bound by anti-ApoE antibody HJ15.30 derived monoclonal F(ab’)2 fragment. Higher resolution of the cryoEM density map allows for better resolution of the electron density of two α-helical chains wrapping around the nanodisc (cyan arrows). Consistent with the lower resolution model, strong electron density is also observed opposite of the F(ab’)2 binding site where the lipoprotein is slightly “pointed” (fuchsia arrow). Micrographs used were collected on a Glacios operating at 200kV at 150,000x magnification (0.928Å/pixel).

C. Cartoon model showing two ApoE wrapping around a nanodisc showing the “double belt” model of lipidated ApoE. Helical domains are represented by cylinders. Epitope of anti-ApoE antibody HJ15.30 F(ab’)2 fragment is represented as a red cylinder. Created with BioRender.com

To further improve resolution, the rApoE4-DMPC-HJ15.30 F(ab’)2 complex was subjected to gradient fixation (Figure S4A and S4B)66,67. This allowed us to achieve a higher resolution of lipidated ApoE4. 2D classification of gradient fixed rApoE4-DMPC-HJ15.30 F(ab’)2 confirmed the presence of two ApoE in an antiparallel orientation (Figure 2C). Using gradient fixation in combination with the HJ15.30 F(ab’)2 fragment allowed us to achieve a nominal resolution of 7.71Å (Figure S5B). The cryoEM density map showed the F(ab’)2 binding to the “top” and “bottom” of the nanodisc (Figure 3B, cyan arrows). Increased electron density is observed opposite of the F(ab’)2 binding site potentially representing a “kink” in the α-helical chain reminiscent to that observed in ApoA168. (Figure 3A and 3B, fuchsia arrow). Overall, these data suggest that ApoE adopts an antiparallel extended α-helical “double-belt” conformation in discoidal lipoprotein (Figure 3C).

Negative stain TEM imaging of rApoE4-DMPC-HJ15.30 F(ab’)2 with additional HJ15.10 Fab fragments bound further supports this model (Figure 2E). ApoE2, ApoE3, and ApoE4 all adopted an antiparallel orientation with two ApoE proteins present in discoidal lipoproteins as visualized by negative stain TEM imaging (Figure 2E). Additionally, TEM imaging of discoidal rApoE-DPPC lipoproteins (Figure S6A) with HJ15.10 Fab (Figure S6B) and HJ15.30 F(ab’)2 (Figure S6C) revealed that ApoE also adopted an antiparallel dimer conformation. This suggests that the conformation adopted by ApoE on discoidal DMPC is not driven by the lipid composition, but by the discoidal nature of the lipoproteins. The cryoEM density maps of rApoE4-DMPC represent the highest resolution to date of lipidated ApoE and suggests a radically different conformation of lipidated ApoE compared to its non-lipidated form.

Discussion:

ApoE is an important protein due to its role in cholesterol and lipid transport as well as its importance as a risk factor for several prominent diseases. It is the strongest genetic risk factor for AD, and it is also a risk factor for macular degeneration and atherosclerosis1,69,70. Due to the importance of ApoE in AD pathogenesis, it is critical to decipher its molecular structure in its native form. We demonstrate that astrocyte-secreted ApoE-containing lipoproteins contain two ApoE proteins that are in an anti-parallel conformation that appears similar to the model proposed for ApoA-I. These findings set the stage for obtaining a high-resolution structural model of lipidated ApoE that will be key in understanding how ApoE interacts with other proteins and lipids as well as understanding the isoform-specific properties of ApoE that greatly influence risk for AD and other diseases.

In this study, we chose to investigate the structure of astrocyte-secreted ApoE lipoprotein as astrocytes secrete the majority of ApoE in the CNS6,9,10,12,71. Characterization of the lipidated ApoE isoforms by native gel analysis shows that ApoE2, ApoE3, and ApoE4 form lipidated ApoE lipoprotein with sizes ranging from ~8nm to 20nm in diameter. We observe that ApoE2 lipoproteins tends to be somewhat larger than ApoE3 which is somewhat larger than ApoE4, with ApoE4 forming the smallest lipoproteins. This is consistent with previous reports from our lab that have characterized astrocyte-secreted ApoE lipoproteins and describe discoidal particles10,12,71.

Previous research on the stoichiometry of ApoE has found that lipoproteins can contain two to four ApoE proteins depending on the size of the lipoprotein72. It was estimated that lipoproteins 13.4nm in diameter contains approximately two ApoE proteins and lipoproteins approximately 21.2nm in diameter contain four ApoE proteins. This is consistent with the findings that we directly demonstrate in this study in which DMPC lipoproteins at a molar ratio of 1:110 ApoE to DMPC form particles approximately 12.5nm in diameter and contain two ApoE proteins. Similarly, previous reports found that the diameter of lipoprotein particles were dependent on both the molar ratio of apolipoprotein to lipid, but also the number of α-helical amphipathic helical segments in each apolipoprotein73. Thus, the size of the lipid-containing particle is constrained by the stoichiometry of protein to lipid, but also by the number of α-helical segments that each apolipoprotein contains. Another consideration for future research is conformation of spherical ApoE lipoprotein. ApoE, and other apolipoproteins like ApoA-I, are initially secreted as discoidal particles. After cholesterol esterification by LCAT, ApoE containing lipoprotein are converted to spherical particles73. As cholesterol is esterified and moves into the core of the lipoprotein particle, this leads to a rearrangement of the nanodisc into a spherical particle. During this process, the hydrophobic tails of the phospholipid are brought into the interior of the lipoprotein. ApoE no longer shields the hydrophobic portion of the phospholipid from solvent, this potentially leads to the self-association of the hydrophobic domains of ApoE leading to the presence of a toroidal horseshoe conformation of ApoE on spherical lipoprotein. Evidence of this was provided by X-ray crystallography of ApoE lipoproteins in an “ellipsoidal” conformation47,50,62. Further research is necessary to understand the conformational transition that occurs as discoidal lipoprotein transitions to spherical lipoproteins.

Previous characterization of the structure of ApoE was focused on the nonlipidated structure of the N-terminal domain of ApoE due to the difficulties in studying lipidated protein by X-ray crystallography37 and the propensity of full length ApoE to aggregate. Crystallization of the ApoE2 N-terminal domain suggested a plausible mechanism for ApoE2’s dramatically reduced binding affinity. The authors found that mutation of Arg158 to Cys158 resulted in salt bridge rearrangements that reduced the positive charge of the ApoE receptor binding region38,40. In support of this, mutation of Asp154 to Ala154 restored ApoE2 receptor binding38,40. Additionally, treatment of ApoE2 with cysteamine to add a positive charge to Cys158 also resulted in restored ApoE2 binding to LDLR74. However, the mechanism of ApoE receptor binding is still unclear as binding of ApoE to LDLR is dependent on lipidation of ApoE41. This study suggests that once ApoE binds lipid, it adopts an extended α-helical, antiparallel conformation on lipoproteins consistent with a “double belt” model. This finding is consistent with previous reports that upon lipidation the four-helix bundle present in nonlipidated ApoE opens, and the hydrophobic portions of the helices associate with lipid. Ellipsoidal DPPC particles are predicted to form a “horseshoe” conformation where the C-terminal domain associates with the N-terminal domain and has reduced binding to the LDLR receptor47. However, the fully extended α-helical conformation proposed by our model would be predicted to have higher binding to LDLR. This is further supported by evidence of large DMPC lipoproteins (containing four ApoE compared to smaller ApoE lipoproteins containing two ApoE) showing a higher binding affinity to LDLR than ellipsoidal DPPC particles47.

