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. Author manuscript; available in PMC: 2021 Sep 3.
Published in final edited form as: Sci Transl Med. 2021 Mar 3;13(583):eaaz4564. doi: 10.1126/scitranslmed.aaz4564

APOE4 disrupts intracellular lipid homeostasis in human iPSC-derived glia

Grzegorz Sienski 1,12,#, Priyanka Narayan 1,2,3,#, Julia Maeve Bonner 1,2,#, Nora Kory 1, Sebastian Boland 4, Aleksandra A Arczewska 5,12, William T Ralvenius 2, Leyla Akay 2, Elana Lockshin 2, Liang He 6, Blerta Milo 2, Agnese Graziosi 2, Valeriya Baru 1,13, Caroline A Lewis 1, Manolis Kellis 9,10, David M Sabatini 1,7,8,9,11, Li-Huei Tsai 2,9,*, Susan Lindquist 1,7,8,†
PMCID: PMC8218593  NIHMSID: NIHMS1706136  PMID: 33658354

Abstract

The E4 allele of the apolipoprotein E gene (APOE) has been firmly established as a genetic risk factor for many diseases including cardiovascular diseases and Alzheimer’s disease (AD), yet its mechanism of action remains poorly understood. APOE is known to function as a lipid transport protein, and the dysregulation of lipids has recently emerged as a key feature of several neurodegenerative diseases including AD. However, it is unclear how APOE4 perturbs the intracellular lipid state. Here, we report that APOE4 disrupted the cellular lipidome in human iPSC-derived astrocytes generated from fibroblasts obtained from APOE4 compared to APOE3 carriers, and in yeast expressing human APOE4 isoforms. We combined lipidomics and unbiased genome-wide screens in yeast with functional and genetic characterization to demonstrate that APOE4 induced widespread changes in lipid homeostasis. These changes resulted in increased unsaturation of fatty acids and an accumulation of intracellular lipid droplets in both yeast and human iPSC-derived astrocytes. Additionally, we identified genetic and chemical modulators of this lipid disruption. In particular, we show that a simple environmental change in the culture medium—supplementation with choline, a soluble phospholipid precursor—was sufficient to restore the cellular lipidome to its basal state in APOE4 expressing astrocytes and in yeast expressing human APOE4. Our study illuminates key molecular disruptions in lipid metabolism that may contribute to the disease risk linked to the APOE4 genotype. Importantly, our study suggests that manipulating lipid metabolism could be a therapeutic approach to help to alleviate the consequences of carrying the APOE4 allele.

One Sentence Summary:

APOE4 disrupts intracellular lipid homeostasis in human and yeast cellular models, which was reversed by promoting phospholipid synthesis via choline supplementation.

Accessible Summary:

APOE4 is a strong genetic risk factor for many diseases, most notably, late-onset Alzheimer’s disease. Sienski, Narayan, & Bonner et al., show that human glia with an APOE4 genotype accumulate unsaturated triglycerides leading to a lipid imbalance. Using genetic screens in yeast, the authors discovered that choline supplementation, by promoting phospholipid synthesis, restores a healthy cellular lipid state in APOE4 cells. The authors then demonstrate that choline supplementation can also restore lipid homeostasis in human APOE4 astrocytes. These findings suggest that rewiring of the metabolic state of glia may reduce APOE4¬-associated disease risk.

Introduction

Genome-wide association studies implicate lipid metabolism in a number of late onset neurodegenerative diseases, including Alzheimer’s Disease (AD) (1–5). The accumulation of lipid (adipose saccules) in glia is a pathologically defining feature of AD (6). The most highly validated genetic risk factor for late-onset AD is the ε4 allele of the APOE gene (APOE4). APOE encodes a lipid-carrier protein that is a key component of many lipoprotein particles (7–9).

The human gene encoding APOE is polymorphic with three common coding variants (isoforms) differing from one another by two amino acids: ε3 (APOE3), ε4 (APOE4) and ε2 (APOE2) (10). The APOE4 genotype is the primary genetic risk factor for late-onset AD (11–13), is associated with cardiovascular diseases (14, 15), increases risk for metabolic syndrome (16), and is linked with decreased lifespan (17). The presence of APOE4 lowers the age of AD onset and increases the lifetime risk for developing the disease in a gene dose-dependent manner (18).

Apolipoprotein E (APOE) is a component of many lipoprotein particles and serves as a ligand for membrane receptors that mediate lipoprotein uptake (7–9). APOE4 has been suggested to contribute to AD pathogenesis by modulating multiple pathways including lipid transport and metabolism (19), however, the ways in which APOE4 alters the lipidome of cells remain unknown. In this study, we characterized the lipidome of APOE4-expressing human astrocytes generated from induced pluripotent stem cells (iPSCs) derived from fibroblasts from both APOE4 and APOE3 carriers (20). We found that iPSC-derived APOE4 astrocytes in vitro accumulated unsaturated triacylglycerides stored in lipid droplets to a greater extent than did their isogenic APOE3 counterparts. Using the conservation of lipid metabolism pathways between yeast and human cells (21–24), we established establish a yeast model where the expression of human APOE4, but not human APOE3, by yeast induced the accumulation of lipid droplets and increased unsaturation of triacylglycerides in a similar manner to iPSC-derived APOE4 astrocytes. Lipid droplet accumulation was accompanied by a specific growth defect in APOE4-expressing yeast. Loss-of-function genetic screens revealed that perturbations in a lipogenic transcriptional program uncoupled APOE4 from its cytotoxic effects. Promoting synthesis of the membrane lipid phosphatidylcholine by supplementing cell cultures with choline reversed the abnormal lipid unsaturation and lipid droplet accumulation in both the APOE4 expressing yeast model and in human iPSC-derived APOE4 astrocytes.

Results

Human iPSC-derived APOE4-expressing astrocytes display intracellular lipid dysregulation

We first characterized the impact of the APOE4 allele on the lipidome of human iPSC-derived astrocytes. In the brain, astrocytes are the major source of APOE (25), and have previously been reported to show APOE4-specific defects (26, 27). We utilized two independent pairs of previously published isogenic iPSC lines, derived from either a parental APOE3 or APOE4 homozygote, that had been edited with CRISPR-Cas9 to make the corresponding APOE3 or APOE4 isogenic line. These lines were differentiated into astrocytes that expressed the astrocyte proteins GFAP and S100β (Fig. 1A, B) (27).

