Fig. 1: Common phospholipid signature between APOEε4/4 and APOEε3/3 human AD brains and APOE3 and E4 native lipoproteins.

a, b The phospholipid compositions of APOEε3/3 (n = 7), APOEε3/4 (n = 8), and APOEε4/4 (n = 7) brains from AD patients and native APOE3 and APOE4 lipoproteins were determined using MDMS-SL. Brain samples were from the inferior parietal lobe. Native lipoproteins were derived from pooled APOE3 or APOE4 astrocyte conditioned media and two samples of each were measured. Bar charts of nine lipid classes analyzed by Shotgun lipidomics in human (a) and native lipoprotein (b) datasets. Analysis is by ANOVA followed by Tukey’s multiple comparison test. PE, p = 0.0256 (E33 vs. E34), p = 0.0021 (E3/3 vs. E4/4); PI, p = 0.0432 (E33 vs. E34), p = 0.0094 (E3/3 vs. E4/4); PS, p = 0.0464 (E33 vs. E34), p = 0.0274 (E3/3 vs. E4/4); SM, p = 0.0357 (E33 vs. E34), p = 0.0007 (E3/3 vs. E4/4); LPE, p = 0.0139 (E33 vs. E34); PG, p = 0.0326 (E3/3 vs. E4/4); PA, p = 0.0177 (E33 vs. E34), p = 0.0039 (E3/3 vs. E4/4). c Correlation matrix of the nine lipid classes analyzed in human and lipoprotein lipidomics data shows correlation scatter plots (bottom-left), histograms of lipid amount (diagonal), and correlation coefficients between lipid classes (top-right). Purple dots denote APOEε3/3, green dots denote APOEε4/4. Axes, pmol for each lipid species. d, e Volcano plots depict all 103 lipid species commonly identified in the human (d) and lipoprotein (e) datasets. Red denotes species which are significantly upregulated in APOEε3/3 brains or APOE3 lipoproteins. X axis, log2 fold change and y axis, –log10P value for all lipid species. e–j Data for native APOE3 or APOE4 lipoproteins derived from astrocyte conditioned media. f–h LC/MS analysis of five Phospholipid classes of APOE3 and E4 lipoproteins. f tSNE plot shows unique clustering of all lipids from lipoprotein samples according to APOE isoform: E3 (purple) and E4 (green). g Bar charts of major lipid classes of E3 and E4 native lipoproteins comprising all the lipids species shown in panel h. Analysis by two-tailed unpaired t-test. PC, p = 0.0152; PE, p = 0.0390; PI, p = 0.0417; PS, p = 0.0003. h Bar chart representing fold change of E3 compared to E4 lipoproteins for individual lipid species from five lipid classes. Significantly impacted species which are found in abundance from the PS and PE classes are labeled. i Top: Native gel electrophoresis probed for APOE. The diameter of native markers (in nm) are shown on the left. The migration of lipidated and non-lipidated APOE is shown on the right indicated with brackets. Bottom: SDS-NuPAGE gel followed by Western blot for APOE. j The size distribution of E3 (purple) and E4 (green) native lipoproteins was assessed by dynamic light scattering using a Zetasizer. Lipidomic analysis was performed on two independent APOE preparations measured in duplicate. The phospholipid content for each APOE isoform was normalized to APOE protein amount as determined by SDS NuPAGE. Bars represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. CL Cardiolipin, LPC lysophosphatidylcholine, LPE lysophosphatidylethanolamine, PA phosphatidic acid, PC phosphatidylcholine, PE phosphatidylethanolamine, PG phosphatidylglycerol, PI phosphatidylinositol, PS phosphatidylserine, and SM sphingomyelin.