Lipidated ApoA-I has been reported to adopt a similar conformation where two ApoA-I proteins wrap around a nanodisc in an antiparallel orientation75. The two proteins in ApoA-I lipoprotein adopt preferential orientations to each other, called registers. ApoA-I preferentially adopts the L5/L5 registry, but is also able to adopt the L4/L5 registry76. Different registries of ApoA-I are known to differentially impact LCAT esterification activity77,78. This leads to the hypothesis that different registries of apolipoproteins can differentially interact with receptors and enzymes throughout the maturation process. In the case of ApoE, our study suggests that ApoE can adopt multiple registries in the lipidated state as evidenced by the different orientations of HJ15.10 Fab bound to ApoE lipoprotein. Of particular interest is how differences in ApoE isoform could impact registry preference and alter the receptor binding properties of ApoE. Previous reports have shown that the disulfide-linked, ApoE3 homodimer has reduced binding to LDLR79. A relatively unexplored area of research is how disulfide linkage of ApoE with another molecule of ApoE or with other apolipoproteins, such as ApoA-II, could alter the binding properties of ApoE.79,80

This study provides a promising methodology for investigating the structure of lipidated apolipoproteins. Future work will aim to achieve a high-resolution model of lipidated ApoE to better characterize isoform specific differences to better understand differences in receptor binding, as well as interactions with other molecules such as heparan sulfate proteoglycans. The complex structure of ApoE with its receptors, specifically LDLR, will also provide better understanding of how the lipidation of ApoE alters its conformation to promote receptor binding. Another important consideration for future studies will be to investigate how different rotamers of ApoE impact ApoE receptor binding properties, lipid content, as well as binding to other molecules believed to be key to disease pathogenesis such as Aβ.

STAR Methods:

Resource Availability:

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. David Holtzman (holtzman@wustl.edu).

Materials Availability

This study did not generate new unique reagents

Data and Code Availability

  • CryoEM electron density maps are deposited in EMDB (EMD-41830 and EMD-41831) and are publicly available at time of publication. Accession numbers are listed in the key resources table.

  • This paper does not report original code.

  • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Mouse monocolonal anti-apoE Antibody (HJ15.3) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.4) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.5) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.6) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.7) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.10) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.12) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.30) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.32) Liao et. al., 201560 N/A
Chemicals, peptides, and recombinant proteins
HBSS, no calcium, no magnesium, no phenol red Gibco Cat#14-175-095
Penicillin-Streptomycin (10,000 U/mL) Gibco Cat#15-140-122
DNaseI MilliporeSigma DN25-100MG
Trypsin MilliporeSigma T4799-5G
DMEM/F12 Gibco Cat#11-320-033
Sodium Pyruvate (100 mM) Gibco Cat#11-360-070
GlutaMAX™ Supplement Gibco Cat#35-050-061
Fetal Bovine Serum Gibco Cat#16-000-044
Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix Gibco Cat#A1413202
N-2 Supplement (100X) Gibco Cat#17-502-048
Sodium Phosphate Monobasic MilliporeSigma SKU: 567545
Sodium Phosphate Dibasic MilliporeSigma SKU: 567550
Sodium Chloride MilliporeSigma SKU: S9888
Sodium Azide Millipore Sigma SKU: S2002
Sodium thiocyanate MilliporeSigma SKU: 251410
Ethylenediaminetetraacetic acid MilliporeSigma SKU: E9884
DL-Dithiothreitol Millipore Sigma SKU: 43815
Sodium Cholate MilliporeSigma SKU: C9282
14:0 PC (DMPC) 1,2-dimyristoyl-sn-glycero-3-phosphocholine Avanti Polar Lipids SKU: 850345C-25mg
16:0 PC (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Avanti Polar Lipids SKU: 850355C-25mg
16:0-18:1 PC (POPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine Avanti Polar Lipids SKU: 850457C-25mg
Recombinant ApoE2 Leinco Product No.: A215
Recombinant ApoE3 Leinco Product No.: A218
Recombinant ApoE4 Leinco Product No.: A219
BS3 (bis(sulfosuccinimidyl)suberate) FisherScientific Cat#PIA39266
HMW Native Marker Kit Cytiva Product: 28403842
Deposited data
CryoEM Map of rApoE4-DMPC-HJ15.30-F(ab’)2 EMDB EMDB: EMD-41830
CryoEM Map of Gradient Fixed rApoE4-DMPC-HJ15.30-F(ab’)2 EMDB EMDB: EMD-41831
Experimental models: Organisms/strains
ApoE2 Knock-In, floxed Huynh et al., 201916 N/A
ApoE3 Knock-In, floxed Huynh et al., 201916 N/A
ApoE4 Knock-In, floxed Huynh et al., 201916 N/A
Software and algorithms
CryoSPARC Structura Biotechnology Inc. Version 4.1.2, https://CryoSPARC.com/
Inkscape Inkscape Developers Inkscape 1.2.2, https://inkscape.org/
FIJI Schindelin, et.al., 201284 https://imagej.net/software/fiji/
SnapGene Dotmatics https://www.snapgene.com/
BioRender BioRender https://www.biorender.com/
Unicorn 5.20 Cytiva https://www.cytivalifesciences.com/en/us/shop/unicorn-5-20-p-04079, Product: 28943244
Excel Microsoft Corporation https://www.microsoft.com/en-us/microsoft-365/excel
Other
Vivaspin 20 MWCO 10 000 Cytiva Product: 28932360
Vivaspin 2 MWCO 10 000 Cytiva Product: 28932247
Slide-A-Lyzer™ Dialysis Cassettes, 10K MWCO FisherScientific Cat#PI66380
Superose 6 10/300 Increase GL Column Cytiva Product: 29091596
Quantifoil 2/2 Copper Grids with GO Electron Microscopy Services SKU: GOQ300R22Cu10
Quantifoil 1.2/1.3 Gold Grids with GO Electron Microscopy Services SKU: GOQ400R1213Au10
Pierce Silver Stain Kit Thermo Scientific Cat#24612
NativePAGE™ Sample Buffer (4X) Invitrogen Cat#BN2003
NativePAGE ® 5% G-250 Sample Additive Invitrogen Cat#BN2004
NativePAGE™ Cathode Buffer Additive (20X) Invitrogen Cat#BN2002
NuPAGE™ LDS Sample Buffer (4X) Invitrogen Cat#NP0007
NativePAGE™ 4 to 16%, Bis-Tris, 1.0 mm, Mini Protein Gels Invitrogen Cat#BN1004BOX
NuPAGE™ 4 to 12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gels Invitrogen Cat#NP0323BOX
PVDF/Filter Paper Sandwich, 0.2 μm, 8.3 x 7.3 cm Invitrogen Cat#LC2002
Lumigen ECL Ultra Lumigen Cat#TMA-100
S.c. EasyComp™ Transformation Kit Invitrogen Cat#K505001
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Experimental Model and Study Participant Details:

Generation and Maintenance of APOE-KI Mice

Mouse models used to generate primary astrocyte cultures are listed in the Key Resources Table. Mice used in these experiments have been previously described16. Briefly, targeting vectors for the human APOE isoforms were transfected into the Taconic Biosciences C57BL/6N Tac ES cell line. Homologous recombinant clones were isolated using double positive (NeoR and PuroR) and negative (Thymidine kinase – Tk) selections. After in vivo Flp-mediated removal of the selection markers the constitutive humanized/conditional knockout alleles were obtained. The human APOE gene is then expressed under the control of the endogenous mouse Apoe promotor. These mice are referred to APOE2-KI, APOE3-KI, or APOE4-KI mice depending on the inserted human APOE isoform. All mice were maintained on a C57BL/6N background. Mice were used for breeding or sacrificed at P2-3 to culture primary glial cultures. Mice were housed in AAALAC accredited facilities with temperature and humidity control with a 12 hour light/dark cycle. Mice had free access to food and water ad libitum.