Fig. 1. Increase in lipid droplets in APOE4 human iPSC-derived astrocytes.

Fig. 1.

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(A) The schematic shows the differentiation of isogenic astrocytes of the APOE3/APOE3 and APOE4/APOE4 genotypes from human iPSCs. (B) Representative fluorescence microscopy images (n=3 replicates) of APOE3/APOE3 and APOE4/APOE4 isogenic astrocytes stained with antibodies against S100β and GFAP (scale bar 100 μm). (C) A heatmap showing the fold change (log2) in abundance of phospholipids (~150 lipid species) and triacylglycerides (~120 species) between isogenic APOE4/APOE4 and APOE3/APOE3 human iPSC-derived astrocytes. (D) Graph shows fold change difference in the number of unsaturated bonds in fatty acids attached to triacylglycerides between isogenic APOE4/APOE4 and APOE3/APOE3 human iPSC-derived astrocytes. For this analysis, we summed the number of unsaturated carbon bonds per triacylglyceride molecule. (E) Representative microscopy images of isogenic APOE4/APOE4 and APOE3/APOE3 human iPSC-derived astrocytes in culture stained with LipidTox. Quantification of the lipid droplet number per cell is shown in the right panel, with each dot representing an average of at least 20 cells in four wells analyzed (n=7 independent replicates). Data are represented as mean ± SD; **** p ≤ 0.0001 by Student’s t-test. The dashed line denotes the boundaries of the cell (scale bar, 20 μm). (F) Representative microscopy images of isogenic APOE4/APOE4 and APOE3/APOE3 human iPSC-derived astrocytes in culture stained with an anti-Perilipin 2 antibody. Magnification is the same as in (E). Quantification of the Perilipin-2 foci per cell is shown in the right panel, with each dot representing an average of four wells with at least 20 cells analyzed (n=4 independent replicates). Data are represented as mean ± SD ; **** p ≤ 0.0001 by Student’s t-test.

We compared the lipid composition of the APOE3 and APOE4 expressing human iPSC-derived astrocytes by performing whole cell lipidomic analysis using liquid chromatography-mass spectrometry (LC-MS) (28, 29). We observed a minor change in phospholipid content and a much larger increase in triacylglycerides in APOE4-expressing human iPSC-derived astrocytes compared to APOE3-expressing human iPSC-derived astrocytes (Fig. 1C). We also observed that the degree of unsaturation of the fatty acids attached to triacylglycerides in APOE4 astrocytes was far higher than that in APOE3 astrocytes (Fig. 1D). Triacylglycerides, along with other neutral lipids such as cholesterol esters are stored in cytoplasmic organelles called lipid droplets (30). We therefore stained our isogenic astrocytes using a lipophilic dye, LipidTox, that labels neutral lipids (31). After 2 weeks in culture, we observed that APOE4 astrocytes accumulated a greater number of lipid droplets than did their APOE3 counterparts (Fig. 1E), which was accompanied by accumulation of a lipid droplet-resident protein, Perilipin-2 (Fig. 1F) (32). We validated these findings in an additional pair of isogenic iPSC lines (Fig. S1A), again observing increased accumulation of lipid droplets in APOE4 astrocytes as compared to APOE3 astrocytes (Fig. S1B).

APOE4 alters the lipid burden in microglia

To address whether the APOE4-associated intracellular lipid accumulation was present in other cell types, we examined microglia, which play a key role in AD (33) and were recently described to accumulate lipids in aged mice (4). We derived APOE3 and APOE4 microglia from our isogenic iPSC lines (Fig. S1C) and measured their lipid droplet content. We observed fewer lipid droplet bearing-microglia in culture than we observed for astrocytes under similar culture conditions. Even so, APOE4 microglia showed more lipid droplet positive cells per well (Fig. S1E, left panel) than did APOE3 microglia. Following 2 weeks of culture in minimal media, APOE4 microglia displayed a trend towards increased lipid droplet numbers per cell when compared to APOE3 microglia (Fig. S1D). Stimulation of the cultures by interferon gamma resulted in increased numbers of lipid droplet bearing cells for both APOE3 and APOE4 cells (Fig. S1E, right panel). These findings suggest that APOE4-associated lipid dysregulation occurred in multiple glial cell types.

Gene expression in brain tissue from individuals with the APOE4 allele suggests dysregulation in lipid metabolism

To determine whether our iPSC-derived human astrocyte cultures reflected APOE4-related dysfunction present in human brains, we examined transcriptomic data from postmortem human brain samples characterized in the Genotype-Tissue Expression (GTEx) project, containing tissue-specific gene expression data for individuals of varying genotype, age, and cause of death. We explored the influence of the APOE4 allele on lipid metabolic pathways, independent of disease state, by comparing the transcriptomic profiles of APOE4 carriers to non-carriers across a Kyoto Encyclopedia of Genes and Genomes (KEGG)-based curated set of 609 lipid metabolism genes (Fig. S1F, Table S1); 529 genes were detected within the GTEx cortex dataset. Of the genes that were significantly differentially expressed (False Discovery Rate (FDR) corrected p-value <0.05) in APOE4 carriers when compared to non-carriers, we identified upregulated genes involved in the metabolism of neutral lipids and cholesterol (FA2H, ACSL1, SQLE, HMGCR, MVK), as well as downregulated genes involved in the metabolism of fatty acids and neutral lipids (OLAH, CNEP1R1, GPAM) (Fig. S1F). This analysis suggested that in the human cortex, APOE4-mediated perturbation of lipid metabolism could be detected in the transcriptome of APOE4 carriers.

Expression of APOE4 in yeast recapitulates lipid defects identified in human astrocytes

In order to explore APOE4-associated lipid defects in an unbiased and genetically tractable manner, we utilized the remarkable conservation of lipid metabolism pathways across eukaryotes including baker’s yeast, Saccharomyces cerevisiae (21, 23, 24, 34).

We first established an APOE model in yeast that captured the lipid dysfunction that we observed in human cells. Our model used the human cDNA of APOE3 and APOE4, directed to the secretory pathway using the signal sequence from S. cerevisiae Kar2p (Fig. S2A), to mirror the localization of APOE in human cells (35, 36). The constructs were driven by tunable transcriptional activation by β-estradiol (37). We found that expression of human APOE4, but not human APOE3, induced the accumulation of lipid droplets in yeast grown in minimal culture media (Fig. 2A), just as we had observed for APOE4-expressing human iPSC-derived astrocytes. The APOE4 yeast strain also accumulated the lipid droplet-resident proteins, Erg6p and Faa4p (Fig. S2B) (38). Yeast cells expressing other neurodegenerative disease-related proteins, Aβ1-42 and TDP-43, showed only a minor increase in accumulation of the neutral lipid dye, BODIPY 493/503, compared to APOE4-expressing yeast (Fig. S2C). The extent of lipid accumulation and dysregulation therefore was a unique feature of APOE4-expressing cells.

