APOE4 disrupts intracellular lipid homeostasis in human iPSC-derived glia

Abstract

The E4 allele of the apolipoprotein E gene (APOE) has been 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 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, but not APOE3, disrupted the cellular lipidomes of human induced pluripotent stem cell (iPSC)-derived astrocytes generated from fibroblasts of APOE4 or APOE3 carriers, and of yeast expressing human APOE isoforms. We combined lipidomics and unbiased genome-wide screens in yeast with functional and genetic characterization to demonstrate that human APOE4 induced altered lipid homeostasis. These changes resulted in increased unsaturation of fatty acids and accumulation of intracellular lipid droplets both in yeast and in APOE4-expressing human iPSC-derived astrocytes. We then identified genetic and chemical modulators of this lipid disruption. We showed that supplementation of the culture medium with choline (a soluble phospholipid precursor) restored the cellular lipidome to its basal state in APOE4-expressing human iPSC-derived 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. Our study suggests that manipulating lipid metabolism could be a therapeutic approach to help alleviate the consequences of carrying the APOE4 allele.

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

(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.

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

(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.

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

(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.

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

(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.