Primary Astrocyte Cell Cultures

Reagents used in the maintenance of primary astrocyte cultures are listed in the Key Resources Table. Generation of primary astrocyte cultures were prepared as previously described10. Pups were sacrificed at age P2-3 for generation of primary glial cultures. Male and female pups were used in all experiments, since cultures used mixed male and female pups we did not assess differences based on sex. Cortex from pups were dissected out and meninges removed. Brains were washed three times with dissection buffer (HBSS with 1X penicillin/streptomycin). A final concentration of 0.125% trypsin and 500μg/mL of DNaseI was added to cortex and tissue was incubated at 37°C for 5 minutes. 5mL of cold dissection media was added to digestion buffer. Tissue was triturated to generate single cell suspension. Cells were centrifuged at 1000×g for 5 minutes at 4°C. Digestion buffer was aspirated and cells were suspended in dissection buffer. A final concentration of 500μg/mL DNaseI was added to cells and cells were incubated at 37°C for 5 minutes. Supernatant was aspirated and cells were resuspended in glia media (DMEM/F12, 1X penicillin/streptomycin 1X sodium pyruvate, 1X GlutaMAX, 10% fetal bovine serum) and plated on Geltrex coated T75 flasks. Cells were cultured until confluent with media changes every 3 days. Microglia were removed from cultures by shaking at 250rpm for 30 minutes. Astrocyte primary cultures were then washed 3 times with PBS. Serum-free media (DMEM/F12, 1X P/S 1X Sodium Pyruvate, 1X GlutaMAX, 1X N2 Neuronal Supplement) was added to cultures. Media was collected after 48 hours and filtered through a 0.22μm filter and sodium azide was added to a final concentration of 0.02%.

Method Details:

Immunopurification of Astrocyte Lipoproteins from Astrocyte Conditioned Media

Reagents used in ApoE immunopurification are listed in the Key Resources Table Astrocyte ApoE lipoproteins were immunopurified as previously described12,71. Serum-free, astrocyte conditioned media was cycled over an anti-ApoE antibody HJ15.4 immunoaffinity column overnight at 1mL/minute at 4°C. The immunoaffinity column was washed with 10 column volumes of buffer A (20mM sodium phosphate buffer, 50mM NaCl, 0.02% sodium azide, pH7.4) at 1mL/minute at 4°C. Nonspecific proteins were eluted with 10 column volumes of buffer B (20mM phosphate buffer, 500mM NaCl, 0.02% sodium azide, pH 7.4). Lipidated ApoE was eluted with 5 column volumes of 3M NaSCN at 1mL/minute at 4°C. Immunopurified ApoE was concentrated using a Cytiva 10kDa MWCO concentrator at 4000×g for 5 minutes at 4°C. Immunopurified ApoE was dialyzed into buffer A with 3 changes overnight using a 10kDa MWCO Slide-A-Lyzer dialysis cassette. Analysis of astrocyte ApoE lipoproteins by size exclusion chromatography (SEC) was on a Superose 6 10/300 Increase GL column at 0.5mL/min with buffer A. UV traces were plotted using Unicorn 5.20 software.

Preparing Lipidated, Recombinant ApoE Lipoproteins

Reagents used for ApoE lipidation are listed in the Key Resources Table. The sodium cholate dialysis method was used to generate lipidated, recombinant ApoE lipoproteins for the studies in this paper. ApoE was suspended in buffer A with 1mM EDTA and 1mM DTT at 10μM final concentration. ApoE was allowed to stand for at least 1 hour prior to lipidation. 10mg/mL DMPC, DPPC, or POPC in chloroform was dried under a stream of nitrogen for 5min or until chloroform was completely evaporated. The dried lipid was then allowed to evaporate under vacuum with desiccant for a minimum of 2 hours. Lipid was resuspended in buffer A at a final concentration of 20mg/mL. Lipid was allowed to hydrate for a minimum of 1 hour. Sodium cholate was added at a 2:1 ratio of sodium cholate to lipid and vortexed. Lipid was then sonicated for 30min (1min on, 1min off) in a water bath sonicator at 30A. Lipid was brought above the phase transition temperature; 45°C for DPPC and 24°C for DMPC, and 4°C for POPC. ApoE at the listed molar ratio of ApoE to lipid was added and incubated overnight. ApoE lipoproteins were dialyzed for 48 hours with 4 changes in buffer A. Samples were then purified on a Superose 6 10/300 Increase GL column at 0.5mL/minute with 1mL fractions collected. Peak fractions were collected and concentrated using an Cytiva 2mL 10kDa MWCO at 4000×g for 5 minutes. Concentration of ApoE particles was determined by A280 and adjusted using an extinction coefficient of 1.31.

Negative Stain Electron Microscopy

For recombinant ApoE lipoproteins, 100nM ApoE lipoproteins with 110nM of anti-ApoE antibody HJ15.10 Fab and/or anti-ApoE antibody HJ15.30 F(ab’)2 were combined overnight at 4°C. For astrocyte ApoE lipoproteins, 200nM astrocyte ApoE lipoproteins were incubated with 220nM HJ15.10 Fab and/or 110nM HJ15.30 F(ab’)2 overnight with rotation at 4°C. 10uL of sample was incubated for 1 min on carbon-coated 200-mesh copper grid (01840-F, Ted Pella) which had been freshly plasma cleaned for 30 seconds in a Solarus 950 plasma cleaner (Gatan). The grid was subsequently washed five times with ultrapure water, then stained with 0.75% uranyl formate for 2 minutes. Excess stain was blotted off with filter paper (Whatman No. 1), then the grid was air dried. Samples were imaged using a JEOL JEM-1400 operating at 120kV at 50,000× magnification (0.222nm/pixel) with a NanoSprint15-MkII 16-megapixel sCMOS camera (Advanced Microscopy Techniques). Micrographs were CTF corrected in CryoSPARC using patch CTF correction81. Blob picker was used to select particles and 2D classes were generated in CryoSPARC.

Gradient Fixation of ApoE Lipoproteins

In order to achieve higher resolution of ApoE lipoproteins gradient fixation was used to fix ApoE without leading to aggregation66,67. 50mM BS3 was diluted in 30% glycerol in 20mM phosphate buffer, 50mM NaCl, pH 7.4 for a final concentration of 0.5mM BS3. 1mL of 30% glycerol with 0.5mM BS3 was overlayed with 23% glycerol in 20mM phosphate buffer, 50mM NaCl, pH 7.4, then 15% glycerol in 20mM phosphate buffer, 50mM NaCl, pH 7.4, and finally 10% glycerol in 20mM phosphate buffer, 50mM NaCl, pH 7.4. Each layer was frozen sequentially then allowed to thaw at 4°C in order to form a continuous gradient. 500μL of 7% glycerol in 20mM phosphate buffer, 50mM NaCl, pH 7.4 was overlayed gradient as a buffer. 100μL of 500nM ApoE lipoproteins were overlayed on buffer. Samples were centrifuged in SW Ti 55 rotor at 55,000rpm for 11h at 8°C. 330μL fractions were collected after ultracentrifugation. Fractions were assessed by SDS-PAGE followed by silver stain following manufacturer’s directions. Fractions containing ApoE with anti-ApoE antibody HJ15.30 F(ab’)2 were combined and dialyzed in 20mM phosphate buffer, 50mM NaCl, pH 7.4 in order to remove glycerol and crosslinker. Sample was concentrated using Cytiva 2mL 10MWCO by centrifuging at 4000g for 5 minutes at 4°C.