Fig. 2. Lipid homeostasis is perturbed in yeast expressing human APOE4.

Fig. 2.

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(A) Representative fluorescence microscopy images of yeast expressing human APOE3 or APOE4 stained with BODIPY 493/503 stain for lipid droplets after growth in synthetic medium (scale bar, 5 μm). Quantification of the number of lipid droplets in yeast is shown in the right panel (n=8 experiments, each with at least 30 yeast cells analyzed). Data are represented as mean ± SD; **** p ≤ 0.0001 by Student’s t-test. The dashed line denotes the boundaries of the yeast cell. (B) Bar graph shows fold change difference in intracellular triacylglycerides between APOE3-expressing and APOE4-expressing yeast cells. Data representsmean ± SD, n=2 independently grown colonies. * p ≤ 0.05 by Student’s t-test. (C) Shown is a yeast growth assay on agar plates containing synthetic complete medium with (+) or without (−) β-estradiol to induce the expression of human APOE3 or APOE4. Representative agar plate shows 5-fold dilutions of yeast cultures. Western blot shows yeast samples collected from the agar plate after 8 hours of β-estradiol induction of APOE3 or APOE4 expression. APOE3 and APOE4 were probed with anti-APOE antibody; anti-PGK1 antibody was used as a loading control. (D) Bar graph shows growth of wildtype yeast expressing GFP or yeast expressing human APOE3 or APOE4 cultured in synthetic complete medium for 24 hours. Data are normalized to the growth rate of the GFP-expressing strain (control) and represented as mean ± SD, n=3 independent yeast colonies with at least two technical replicates. ns, nonsignificant p > 0.05; **** p ≤ 0.0001 by Student’s t-test. (E) A scatter plot from two independent loss-of-function screens showing the average colony size of yeast deletion strains with loss of individual non-essential genes and expressing human APOE4. Each circle represents data averaged from four technical replicates. Yeast strains with variability higher than SD>25% between technical replicates were removed resulting in ~2800 genes assayed). (F) Bar graph shows the growth of yeast expressing APOE4 compared to those expressing APOE3 for a wildtype strain and mga2Δ, ubx2Δ and opi1Δ strains. Data are represented as mean ± SD, n=3 independent yeast colonies with at least two technical replicates each. p ≤ 0.05; **** p ≤ 0.0001. (G) A Western blot of whole cell extracts of wildtype and MGA2-null yeast (top) and wildtype and OPI1-null yeast (bottom) showing expression of human APOE3 and APOE4, with PGK1 as loading control. (H) Bar graph shows the growth rate of APOE3-expressing and APOE4-expressing yeast treated with 10, 20 or 40 μM of ECC145 (an Ole1p inhibitor) or vehicle (DMSO). The data are normalized to the growth of a control untreated yeast strain expressing GFP. Data are represented as mean ± SD, n=3 independent yeast colonies with at least two technical replicates each). (I) Bar graph shows the mean signal of BODIPY 498/503 staining measured by fluorescence-based cell cytometry in yeast expressing human APOE3 or APOE4 for a wildtype strain or a MGA2-null strain. Data represent mean ± SD, n=3 independent yeast colonies with at least two technical replicates each. *** p ≤ 0.001 by Student’s t-test.

We performed lipid extraction and lipidomic analyses of APOE3 and APOE4-expressing yeast, observing an increase in levels of triacylglycerides (Fig. 2B) and the unsaturated fatty acids attached to them (Fig. S2D), similar to what we observed in APOE4-expressing human iPSC-derived astrocytes. Thus, expression of APOE4 in our yeast model induced similar lipid defects to those observed in iPSC-derived astrocytes and therefore could be used to probe the genetics underlying this effect.

Loss-of-function genetic screens in yeast reveal genetic suppressors of APOE4-mediated lipid defects

The disruption in lipid homeostasis in APOE4-expressing yeast was accompanied by an isoform-specific growth defect when the yeast were grown on either solid media (Fig. 2C) or in liquid synthetic Complete Supplement Mixture media (Fig. 2D, S3A). This growth defect scaled with expression of APOE4, where increasing amounts of APOE4 protein correlated with a reduction in the growth rate (Fig. S3B). This effect was not observed for APOE3-expressing yeast (Fig. S3C).

Given that APOE4-expressing yeast exhibited the lipid homeostasis defects observed in APOE4-expressing human iPSC-derived astrocytes, we performed loss-of-function genetic screens among the non-essential genes in the yeast genome to identify genetic suppressors of the APOE4-induced growth phenotype (39, 40). We performed two independent screens with the APOE4 gene expressed either from a cassette integrated into the yeast genome or from a centromeric plasmid (Fig. S3D). The gene deletion strains that exhibited unperturbed growth despite the expression of APOE4 (Fig. 2E, S3E) were enriched for genes whose protein products are associated with the endoplasmic reticulum (Fig. S3F), with many genes reported to genetically interact with each other (41). Our top hits were OPI1, MGA2 and UBX2, which all encode proteins that regulate lipid metabolism (42, 43). Mga2p and Ubx2p act as sensors for fatty acid saturation (42, 44–46) and Opi1p works as a sensor of phospholipid composition (47–49). We focused on MGA2 and OPI1 and found that deletion of neither MGA2 nor OPI1 altered the amounts of APOE4 protein (Fig. 2G). Given the established role of Mga2p and Opi1p in yeast lipid metabolism (42, 47, 48, 50), our results reaffirm that the APOE4-associated growth impediment in yeast stemmed from abnormal lipid metabolism.