Cryogenic Electron Microscopy

Samples were prepared on Quantifoil 2/2 copper grids with graphene oxide (GO) or to gold 1.2/1.3 Quantifoil girds coated with graphene oxide (EMS) and were plunge-frozen using a Vitrobot Mark IV (Thermo Fisher Scientific). Prior to freezing, grids were freshly glow-discharged for 20 seconds at 15mA with a GloQube glow-discharger (Quorum Technologies). 3μL of sample was applied to the Quantifoil 2/2 copper grids with GO. After a 1 minute wait, the grids were blotted for 2.5 seconds with a blot force of −1 and plunge frozen in liquid ethane. For gradient fixed samples, 30μL of gradient fixed samples was placed onto parafilm. Gold Quantifoil 1.2/1.3 grids with GO were then incubated on sample for 1 hour by floating on droplet. Samples were then frozen using Vitrobot Mark IV. An additional 3μL of sample were adsorbed onto grid to facilitate blotting. Sample was incubated for 5 seconds, blotted for 2.5 seconds with blot force of −1, and plunge frozen into liquid ethane. Sample was imaged on either a 200kV Glacios or 300kV Krios cryo-electron microscope (Thermo Fisher Scientific) located at Washington University Center for Cellular Imaging (WUCCI), both equipped with a Falcon IV direct electron detector (Thermo Fisher Scientific). For the Glacios cryo-TEM, the nominal magnification was 150,000× magnification, resulting in a pixel size of 0.928Å/pixel. For density map shown in Figure 3B, total dose was 52.8/Å2, with 48 frames, with a defocus range from −1.0 to −2.4μm. For data in figure 2A, the Krios cryo-TEM, the nominal magnification was 59,000μ magnification, resulting in a pixel size of 1.081Å/pixel. Total dose for this dataset was 72.3e/Å2, with 48 frames, and a defocus range of −1.0¼m to −2.4¼m. For data in figure 2B and figure 3A, the Krios cryo-TEM, the nominal magnification was 75,000× magnification, resulting in a pixel size of 0.842Å/pixel. Total dose for this dataset was 58.1e/Å2, with 48 frames, and a defocus range of −1.0¼m to −2.4¼m. Movies were motion corrected using patch motion correction in CryoSPARC81. The CTF parameters were estimated using Patch CTF estimation in CryoSPARC. The particles were first selected automatically using Blob picker, followed by 2D classification in CryoSPARC. Good 2D classes were selected for ab intio modeling. Ab inito model was used to generate templates (projections) used for template picking in picking CryoSPARC, followed by 2D classification. Selected classes were used to generate an ab intio model. A refined model was generated using the ab intio model and non-uniform refinement in CryoSPARC82.

Blue Native PAGE

Reagents for blue native PAGE are listed in the Key Resources Table To assess particle size, Blue Native PAGE was performed83. Samples were incubated with DDM at a 1g:1g ratio for 10 minutes after dilution in NuPAGE 4X Native Buffer. Coomassie R-250 was then added at a 1g:8g ratio of DDM to Coomassie R-250. Samples were loaded on a 4-16% NativePAGE gel. Cathode chamber was filled with 1X Native PAGE running buffer with 1X NativePAGE cathode buffer additive. Anode chamber was filled with 1X NativePAGE running buffer. Gel was run at 150V for 2 hours. Gel was stained with SimplyBlue stain and imaged on a Bio-Rad imager.

SDS-PAGE, Native PAGE, and Western Blot

Reagents used for SDS-PAGE, native PAGE, and western blot are listed in the Key Resources Table. SDS-PAGE was performed under reducing with a final concentration of 1% BME as the reducing agent. NuPAGE 4X LDS loading dye was used to prepare the samples. Samples were run on a 4-16% Bis-Tris gel for 50V for 15 minutes followed by 200V for 30 minutes. For Native PAGE gels, samples were diluted in 4X Native buffer. Cytiva HMW protein markers were diluted in the same buffer to estimate radii of ApoE lipoproteins. Samples were loaded onto a 4-16% Bis-Tris gel with native running buffer and run at 100V for 16 hours at 4°C. Gels were then transferred to PVDF membranes using the XCell II blot system at 30V for 1 hour. Samples were then blocked in 5% milk in TBST for 1 hour and then probed with primary antibody overnight. Following day, blots were washed with TBS, incubated with secondary for 1h, washed with TBS, then developed with Lumigen ECL Ultra. Blots were imaged on Bio-Rad imager.

Epitope Mapping of Antibodies

Epitope mapping was performed using yeast display. Plasmids containing truncated versions of human APOE were cloned into the pYD2 display vector. The vector was transfected into yeast using the S.c. EasyComp™ Transformation Kit. Antibodies were incubated with cells and detected using an anti-mouse AlexaFluor-488 secondary. Fluorescence was detected using a Cytation 5 plate reader.

Quantification and Statistical Analysis:

FIJI Analysis of Micrographs

FIJI was used to estimate the diameter of ApoE lipoproteins from native and blue native PAGE84. A standard curve was generated from the high molecular weight standards, fitted with a second-order polynomial, then diameter was estimated based on the relative migration distance. FIJI was also used to measure angle between HJ15.10 Fab fragments from 2D class averages. FIJI was used to measure lipoprotein diameter as previously described85. Briefly, TEM micrographs were converted to 8-bit, an auto-threshold was applied using the Otsu algorithm, and the minimum Feret’s diameter was used as an estimate of lipoprotein diameter.

Supplementary Material

1

Highlights:

  • Astrocyte ApoE adopts an antiparallel dimer in discoidal lipoproteins

  • Recombinant ApoE adopts an antiparallel dimer in discoidal lipoproteins

  • Lipid composition affects lipoprotein diameter

Acknowledgements:

We thank Mary G. Sorci-Thomas at the Medical College of Wisconsin for her valuable advice. We thank Chen Sun at Washington University in Saint Louis for providing homemade graphene oxide grids for cryo-EM. We thank Chanung Wang and Xiaoying Chen at Washington University in Saint Louis for their discussions on this work. Funding provided by the NIH T32 Fellowship (T32AG058518, MRS), NSF GRFP Fellowship (DGE-1745038, MRS), NIH RF1NS090934 (DMH), NIH 1U19AG069701 (DMH), and the JPB Foundation (DMH).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Declaration of Interests:

D.M.H. is an inventor on a patent licensed by Washington University to NextCure on the therapeutic use of anti-apoE antibodies. D.M.H. co-founded and is on the scientific advisory board of C2N Diagnostics. D.M.H. is on the scientific advisory board of Denali, Genentech, and Cajal Neuroscience and consults for Asteroid, and is on the Advisory Board for Cell.

References:

  • 1.Mahley RW, Weisgraber KH, and Huang Y (2009). Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer’s disease to AIDS. J. Lipid Res 50, S183–S188. 10.1194/jlr.R800069-JLR200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Huang Y, and Mahley RW (2014). Apolipoprotein E: Structure and function in lipid metabolism, neurobiology, and Alzheimer’s diseases. Neurobiol. Dis 72, 3–12. 10.1016/j.nbd.2014.08.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Chen Y, Strickland MR, Soranno A, and Holtzman DM (2021). Apolipoprotein E: Structural Insights and Links to Alzheimer Disease Pathogenesis. Neuron 109, 205–221. 10.1016/j.neuron.2020.10.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Boyles JK, Pitas RE, Wilson E, Mahley RW, and Taylor JM (1985). Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J. Clin. Invest 76, 1501–1513. 10.1172/JCI112130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Pitas RE, Boyles JK, Lee SH, Hui D, and Weisgraber KH (1987). Lipoproteins and their receptors in the central nervous system. Characterization of the lipoproteins in cerebrospinal fluid and identification of apolipoprotein B,E(LDL) receptors in the brain. J. Biol. Chem 262, 14352–14360. 10.1016/s0021-9258(18)47945-8. [DOI] [PubMed] [Google Scholar]
  • 6.Pitas RE, Boyles JK, Lee SH, Foss D, and Mahley RW (1987). Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim. Biophys. Acta (BBA)/Lipids Lipid Metab 917, 148–161. 10.1016/0005-2760(87)90295-5. [DOI] [PubMed] [Google Scholar]
  • 7.Koch S, Donarski N, Goetze K, Kreckel M, Stuerenburg HJ, Buhmann C, and Beisiegel U (2001). Characterization of four lipoprotein classes in human cerebrospinal fluid. J. Lipid Res 42, 1143–1151. 10.1016/s0022-2275(20)31605-9. [DOI] [PubMed] [Google Scholar]
  • 8.Wahrle SE, Jiang H, Parsadanian M, Legleiter J, Han X, Fryer JD, Kowalewski T, and Holtzman DM (2004). ABCA1 is required for normal central nervous system apoE levels and for lipidation of astrocyte-secreted apoE. J. Biol. Chem 279, 40987–40993. 10.1074/jbc.M407963200. [DOI] [PubMed] [Google Scholar]
  • 9.LaDu MJ, Gilligan SM, Lukens JR, Cabana VG, Reardon CA, Van Eldik LJ, and Holtzman DM (1998). Nascent astrocyte particles differ from lipoproteins in CSF. J. Neurochem 70, 2070–2081. 10.1046/j.1471-4159.1998.70052070.x. [DOI] [PubMed] [Google Scholar]
  • 10.Fagan AM, Holtzman DM, Munson G, Mathur T, Schneider D, Chang LK, Getz GS, Reardon CA, Lukens J, Shah JA, et al. (1999). Unique lipoproteins secreted by primary astrocytes from wild type, apoE (−/−), and human apoE transgenic mice. J. Biol. Chem 274, 30001–30007. 10.1074/jbc.274.42.30001. [DOI] [PubMed] [Google Scholar]
  • 11.Hirsch-Reinshagen V, Donkin J, Stukas S, Chan J, Wilkinson A, Fan J, Parks JS, Kuivenhoven JA, Lütjohann D, Pritchard H, et al. (2009). LCAT synthesized by primary astrocytes esterifies cholesterol on glia-derived lipoproteins. J. Lipid Res 50, 885–893. 10.1194/jIrM800584-JLR200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.DeMattos RB, Brendza RP, Heuser JE, Kierson M, Cirrito JR, Fryer J, Sullivan PM, Fagan AM, Han X, and Holtzman DM (2001). Purification and characterization of astrocyte-secreted apolipoprotein E and J-containing lipoproteins from wild-type and human apoE transgenic mice. Neurochem. Int 39, 415–425. 10.1016/S0197-0186(01)00049-3. [DOI] [PubMed] [Google Scholar]
  • 13.Keren-Shaul H, Spinrad A, Weiner A, Matcovitch-Natan O, Dvir-Szternfeld R, Ulland TK, David E, Baruch K, Lara-Astaiso D, Toth B, et al. (2017). A Unique Microglia Type Associated with Restricting Development of Alzheimer’s Disease. Cell 169, 1276–1290.e17. 10.1016/j.cell.2017.05.018. [DOI] [PubMed] [Google Scholar]
  • 14.Philips T, and Robberecht W (2011). Neuroinflammation in amyotrophic lateral sclerosis: Role of glial activation in motor neuron disease. Lancet Neurol. 10, 253–263. 10.1016/S1474-4422(11)70015-1. [DOI] [PubMed] [Google Scholar]
  • 15.Krasemann S, Madore C, Cialic R, Baufeld C, Calcagno N, El Fatimy R, Beckers L, O’Loughlin E, Xu Y, Fanek Z, et al. (2017). The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases. Immunity 47, 566–581.e9. 10.1016/j.immuni.2017.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Huynh T-PV, Wang C, Tran AC, Tabor GT, Mahan TE, Francis CM, Finn MB, Spellman R, Manis M, Tanzi RE, et al. (2019). Lack of hepatic apoE does not influence early Aβ deposition: observations from a new APOE knock-in model. Mol. Neurodegener 14, 37. 10.1186/s13024-019-0337-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Bu G. (2009). Apolipoprotein e and its receptors in Alzheimer’s disease: Pathways, pathogenesis and therapy. Nat. Rev. Neurosci 10, 333–344. 10.1038/nrn2620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Holtzman DM, Herz J, and Bu G (2012). Apolipoprotein E and apolipoprotein E receptors: normal biology and roles in Alzheimer disease. Cold Spring Harb. Perspect. Med 2, a006312. 10.1101/cshperspect.a006312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.William Rebeck G, Reiter JS, Strickland DK, and Hyman BT (1993). Apolipoprotein E in sporadic Alzheimer’s disease: Allelic variation and receptor interactions. Neuron 11, 575–580. 10.1016/0896-6273(93)90070-8. [DOI] [PubMed] [Google Scholar]
  • 20.Coon KD, Myers AJ, Craig DW, Webster JA, Pearson JV, Lince DH, Zismann VL, Beach TG, Leung D, Bryden L, et al. (2007). A high-density whole-genome association study reveals that APOE is the major susceptibility gene for sporadic late-onset Alzheimer’s disease. J. Clin. Psychiatry 68, 613–618. 10.4088/JCP.v68n0419. [DOI] [PubMed] [Google Scholar]
  • 21.Corder EH, Saunders AM, Risch NJ, Strittmatter WJ, Schmechel DE, Gaskell PC, Rimmler JB, Locke PA, Conneally PM, Schmader KE, et al. (1994). Protective effect of apolipoprotein E type 2 allele for late onset Alzheimer disease. Nat. Genet 7, 180–184. 10.1038/ng0694-180. [DOI] [PubMed] [Google Scholar]
  • 22.Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, and Pericak-Vance MA (1993). Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science (80-. ) 261, 921–923. 10.1126/science.8346443. [DOI] [PubMed] [Google Scholar]
  • 23.Reiman EM, Arboleda-Velasquez JF, Quiroz YT, Huentelman MJ, Beach TG, Caselli RJ, Chen Y, Su Y, Myers AJ, Hardy J, et al. (2020). Exceptionally low likelihood of Alzheimer’s dementia in APOE2 homozygotes from a 5,000-person neuropathological study. Nat. Commun 11. 10.1038/s41467-019-14279-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Farrer LA, Cupples LA, Haines JL, Hyman B, Kukull WA, Mayeux R, Myers RH, Pericak-Vance MA, Risch N, and Van Duijn CM (1997). Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease: A meta-analysis. J. Am. Med. Assoc 278, 1349–1356. 10.1001/jama.278.16.1349. [DOI] [PubMed] [Google Scholar]
  • 25.UTERMANN G, HEES M, and STEINMETZ A (1977). Polymorphism of apolipoprotein E and occurrence of dysbetalipoproteinaemia in man. Nature 269, 604–607. 10.1038/269604a0. [DOI] [PubMed] [Google Scholar]