An imbalance in lipid saturation is the primary contributor to APOE4-associated lipid defects in yeast

Both Ubx2p and Mga2p control the stability and expression of the only fatty acyl-CoA desaturase in yeast, OLE1 (42, 51, 52). Since the Ole1p desaturase is an essential gene in yeast, it was not present in the library we used for our loss-of-function genetic screens. We engineered an mga2Δ yeast strain in which APOE4 expression was no longer associated with a growth defect. We observed that complementation of the mga2Δ strains with either MGA2 or OLE1 under control of a constitutively active promoter rendered yeast cells sensitive again to APOE4 expression (Fig. S3G). Chemical inhibition of Ole1p with a small molecule (ECC145) (53), restored the growth of APOE4-expressing yeast cells in a dose-dependent manner without impacting the growth of GFP-expressing or APOE3-expressing cells (Fig. 3H). Lastly, we asked if MGA2 is involved in the APOE4-mediated accumulation of LDs. We measured the BODIPY 493/503 signal in the mga2Δ strain and found that the signal does not accumulate (Fig. 2I).

Fig. 3. Choline supplementation rescues APOE4-mediated lipid defects in yeast.

Fig. 3.

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(A) Shown is relative growth of APOE4-expressing yeast in synthetic media supplemented with ethanolamine (1 mM), choline chloride (1 mM) or choline bitartrate (100 μg/ml). Data are shown relative to the growth of the APOE3-expressing strain. Data are represented as mean ± SD, n=3 independent yeast colonies with at least two technical replicates each. ns, nonsignificant p > 0.05; *** p ≤ 0.001, Student’s t-test. (B) Graph shows growth of APOE4-expressing and APOE3-expressing yeast strains normalized to a wildtype strain after culture in synthetic media supplemented with choline chloride. Data represent measurements of three independent yeast colonies with at least two technical replicates each. (C) Western blot of whole cell extracts of yeast expressing APOE3 or APOE4 cultured in synthetic media with or without choline supplementation (1 mM), with PGK1 serving as loading control. (D) Bar graph shows intracellular triacylglycerides in APOE3-expressing and APOE4-expressing yeast cells grown in synthetic media with choline (1 mM) supplementation (square pattern) or vehicle (fill) as control. Data represent mean ± SD, n=3 independent yeast colonies. ns, nonsignificant p > 0.05; * p ≤ 0.05 by Student’s t-test. (E) Bar graph shows the fold change difference in intracellular triacylglycerides in APOE3-expressing and APOE4-expressing yeast cells grown in synthetic medium supplemented with choline (1mM) or vehicle as control. Triacylglycerides were stratified by the total number of unsaturated bonds present in the attached fatty acids. Data represent mean ± SD, n=3 independent yeast colonies measured. ns nonsignificant p > 0.05; * p ≤ 0.05 by Student’s t-test. (F) Representative fluorescence microscopy images of APOE3-expressing and APOE4-expressing yeast stained with BODIPY 493/503 stain for lipid droplets after growth in synthetic media supplemented with choline (1mM) or vehicle as control. Scale bar, 5μm. Dashed yellow lines demarcate individual yeast cells. Quantification of the number of lipid droplets is shown in the right panel (n=4 experiments, each with at least 30 yeast cells analyzed). Data are represented as mean ± SD , *** p ≤ 0.001, **** p ≤ 0.0001 by ANOVA.

Lipid droplets act to buffer against lipotoxicity by sequestering free fatty acids (FFAs) as triacylglycerides (54). To test whether a high burden of triacylglycerides altered the lipid buffering capacity of APOE4-expressing yeast cells, we treated this strain with oleic acid (C18:1), an unsaturated FFA. We found that whereas the growth of both wildtype and APOE3-expressing yeast was not affected by the addition of FFAs, the growth defect in the APOE4-expressing yeast was exacerbated by addition of oleic acid (Fig. S3H).

Choline supplementation is sufficient to rescue APOE4-induced lipid defects in yeast

Cellular lipid metabolism is shaped by both the genes and the environment (55). We asked whether yeast media differing in nutrient content could modify the growth of APOE4-expressing or APOE3-expressing yeast. Whereas yeast grown in synthetic media displayed a growth difference between APOE3-expressing and APOE4-expressing cells, yeast grown in a rich media (yeast extract-peptone) did not (Fig. S4A, S4B). This observation suggested that the APOE4 slow-growth phenotype depended on nutrient availability. This effect was specific to APOE4 as other proteins (Aβ1-42, TDP43) displayed a clear growth disadvantage in both synthetic media and yeast extract-peptone (YP) media (Fig. S4A). This finding suggested that environmental factors could modify APOE4 phenotypes in yeast.

Given that one of our top genetic screen hits, OPI1 is a negative regulator of phospholipid synthesis (48, 49, 56), we asked whether the soluble precursors of phospholipid synthesis, ethanolamine and choline (57), could modulate the APOE4-related growth defect (58). While ethanolamine did not influence growth of yeast expressing APOE4, addition of choline salts (choline chloride or choline bitartrate) to synthetic yeast media was sufficient to suppress the APOE4-associated growth defect (Fig. 3A) in a dose-dependent manner (Fig. 3B). Similar to the genetic rescue observed upon deletion of MGA2, choline supplementation did not simply decrease APOE4 protein (Fig. 3C), but rather uncoupled the presence of APOE4 from its growth-perturbing effects. Furthermore, the observed rescue was specific to APOE4, as synthetic medium supplemented with choline did not rescue the growth defect in yeast expressing Aβ1-42 (Fig. S4C). Given that choline is a growth-limiting factor in APOE4-expressing yeast cultured in synthetic medium, we measured intracellular levels of choline and the choline derivatives, phosphocholine and glycerophosphocholine. We found that, compared to APOE3-expressing yeast, APOE4-expressing yeast grown in synthetic media contained elevated concentrations of these molecules (Fig. S4D).

Choline (trimethylamine) serves as the head group for phosphatidylcholines and sphingomyelins, and functions as a signaling molecule and methyl donor (59). Both yeast and human cells can generate phosphatidylcholine by the choline-dependent Kennedy pathway or the choline-independent phosphatidylethanolamine methylation pathway (Fig. S4E) (57, 60). To understand which of these biosynthesis pathways was relevant to choline-dependent rescue of the APOE4 phenotypes, we deleted every enzyme in the phosphatidylcholine synthesis or phosphatidylethanolamine synthesis pathways (57). As expected, the genetic ablation of the phosphatidylethanolamine synthesis pathway did not impact the growth of APOE3-expressing or APOE4-expressing strains cultured in nutrient-rich yeast extract-peptone media (Fig. S4F). In contrast, the deletion of every enzyme acting in the phosphatidylcholine synthesis pathway rendered APOE4-expressing strains slow-growing, despite being grown in the nutrient-rich yeast extract-peptone media (Fig. S4F). We also observed differential growth between APOE3-expressing and APOE4-expressing strains upon deletion of HNM1, a transporter that carries choline and ethanolamine into yeast cells (61). We therefore concluded that without HNM1, CKI1, PCT1 and CPT1, yeast cells could not process the excess choline delivered by the yeast extract-peptone media to support the growth of APOE4-expressing yeast. Thus, the Kennedy pathway was necessary to sustain the growth of APOE4-expressing yeast.