  • 26.Holtzman D, and Herz J (2012). Apolipoprotein E and apolipoprotein receptors: normal biology and roles in Alzheimer’s disease. Cold Spring Harb. Perspect. Med 2, a006312. 10.1101/cshperspect.a006312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Morris JC, Roe CM, Xiong C, Fagan AM, Goate AM, Holtzman DM, and Mintun MA (2010). APOE predicts amyloid-beta but not tau Alzheimer pathology in cognitively normal aging. Ann. Neurol 67, 122–131. 10.1002/ana.21843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Reiman EM, Chen K, Liu X, Bandy D, Yu M, Lee W, Ayutyanont N, Keppler J, Reeder SA, Langbaum JBS, et al. (2009). Fibrillar amyloid-beta burden in cognitively normal people at 3 levels of genetic risk for Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A 106, 6820–6825. 10.1073/pnas.0900345106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Huynh T-PV, Liao F, Francis CM, Robinson GO, Serrano JR, Jiang H, Roh J , Finn MB, Sullivan PM, Esparza TJ, et al. (2017). Age-Dependent Effects of apoE Reduction Using Antisense Oligonucleotides in a Model of β-amyloidosis. Neuron 96, 1013–1023.e4. 10.1016/j.neuron.2017.11.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Holtzman DM, Fagan AM, Mackey B, Tenkova T, Sartorius L, Paul SM, Bales K , Ashe KH, Irizarry MC, and Hyman BT (2000). Apolipoprotein E facilitates neuritic and cerebrovascular plaque formation in an Alzheimer’s disease model. Ann. Neurol 47, 739–747. [PubMed] [Google Scholar]
  • 31.Holtzman DM, Bales KR, Wu S, Bhat P, Parsadanian M, Fagan AM, Chang LK, Sun Y, and Paul SM (1999). Expression of human apolipoprotein E reduces amyloid-beta deposition in a mouse model of Alzheimer’s disease. J. Clin. Invest 103, R15–R21. 10.1172/JC16179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Fagan AM, Watson M, Parsadanian M, Bales KR, Paul SM, and Holtzman DM (2002). Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer’s disease. Neurobiol. Dis 9, 305–318. 10.1006/nbdi.2002.0483. [DOI] [PubMed] [Google Scholar]
  • 33.Castellano JM, Kim J, Stewart FR, Jiang H, DeMattos RB, Patterson BW, Fagan AM, Morris JC, Mawuenyega KG, Cruchaga C, et al. (2011). Human apoE isoforms differentially regulate brain amyloid-β peptide clearance. Sci. Transl. Med 3, 89ra57. 10.1126/scitranslmed.3002156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Bales KR, Verina T, Dodel RC, Du Y, Altstiel L, Bender M, Hyslop P, Johnstone EM, Little SP, Cummins DJ, et al. (1997). Lack of apolipoprotein E dramatically reduces amyloid beta-peptide deposition. Nat. Genet 17, 263–264. 10.1038/ng1197-263. [DOI] [PubMed] [Google Scholar]
  • 35.Bales KR, Verina T, Cummins DJ, Du Y, Dodel RC, Saura J, Fishman CE, DeLong CA, Piccardo P, Petegnief V, et al. (1999). Apolipoprotein E is essential for amyloid deposition in the APP(V717F) transgenic mouse model of Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 96, 15233–15238. 10.1073/pnas.96.26.15233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Shi Y, Yamada K, Liddelow SA, Smith ST, Zhao L, Luo W, Tsai RM, Spina S, Grinberg LT, Rojas JC, et al. (2017). ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy. Nature 549, 523–527. 10.1038/nature24016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Wilson C, Wardell MR, Weisgraber KH, Mahley RW, and Agard DA (1991). Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E. Science (80-. ) 252, 1817–1822. 10.1126/science.2063194. [DOI] [PubMed] [Google Scholar]
  • 38.Wilson C, Mau T, Weisgraber KH, Wardell MR, Mahley RW, and Agard DA (1994). Salt bridge relay triggers defective LDL receptor binding by a mutant apolipoprotein. Structure 2, 713–718. 10.1016/S0969-2126(00)00072-1. [DOI] [PubMed] [Google Scholar]
  • 39.Dong LM, Wilson C, Wardell MR, Simmons T, Mahley RW, Weisgraber KH, and Agard DA (1994). Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms. J. Biol. Chem 269, 22358–22365. 10.2210/pdb1le4/pdb. [DOI] [PubMed] [Google Scholar]
  • 40.Dong L, Parkin S, Trakhanov SD, Rupp B, Simmons T, Arnold KS, Newhouse YM, Innerarity TL, and Weisgraber KH (1996). Novel mechanism for defective receptor binding of apolipoprotein E2 in type III hyperlipoproteinemia. Nat. Struct. Mol. Biol 3, 718–722. 10.1038/nsb0896-718. [DOI] [PubMed] [Google Scholar]
  • 41.Yamamoto T, Choi HW, and Ryan RO (2008). Apolipoprotein E isoform-specific binding to the low-density lipoprotein receptor. Anal. Biochem 372, 222–226. 10.1016/j.ab.2007.09.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Weisgraber KH, Innerarity TL, and Mahley RW (1982). Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine-arginine interchange at a single site. J. Biol. Chem 257, 2518–2521. [PubMed] [Google Scholar]
  • 43.Yamamoto T, and Ryan RO (2007). Anionic phospholipids inhibit apolipoprotein E-Low-density lipoprotein receptor interactions. Biochem. Biophys. Res. Commun. 354, 820–824. 10.1016/j.bbrc.2007.01.066. [DOI] [PubMed] [Google Scholar]
  • 44.Innerarity TL, Weisgraber KH, Arnold KS, Rail SCJ, and Mahley RW (1984). Normalization of receptor binding of apolipoprotein E2. Evidence for modulation of the binding site conformation. J. Biol. Chem 259, 7261–7267. [PubMed] [Google Scholar]
  • 45.Lu B, Morrow JA, and Weisgraber KH (2000). Conformational Reorganization of the Four-helix Bundle of Human Apolipoprotein E in Binding to Phospholipid. J. Biol. Chem 275, 20775–20781. 10.1074/jbc.M003508200. [DOI] [PubMed] [Google Scholar]
  • 46.Fisher CA, and Ryan RO (1999). Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E. J. Lipid Res 40, 93–99. [PubMed] [Google Scholar]
  • 47.Peters-Libeu CA, Newhouse Y, Hatters DM, and Weisgraber KH (2006). Model of biologically active apolipoprotein E bound to dipalmitoylphosphatidylcholine. J. Biol. Chem 281, 1073–1079. 10.1074/jbc.M510851200. [DOI] [PubMed] [Google Scholar]
  • 48.Hatters DM, Voss JC, Budamagunta MS, Newhouse YN, and Weisgraber KH (2009). Insight on the Molecular Envelope of Lipid-Bound Apolipoprotein E from Electron Paramagnetic Resonance Spectroscopy. J. Mol. Biol 386, 261–271. 10.1016/j.jmb.2008.12.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Gupta V, Narayanaswami V, Budamagunta MS, Yamamato T, Voss JC, and Ryan RO (2006). Lipid-induced extension of apolipoprotein E helix 4 correlates with low density lipoprotein receptor binding ability. J. Biol. Chem 281, 39294–39299. 10.1074/jbc.M608085200. [DOI] [PubMed] [Google Scholar]
  • 50.Newhouse Y, Peters-Libeu C, and Weisgraber KH (2005). Crystallization and preliminary X-ray diffraction analysis of apolipoprotein E-containing lipoprotein particles. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun 61, 981–984. 10.1107/S1744309105032410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Autzen HE, Julius D, and Cheng Y (2019). Membrane mimetic systems in CryoEM: keeping membrane proteins in their native environment. Curr. Opin. Struct. Biol 58, 259–268. 10.1016/j.sbi.2019.05.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Frauenfeld J, Gumbart J, Sluis E.O.V. Der, Funes S, Gartmann M, Beatrix B, Mielke T, Berninghausen O, Becker T, Schulten K, et al. (2011). Cryo-EM structure of the ribosome-SecYE complex in the membrane environment. Nat. Struct. Mol. Biol 18, 614–621. 10.1038/nsmb.2026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Yu I, Nguyen L, Avaylon J, Wang K, Lai M, and Zhou ZH (2018). Building atomic models based on near atomic resolution cryoEM maps with existing tools. J. Struct. Biol 204, 313–318. 10.1016/j.jsb.2018.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Cheng Y. (2018). Single-particle cryo-EM-How did it get here and where will it go. Science (80-. ) 361, 876–880. 10.1126/science.aat4346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Brussel VU, Efremov RG, Gatsogiannis C, and Raunser S (2017). Lipid Nanodiscs as a Tool for High-Resolution Structure Determination of Membrane Proteins by Single-Particle Cryo-EM 1st ed. (Elsevier Inc.) 10.1016/bs.mie.2017.05.007. [DOI] [PubMed] [Google Scholar]
  • 56.Cheng Y. (2015). Single-particle Cryo-EM at crystallographic resolution. Cell 161, 450–457. 10.1016/j.cell.2015.03.049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Nakane T, Kotecha A, Sente A, McMullan G, Masiulis S, Brown PMGE, Grigoras IT, Malinauskaite L, Malinauskas T, Miehling J, et al. (2020). Single-particle cryo-EM at atomic resolution. Nature 587, 152–156. 10.1038/s41586-020-2829-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Nogales E, and Scheres SHW (2015). Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity. Mol. Cell 58, 677–689. 10.1016/j.molcel.2015.02.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Wu S, Avila-Sakar A, Kim J, Booth DS, Greenberg CH, Rossi A, Liao M, Li X, Alian A, Griner SL, et al. (2012). Fabs enable single particle cryoEM studies of small proteins. Structure 20, 582–592. 10.1016/j.str.2012.02.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Liao F, Zhang TJ, Jiang H, Lefton KB, Robinson GO, Vassar R, Sullivan PM, and Holtzman DM (2015). Murine versus human apolipoprotein E4: differential facilitation of and co-localization in cerebral amyloid angiopathy and amyloid plaques in APP transgenic mouse models. Acta Neuropathol. Commun 3, 70. 10.1186/s40478-015-0250-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Charles A Janeway Jr, Travers Paul, Walport Mark, and J.S M. (2001). The structure of a typical antibody molecule. In Immunobiology: The Immune System in Health and Disease. (New York: Garland Science). [Google Scholar]
  • 62.Peters-Libeu CA, Newhouse Y, Hall SC, Witkowska HE, and Weisgraber KH (2007). Apolipoprotein E·dipalmitoylphosphatidylcholine particles are ellipsoidal in solution. J. Lipid Res 48, 1035–1044. 10.1194/jlr.M600545-JLR200. [DOI] [PubMed] [Google Scholar]
  • 63.Garai K, Baban B, and Frieden C (2011). Dissociation of apolipoprotein e oligomers to monomer is required for high-affinity binding to phospholipid vesicles. Biochemistry 50, 2550–2558. 10.1021/bi1020106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Verghese PB, Castellano JM, Garai K, Wang Y, Jiang H, Shah A, Bu G, Frieden C, and Holtzman DM (2013). ApoE influences amyloid-$β$ (A$β$) clearance despite minimal apoE/A$β$ association in physiological conditions. Proc. Natl. Acad. Sci. U. S. A 110, E1807–16. 10.1073/pnas1220484110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Segrest JP, Jackson RL, Morrisett JD, and Gotto AM (1974). A molecular theory of lipid-protein interactions in the plasma lipoproteins. FEBS Lett. 38, 247–253. 10.1016/0014-5793(74)80064-5. [DOI] [PubMed] [Google Scholar]
  • 66.Stark H. (2010). GraFix: Stabilization of fragile macromolecular complexes for single particle Cryo-EM (Elsevier Masson SAS; ) 10.1016/S0076-6879(10)81005-5. [DOI] [PubMed] [Google Scholar]
  • 67.Kastner B, Fischer N, Golas MM, Sander B, Dube P, Boehringer D, Hartmuth K, Decked J, Hauer F, Wolf E, et al. (2008). GraFix: Sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55. 10.1038/nmethl139. [DOI] [PubMed] [Google Scholar]
  • 68.Borhani DW, Rogers DP, Engler JA, and Brouillette CG (1997). Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation. Proc. Natl. Acad. Sci. U. S. A 94, 12291–12296. 10.1073/pnas.94.23.12291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Hu ML, Quinn J, and Xue K (2021). Interactions between apolipoprotein e metabolism and retinal inflammation in age-related macular degeneration. Life 11, 1–15. 10.3390/life11070635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Rasmussen KL, Tybjærg-Hansen A, Nordestgaard BG, and Frikke-Schmidt R (2023). Associations of Alzheimer Disease-Protective APOE Variants with Age-Related Macular Degeneration. JAMA Ophthalmol. 141, 13–21. 10.1001/jamaophthalmol.2022.4602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Morikawa M, Fryer JD, Sullivan PM, Christopher EA, Wahrle SE, DeMattos RB, O’Dell MA, Fagan AM, Lashuel HA, Walz T, et al. (2005). Production and characterization of astrocyte-derived human apolipoprotein E isoforms from immortalized astrocytes and their interactions with amyloid-β. Neurobiol. Dis 19, 66–76. 10.1016/j.nbd.2004.11.005. [DOI] [PubMed] [Google Scholar]
  • 72.Zorich N, Jonas A, and Pownall HJ (1985). Activation of lecithin cholesterol acyltransferase by human apolipoprotein E in discoidal complexes with lipids. J. Biol. Chem 260, 8831–8837. 10.1016/s0021-9258(17)39427-9. [DOI] [PubMed] [Google Scholar]
  • 73.Jonas A, Steinmetz A, and Churgay L (1993). The number of amphipathic α-helical segments of apolipoproteins A-I, E, and A-IV determines the size and functional properties of their reconstituted lipoprotein particles. J. Biol. Chem 268, 1596–1602. 10.1016/s0021-9258(18)53895-3. [DOI] [PubMed] [Google Scholar]
  • 74.Rall SC, Weisgraber KH, Innerarity TL, and Mahley RW (1983). Identical structural and receptor binding defects in apolipoprotein E2 in hypo-, normo-, and hypercholesterolemic dysbetalipoproteinemia. J. Clin. Invest 71, 1023–1031. 10.1172/JCI110829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75.Bedi S, Morris J, Shah A, Hart RC, Gray Jerome W, Aller SG, Tang C, Vaisar T, Bornfeldt KE, Segrest JP, et al. (2022). Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux. J. Lipid Res 63, 100168. 10.1016/j.jlr.2022.100168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76.He Y, Song HD, Anantharamaiah GM, Palgunachari MN, Bornfeldt KE, Segrest JP, and Heinecke JW (2019). Apolipoprotein A1 Forms 5/5 and 5/4 Antiparallel Dimers in Human High-density Lipoprotein. Mol. Cell. Proteomics 18, 854–864. 10.1074/mcp.RA118.000878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77.Melchior JT, Street SE, Vaisar T, Hart R, Jerome J, Kuklenyik Z, Clouet-Foraison N, Thornock C, Bedi S, Shah AS, et al. (2021). Apolipoprotein A-I modulates HDL particle size in the absence of apolipoprotein A-II. J. Lipid Res 62, 100099. 10.1016/JJLR.2021.100099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 78.Cooke AL, Morris J, Melchior JT, Street SE, Gray Jerome W, Huang R, Herr AB, Smith LE, Segrest JP, Remaley AT, et al. (2018). A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL. J. Lipid Res 59, 1244–1255. 10.1194/jlr.M085332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 79.Weisgraber KHH, and Shinto LHH (1991). Identification of the disulfide-linked homodimer of apolipoprotein E3 in plasma. Impact on receptor binding activity. J. Biol. Chem 266, 12029–12034. 10.1016/S0021-9258(18)99060-5. [DOI] [PubMed] [Google Scholar]
  • 80.Elliott DA, Halliday GM, and Garner B (2010). Apolipoprotein-E forms dimers in human frontal cortex and hippocampus. BMC Neurosci. 11. 10.1186/1471-2202-11-23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 81.Punjani A, Rubinstein JL, Fleet DJ, and Brubaker MA (2017). CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296. 10.1038/nmeth.4169. [DOI] [PubMed] [Google Scholar]
  • 82.Punjani A, Zhang H, and Fleet DJ (2020). Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221. 10.1038/s41592-020-00990-8. [DOI] [PubMed] [Google Scholar]
  • 83.Wittig I, Braun HP, and Schägger H (2006). Blue native PAGE. Nat. Protoc 1, 418–428. 10.1038/nprot.2006.62. [DOI] [PubMed] [Google Scholar]
  • 84.Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al. (2012). Fiji: An open-source platform for biological-image analysis. Nat. Methods 9, 676–682. 10.1038/nmeth.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 85.Cashikar AG, Toral-Rios D, Timm D, Romero J, Strickland M, Long JM, Han X, Holtzman DM, and Paul SM (2023). Regulation of astrocyte lipid metabolism and ApoE secretionby the microglial oxysterol, 25-hydroxycholesterol. J. Lipid Res 64, 100350. 10.1016/JJLR.2023.100350. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1960906-supplement-1.pdf (2.9MB, pdf)