APOE4-expressing yeast cells show elevated levels of phosphatidylcholine

To characterize the broader impact of choline supplementation on cellular lipid composition, we used LC-MS-based lipidomics to analyze lipids from APOE3-expressing and APOE4-expressing yeast grown in either synthetic medium alone or synthetic medium supplemented with choline. Along with the previously observed increases in triacylglycerides and unsaturated fatty acids upon APOE4 expression (Fig. 2B, S2D), APOE4-expressing yeast cells also displayed elevated total phosphatidylcholine when cultured in minimal synthetic medium (Fig. S4G). These data suggested that APOE4-expressing cells may have engaged the phosphatidylcholine synthesis pathway to provide an alternate sink for unsaturated fatty acids that would otherwise have accumulated in triglycerides and lipid droplets, resulting in an increased requirement for choline. Indeed, choline supplementation decreased both intracellular triacylglycerides (Fig. 3D) and the quantities of unsaturated fatty acids attached to triacylglycerides in APOE4-expressing yeast to levels found in APOE3-expressing cells (Fig. 3E). In line with this result, choline supplementation also prevented lipid droplet accumulation in APOE4-expressing yeast (Fig. 3F).

Targeting of lipid desaturation and triacylglyceride synthesis modifies APOE4-mediated lipid accumulation in iPSC-derived astrocytes

We then investigated whether our findings in yeast could be reproduced in human iPSC-derived astrocytes. Examining previously published transcriptomes of human iPSC-derived astrocytes (27), we noted that APOE4-expressing human iPSC-derived astrocytes showed a decrease in transcripts of fatty acid desaturases including SCD and FADS2 (Fig. S5A), suggesting that, as in yeast, fatty acid saturation enzymes could be mediating APOE4-associated lipid defects. We chemically interrogated this pathway by treating APOE4-expressing human iPSC-derived astrocytes in culture with a small molecule inhibitor of SCD1 (A939572) (62), the human homolog of yeast Ole1p. Inhibition of SCD1 resulted in a reduced number of lipid droplets (Fig. 4A) analogous to our finding in APOE4-expressing yeast (Fig. 2I).

Figure 4. Modification of APOE4-induced lipid droplet accumulation in human iPSC-derived astrocytes.

Figure 4.

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(A) Shown are representative microscopy images of APOE4/APOE4 iPSC-derived astrocytes treated with A939572 (inhibitor of Stearoyl-CoA desaturase-1, 100nM) or DMSO vehicle control, stained with LipidTox. Quantification of the lipid droplet number per cell is shown in the right panel. Each dot is an average of four wells with at least 20 cells measured (n=4 independent replicates). Data are represented as mean ± SD , ** p ≤ 0.01 by Student’s t-test. Scale bar, 25μm. The dashed red lines denote the boundaries of the cells. (B) Quantification of the lipid droplet number per cell in APOE4/APOE4 iPSC-derived astrocytes treated with a combination of small molecule inhibitors of Diacylglycerol O-acyltransferase 1 and 2 (iDGATs, 1μM) or DMSO as vehicle control. Each dot is an average of ~50 cells analyzed. Data represent mean ± SD, * p ≤ 0.05 by Student’s t-test. (C) Quantification of the number of lipid droplets in APOE4 or APOE3 iPSC-derived astrocytes treated with oleic acid (20μM) or vehicle control (n=4 experiments, and each experiment imaged at least 20 cells). Data are represented as mean ± SD , **** p ≤ 0.0001, ANOVA with multiple comparisons. (D) Representative microscopy images of APOE4 or APOE3 iPSC-derived astrocytes stained with LipidTox after culture in media supplemented with CDP-choline (100μM) or vehicle as control. Quantification of the lipid droplet number per cell is shown in the right panel (n=4 experiments, with at least 20 cells measured per experiment). Data represent mean ± SD, *** p ≤ 0.001 by ANOVA. (E) Bar graph shows intracellular triacylglycerides extracted from APOE3 or APOE4 iPSC-derived astrocytes grown in astrocyte media supplemented with CDP-choline (100μM) or vehicle as control. Data represent mean ± SD,with n=3 independent yeast colonies measured. ** p ≤ 0.01 by Student’s t-test. (F) Fold change in number of unsaturated bonds in fatty acids attached to triacylglycerides . Lipids were extracted from APOE3 and APOE4 iPSC-derived astrocytes grown in astrocyte media supplemented with CDP-choline (100μM) or vehicle as control. Data represent mean ± SD, n=3 experiments. (G) Representative microscopy images of APOE3 and APOE4 iPSC-derived astrocytes stained with Filipin III probe after culture in astrocyte media supplemented with CDP-choline (100μM) or vehicle as control. Quantification of Filipin III staining per cell (n=6-7 experiments, with at least 20 cells imaged) is shown in the right panel. Data represent mean ± SD, ** p ≤ 0.01, *** p ≤ 0.001 by ANOVA.

Triacylglyceride and phosphatidylcholine synthesis pathways share a common substrate, diacylglycerol (Fig. S5B). We hypothesized that chemically impairing the synthesis of triacylglycerides so that only diacylglycerol served as a substrate only for the synthesis of phospholipids would ameliorate the lipid droplet burden in APOE4-expressing human iPSC-derived astrocytes. We treated APOE4-expressing human iPSC-derived astrocytes with small molecule inhibitors of diglyceride acyltransferases 1 and 2 (63, 64), and observed a decrease in the lipid droplet number in the inhibitor-treated astrocytes compared to control untreated astrocytes (Fig. 4B, S5C). Therefore, APOE4-associated lipid defects may have arisen from the accumulation of endogenously synthesized lipids. Higher endogenous triacylglycerides and lipid droplet burden also suggested that APOE4-expressing human iPSC-derived astrocytes might present a genotype-specific sensitivity to challenge with exogenous lipids, as lipid droplets sequester FFAs into triacylglycerides to prevent lipotoxicity (54). Addition of the unsaturated fatty acid oleic acid to APOE4-expressing human iPSC-derived astrocytes in culture exacerbated lipid droplet accumulation (~3-fold) compared to APOE3-expressing human iPSC-derived astrocytes (Fig. 4C) (26), suggesting that APOE4-expressing human iPSC-derived astrocytes had reduced capacity to buffer excess lipids.