Data Availability Statement

  • CryoEM electron density maps are deposited in EMDB (EMD-41830 and EMD-41831) and are publicly available at time of publication. Accession numbers are listed in the key resources table.

  • This paper does not report original code.

  • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Mouse monocolonal anti-apoE Antibody (HJ15.3) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.4) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.5) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.6) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.7) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.10) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.12) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.30) Liao et. al., 201560 N/A
Mouse monocolonal anti-apoE Antibody (HJ15.32) Liao et. al., 201560 N/A
Chemicals, peptides, and recombinant proteins
HBSS, no calcium, no magnesium, no phenol red Gibco Cat#14-175-095
Penicillin-Streptomycin (10,000 U/mL) Gibco Cat#15-140-122
DNaseI MilliporeSigma DN25-100MG
Trypsin MilliporeSigma T4799-5G
DMEM/F12 Gibco Cat#11-320-033
Sodium Pyruvate (100 mM) Gibco Cat#11-360-070
GlutaMAX™ Supplement Gibco Cat#35-050-061
Fetal Bovine Serum Gibco Cat#16-000-044
Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix Gibco Cat#A1413202
N-2 Supplement (100X) Gibco Cat#17-502-048
Sodium Phosphate Monobasic MilliporeSigma SKU: 567545
Sodium Phosphate Dibasic MilliporeSigma SKU: 567550
Sodium Chloride MilliporeSigma SKU: S9888
Sodium Azide Millipore Sigma SKU: S2002
Sodium thiocyanate MilliporeSigma SKU: 251410
Ethylenediaminetetraacetic acid MilliporeSigma SKU: E9884
DL-Dithiothreitol Millipore Sigma SKU: 43815
Sodium Cholate MilliporeSigma SKU: C9282
14:0 PC (DMPC) 1,2-dimyristoyl-sn-glycero-3-phosphocholine Avanti Polar Lipids SKU: 850345C-25mg
16:0 PC (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Avanti Polar Lipids SKU: 850355C-25mg
16:0-18:1 PC (POPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine Avanti Polar Lipids SKU: 850457C-25mg
Recombinant ApoE2 Leinco Product No.: A215
Recombinant ApoE3 Leinco Product No.: A218
Recombinant ApoE4 Leinco Product No.: A219
BS3 (bis(sulfosuccinimidyl)suberate) FisherScientific Cat#PIA39266
HMW Native Marker Kit Cytiva Product: 28403842
Deposited data
CryoEM Map of rApoE4-DMPC-HJ15.30-F(ab’)2 EMDB EMDB: EMD-41830
CryoEM Map of Gradient Fixed rApoE4-DMPC-HJ15.30-F(ab’)2 EMDB EMDB: EMD-41831
Experimental models: Organisms/strains
ApoE2 Knock-In, floxed Huynh et al., 201916 N/A
ApoE3 Knock-In, floxed Huynh et al., 201916 N/A
ApoE4 Knock-In, floxed Huynh et al., 201916 N/A
Software and algorithms
CryoSPARC Structura Biotechnology Inc. Version 4.1.2, https://CryoSPARC.com/
Inkscape Inkscape Developers Inkscape 1.2.2, https://inkscape.org/
FIJI Schindelin, et.al., 201284 https://imagej.net/software/fiji/
SnapGene Dotmatics https://www.snapgene.com/
BioRender BioRender https://www.biorender.com/
Unicorn 5.20 Cytiva https://www.cytivalifesciences.com/en/us/shop/unicorn-5-20-p-04079, Product: 28943244
Excel Microsoft Corporation https://www.microsoft.com/en-us/microsoft-365/excel
Other
Vivaspin 20 MWCO 10 000 Cytiva Product: 28932360
Vivaspin 2 MWCO 10 000 Cytiva Product: 28932247
Slide-A-Lyzer™ Dialysis Cassettes, 10K MWCO FisherScientific Cat#PI66380
Superose 6 10/300 Increase GL Column Cytiva Product: 29091596
Quantifoil 2/2 Copper Grids with GO Electron Microscopy Services SKU: GOQ300R22Cu10
Quantifoil 1.2/1.3 Gold Grids with GO Electron Microscopy Services SKU: GOQ400R1213Au10
Pierce Silver Stain Kit Thermo Scientific Cat#24612
NativePAGE™ Sample Buffer (4X) Invitrogen Cat#BN2003
NativePAGE ® 5% G-250 Sample Additive Invitrogen Cat#BN2004
NativePAGE™ Cathode Buffer Additive (20X) Invitrogen Cat#BN2002
NuPAGE™ LDS Sample Buffer (4X) Invitrogen Cat#NP0007
NativePAGE™ 4 to 16%, Bis-Tris, 1.0 mm, Mini Protein Gels Invitrogen Cat#BN1004BOX
NuPAGE™ 4 to 12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gels Invitrogen Cat#NP0323BOX
PVDF/Filter Paper Sandwich, 0.2 μm, 8.3 x 7.3 cm Invitrogen Cat#LC2002
Lumigen ECL Ultra Lumigen Cat#TMA-100
S.c. EasyComp™ Transformation Kit Invitrogen Cat#K505001
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