Choline supplementation reverses defective lipid homeostasis in human APOE4-expressing iPSC-derived astrocytes

Having shown that choline-stimulated synthesis of phosphatidylcholine restored lipid metabolism in APOE4-expressing yeast, we examined choline derivatives in metabolites extracted from our human iPSC-derived astrocytes. Similar to our observations in yeast, we found an increase in choline and glycerophosphocholine in APOE4 astrocytes, suggesting an increased demand for uptake of choline and its metabolites (Fig. S5D). Betaine, a choline derivative used in one-carbon metabolism but not in phosphatidylcholine synthesis, did not show changes in abundance between APOE genotypes (Fig. S5D) suggesting that exogenously acquired choline metabolites were preferentially channeled towards phosphatidylcholine synthesis in APOE4 astrocytes.

We then explored the effect of choline supplementation on APOE4-associated lipid perturbations in human iPSC-derived astrocytes in vitro. We cultured APOE3 and APOE4 isogenic astrocytes in either standard media or media supplemented with cytidine 5’-diphosphocholine (CDP-choline), a direct precursor of phosphatidylcholine synthesis by the Kennedy pathway (65). APOE4-expressing astrocytes treated with CDP-choline showed a decrease in lipid droplet number to that found in APOE3-expressing astrocytes (Fig. 4D). Further, lipidomic profiling showed that CDP-choline treatment resulted in the reduction of total triacylglycerides in APOE4 astrocytes (Fig. 4E). CDP-choline treatment also reduced the enrichment of unsaturated fatty acids in APOE4 astrocytes (Fig. 4F). These results confirmed that choline supplementation ameliorated the APOE4-induced lipid defects in iPSC-derived human astrocytes in vitro.

Choline supplementation rescues cholesterol accumulation associated with APOE4

Lipid droplets are composed not only of triacylglycerides but also of cholesterol and cholesterol esters (30, 54). Cholesterol regulation plays a key role in APOE-related diseases including both cardiovascular disease and AD (3, 66). We previously reported using a fluorescent probe Filipin III (67) that APOE4 astrocytes accumulate cholesterol (27). Cholesterol metabolism, including biosynthesis, efflux and cholesterol storage in lipid droplets, is tuned to the cholesterol/phosphatidylcholine ratio in mammalian cells (68). We therefore used Filipin III to examine intracellular cholesterol in human iPSC-derived astrocytes treated with CDP-choline or vehicle in vitro. While vehicle-treated APOE4-expressing astrocytes accumulated more cellular cholesterol than did APOE3-expressing astrocytes, APOE4 astrocyte cultures supplemented with CDP-choline did not show this defect (Fig. 4G).

Discussion

Relating genetic polymorphisms to their phenotypic outcomes is key to understanding the molecular bases of disease risk and pathogenesis. In this study, we investigated in human iPSC-derived astrocytes the molecular basis of APOE4-associated lipid defects including an aberrant accumulation of unsaturated fatty acids and their storage in triglyceride-rich lipid droplets in human iPSC- derived astrocytes. Previous results in astrocytes from mice with human APOE targeted replacement showed an increase in lipid droplets associated with the APOE4 genotype (26). Another study (3) specifically examined the transcriptomic profiles of human iPSC-derived glia, human glia from postmortem AD patient brain tissue, as well as glia from mice with human APOE targeted replacement. This study affirmed the central role of APOE4 in perturbing lipid metabolism pathways in both humans and mice.

In our cellular models, we found several lipid metabolic nodes that potentially could be manipulated to improve lipid regulation in APOE4-expressing yeast cells and human iPSC-derived astrocytes. In particular, we discovered that stimulating phosphatidylcholine synthesis via the Kennedy pathway using exogenous choline supplementation was sufficient to reverse the APOE4-induced lipid defects in both yeast and astrocytes while not impacting APOE4 expression. Compromised activity of the Kennedy pathway has been shown to result in accumulation of intracellular fats in humans and in mouse models bearing mutations in the PCYT1A gene (69, 70). Interestingly, a genetic polymorphism in the PEMT gene that functions in an alternative phosphatidylcholine synthesis pathway has been associated with AD in a Chinese population (71).

A number of APOE4-associated diseases have been linked to alterations in glucose and fatty acid metabolism (72, 73). Lipid metabolism is an area of active investigation in research on aging and neurodegenerative diseases (1–4, 6, 27). Indeed, AD-associated intracellular lipid accumulation has been reported in the postmortem brain tissue of AD patients (5), human fibroblasts (74), transgenic mouse models of AD (5), mouse primary culture of neurons (26), and flies (75) expressing APOE4.

Many lipid species have been implicated in neurotoxicity or selected as markers for early diagnosis of AD (76). Examination of a number of postmortem AD brains revealed a reduction in choline, ethanolamine, phosphatidylcholine and phosphatidylethanolamine and an increase in their degradation products (77). Our data suggest that the APOE4 genotype may impose additional choline dependency thus exacerbating the cholinergic deficit found in AD brains. In fact, our analysis of transcriptomes in postmortem brain tissue from APOE4 carriers revealed that these individuals had increased expression of the high-affinity choline transporter SLC44A1 (Fig. S1F).

Over the last three decades, the link between age-related cognitive decline and compromised lipid metabolism has been increasingly appreciated. As a result, nutritional interventions aimed at elevating the synthesis of phospholipids became attractive as a noninvasive therapeutic strategy (78, 79). CDP-choline itself has been previously proposed as a dietary supplement (80, 81). In general, the demand for dietary choline may depend on multiple factors like age, ethnicity, and sex (82). For example, women tend to develop choline deficiency more often than do men (65). Intriguingly, women with an APOE4 genotype show an increased risk of AD (83, 84). Although most clinical trials for choline derivatives supplementation in AD did not stratify individuals by APOE genotype, those that did noted greater efficacy of choline supplementation in APOE4 populations than in those without the APOE4 genotype (85). Our findings could point to a therapeutic strategy where choline supplementation is targeted to those with the APOE4 genotype. Given our study is limited to in vitro cell models, future research is required to expand the findings in animal models and APOE4 carriers. For example, it would be interesting to examine how specific dietary changes impact brain metabolites and lipids in APOE4 models and carriers and whether this impacts progression of APOE4-associated diseases. Such lines of research could identify the early-stage biomarkers of APOE4-dependent pathological states.

Lipid homeostasis impacts many cellular processes, including membrane synthesis, vesicular trafficking, protein turnover and cell proliferation (86). Our study suggests that APOE4’s status as a genetic risk factor for AD and other distinct diseases originates from the pleiotropic consequences of perturbed lipid metabolism characterized by increases in unsaturated triacylglycerides and a higher phosphatidylcholine requirement compared to the APOE3 genotype. Our work provides a framework for understanding APOE4 function in disease risk and provides a rationale for genotype-specific dietary supplementation to diminish the detrimental consequences of the APOE4 genetic polymorphism.

Materials and Methods

The objective of this study was to establish the molecular consequences of the APOE4 allele in yeast and human iPSC-derived astrocytes in vitro and to modulate theses effects using chemical and genetic interventions. The study used APOE3 and APOE4 isogenic pairs of human iPSC-derived astrocytes and microglia, and yeast expressing human APOE4 or APOE3. We measured the intracellular lipid content of yeast and human astrocytes using fluorescent probes and mass spectroscopy, and yeast growth after chemical and genetic perturbations. The sample sizes were selected based on previous literature. The number of replications varied and is indicated in each figure legend. The study was not blinded.

Human iPSC-derived cell culture

iPSC-derived lines were generated from the parent line Coriell #AG09173 (female APOE3/3 parental) and #AG10788 (female APOE4/4 parental). Differentiation into astrocytes was performed as previously described (27). iPSCs were derived into microglia using a previously described protocol (87) with minor modifications.

Lipid droplet analysis

To assay the number of lipid droplets in astrocytes, equal number of cells were plated on the 96-well μ-Plate (Ibidi, 89626) in astrocyte medium or astrocyte medium supplemented with SCD1 inhibitor (A939572, 100nM), inhibitors of DGAT1 (PF-04620110, 1μM) and DGAT2 (PF-06424439, 1μM), or CDP-choline (Sigma-Aldrich, 100μM). After 12-14 days cells were stained with LipidTox (ThermoFisher/Molecular Probes) or Filipin III (Sigma-Aldrich) according to manufacturer’s protocols. To induce lipid droplet formation, astrocytes were incubated in the presence of oleic acid (Sigma-Aldrich, 20μM) or vehicle (control). After 6 hours, the cells were carefully washed and stained with LipidTox, and proceeded as above. For lipid droplet count analysis in microglia, cells were plated at a density of 10,000 cells per well in a 96-well glass bottom plate (IBIDI) in iMBM media prepared with custom DMEM/F12 lacking choline (Gibco), and supplemented with a low amount of choline chloride (Sigma-Aldrich, 15 μM). The cells were cultured for two weeks following plating without media changes and were then fixed and stained using LipidTox Red (Thermo Fischer/Molecular Probes).

GTEx transcriptomics analysis

The genotypes of APOE ε4 (rs429358) for 838 subjects in the GTEx project (version 8) were extracted from vcf files (hg38) called from whole genome sequencing data. Differential gene expression analysis of APOE ε4 (dichotomized into two groups, APOE ε4+ and APOE ε4−) was performed using DESeq2 (88) adjusted for RIN, age, sex and five remove unwanted variation (RUV) components. The mean age of death of the sample is 57.56 +/− 10.34(S.D). 68.8% of the individuals are male, and 89.8% are white.

Yeast

Saccharomyces cerevisiae strains were isogenic to the BY4741 background (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0). For the deletion of genes, a PCR product containing a selection cassette and short sequences for site-specific homologous recombination was transformed to yeast as described (89). Yeast were grown with at 30°C in synthetic media (SC) with YNB or nutrient-rich (YP) media supplemented with 2% glucose (or 1% galactose only in Fig. S2A and genetic screens). Yeast growth was measured using Epoch 2 Microplate Spectrophotometer (BioTek) and quantified as an area under the growth curve within the 24 hours post induction (starting at OD 600 nm =0.1).

Ole1p inhibitor ECC145 was produced by ChemBridge Co. (San Diego, CA, USA) and was added at indicated concentrations (10, 20, 40μM) to the synthetic media containing β-estradiol (100nM).

For genetic screens, we expressed APOE variants under galactose-inducible promoter. For screen validations and subsequent follow-up experiments, we adopted β-estradiol-inducible system that is independent on carbon source (37, 90). We utilized this system to express APOE variants, TDP-43 and Aβ1-42. For the vast majority of experiments, we used β-estradiol at the 100 nM concentration, unless stated otherwise (e.g. to elicit a mild growth defect, then 10nM of β-estradiol was applied). All the plasmids used in this study were deposited at Addgene (#78653).

Synthetic Genetic Array (SGA)

Bait strains expressing APOE variants under galactose promoter, either integrated in the LEU2 locus or on a centromeric plasmid, were engineered in the screening strain y7092 (gift from the Boone laboratory). Screens were performed as previously described (39, 91).

Western blotting

The standard trichloroacetic acid (TCA) precipitation-based method was used to extract protein from yeast. Precipitates were resuspended in HU buffer (8 M urea; 5% SDS; 1mM EDTA; 1,5% DTT; 1% bromophenol blue; 200mM Tris-HCl pH 6,8) and incubated at 70°C for 15 min prior to loading on SDS-PAGE gels. To separate proteins by SDS-PAGE, the 4-12% gradient Bis-Tris NuPAGE gels (Invitrogen) run in MES buffer were used. Gels were transferred to a nitrocellulose membrane using the iBlot2 system (ThermoFisher). Primary antibodies were probed overnight followed by several washing steps and incubation with fluorescent secondary antibodies. Signal was measured using the Li-COR (Odyssey) system. Antibodies used in this study: Anti-APOE (mouse monoclonal E8, Santa Cruz), anti-PGK1 (rabbit polyclonal, ABIN568371), anti-mouse secondary 800CW dye conjugate (LI-COR, 926-32212), anti-rabbit secondary 680CW dye conjugate (LI-COR, 926-68073).

Lipidomics analysis

For lipid analysis, iPSC-derived astrocytes (derived from parental line Coriell #AG09173) were seeded in 10cm dish for 12-14 days and grown in the astrocyte medium with 2% dialyzed FBS and supplemented with vehicle or CDP-choline (100μM). Yeast were grown in synthetic media or synthetic media supplemented with choline chloride (1mM). The expression of APOE variants was induced with β-estradiol (100nM) for 8 hours. 10 ODs equivalent of yeast were collected, washed twice with ice-cold ammonium bicarbonate, and resuspended in 1ml of ammonium bicarbonate. Zirconia beads (Sigma) were added to mechanically disrupts cells using TissueLyser (Qiagen) (28). For both yeast and astrocytes, lipids were extracted using the Folch method (92).

Cytometry analysis after BODIPY 493/503 staining

Yeast strains were grown to saturation in synthetic media with 2% glucose and subsequently diluted to OD600 = 0.1 in the synthetic media containing β-estradiol (100nM) for 8 hours. MACSQuant VYB cytometer with a 96-well plate platform (Miltenyi Biotech) was used to acquire 10,000 events in biological triplicates. The BODIPY 493/503 signal (green fluorescence) was measured with the B1 channel (525/50 filter) and median fluorescence values were calculated. Data was processed using FlowJo (FlowJo LLC).

Fluorescence microscopy

Yeast strains were grown to saturation in synthetic media with 2% glucose, diluted to OD600 = 0.1 in the synthetic media containing β-estradiol (100nM) for 8 hours. Cells were diluted to OD600 of 0.2 in the same media and transferred to a concanavalin A-coated 96-well plate. All images were acquired using a Nikon Eclipse Ti-E inverted microscope equipped with a Nikon Plan Apo 100x oil objective (NA 1.4) and a CCD camera (Andor technology). For all iPSC-derived cell type imaging, images were acquired using a Nikon Plan Apo 63x oil objective (NA 1.4) using a Nikon Eclipse Ti-E inverted microscope and a CCD camera (Andor technology) or a Zeiss LSM710 inverted confocal microscope with a 40x water immersion objective and Z-stacks with a stack height of 0.5μm. Imaging was analyzed using Imaris (Bitplane) spot counting and area functions.

Statistical Analysis

GraphPad Prism software was used to process data, calculate statistics and prepare graphs. For determining statistical significance, we used the unpaired versions of t-tests, unless stated otherwise. FlowJo was used to process FACS data. Images were processed using Imaris and Fiji (93).

Supplementary Material

Figure S1

Fig. S1. Increase in lipid droplets in human iPSC-derived APOE4/APOE4 astrocytes and microglia.

Figure S2

Fig. S2. Yeast accumulate lipid droplets and lipid droplet-resident proteins upon expression of human APOE4.

Figure S3

Fig. S3. APOE4 expression in yeast leads to a growth defect in a dose-dependent manner.

Figure S4

Fig. S4. Choline supplementation rescues the APOE4-mediated growth defect in yeast.

Data file S1

Data file S1. Individual level data for all figures.

Figure S5

Fig. S5. Analysis of lipid metabolism in the iPSC-derived APOE4/APOE4 astrocytes.

Supplementary materials

Acknowledgments:

This work is dedicated to our inspiring mentor Susan Lindquist. We thank members of the Lindquist, Tsai, and Sabatini labs for helpful suggestions. We thank Prathapan Thiru for technical assistance with bioinformatics, Yuan-Ta Lin for help with iPSC lines and sharing of transcriptomics data and Jose Valderrain for analysis of large-scale datasets. We thank Daniel Jarosz, David Pincus, Peter Tsvetkov, Justin Rettenmaier, Marcello Maresca, Mohammad Bohlooly, Linda Clayton, and Brooke Bevis for suggestions.

Funding:

GS was supported by an EMBO Fellowship (ALTF 829-2015) and was a HHMI fellow of the Helen Hay Whitney Foundation. PN was supported by the Helen Hay Whitney Foundation, by a NIH/NIA K99 award (AG055697-03) and by the Intramural Research Program of the NIH/NIDDK. NK is an HHMI fellow of the Damon Runyon Foundation. DMS is supported by an NIH/NCI grant (R01 CA103866). He is an investigator of the Howard Hughes Medical Institute and an American Cancer Society Research Professor. The work in the Lindquist and Tsai labs was supported by the Neurodegeneration Consortium, the Robert A. and Renee E. Belfer Foundation, the Howard Hughes Medical Institute, the Ludwig Family Foundation, support from Kara and Stephen Ross, and NIH grants: RF1 AG048029, RF1 AG062377, U01 NS110453, R01 AG062335 and R01 AG058002 (to L-H. T.). The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the NIH, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS.

Footnotes

Competing interests: SL was a co-founder of Yumanity Therapeutics. LHT is a member of the Scientific Advisory Board of Yumanity Therapeutics. PN and SL are co-inventors on US Patent PCT/US2015/049674 (Cells expressing Apolipoprotein E and uses thereof). GS, PN, JMB, LHT are co-inventors on patent application 63/023,698 (Use of choline supplementation as therapy for APOE4-related disorders). GS and AAA are currently employees and shareholders of AstraZeneca. CAL is a paid consultant for ReviveMed Inc.

Data and materials availability: Plasmids used in the study have been deposited at Addgene with accession number #78653.

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Associated Data

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

Supplementary Materials

Figure S1

Fig. S1. Increase in lipid droplets in human iPSC-derived APOE4/APOE4 astrocytes and microglia.

NIHMS1706136-supplement-Figure_S1.ai (623.9KB, ai)
Figure S2

Fig. S2. Yeast accumulate lipid droplets and lipid droplet-resident proteins upon expression of human APOE4.

NIHMS1706136-supplement-Figure_S2.ai (574KB, ai)
Figure S3

Fig. S3. APOE4 expression in yeast leads to a growth defect in a dose-dependent manner.

NIHMS1706136-supplement-Figure_S3.ai (3.5MB, ai)
Figure S4

Fig. S4. Choline supplementation rescues the APOE4-mediated growth defect in yeast.

NIHMS1706136-supplement-Figure_S4.ai (3.4MB, ai)
Data file S1

Data file S1. Individual level data for all figures.

NIHMS1706136-supplement-Data_file_S1.xlsx (129.7KB, xlsx)
Figure S5

Fig. S5. Analysis of lipid metabolism in the iPSC-derived APOE4/APOE4 astrocytes.

NIHMS1706136-supplement-Figure_S5.ai (5.4MB, ai)
Supplementary materials
NIHMS1706136-supplement-Supplementary_materials.docx (2.8MB, docx)

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