Mechanisms of reduced Apolipoprotein E4 lipidation in iPSC‐derived astrocytes

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AbstractBackgroundCarrying the apolipoprotein E ε4 (APOE4) allele is the highest known genetic risk factor for late‐onset Alzheimer’s disease (LOAD). Although how APOE4 leads to LOAD is not known, dysregulated endo‐lysosomal trafficking and lipid metabolism are key defects associated with APOE4. Cholesterol accumulation in glia instigates intracellular trafficking defects, increases cellular stress, and disrupts secretion. Additionally, enlarged endosomes were observed in the frontal lobes of APOE4‐carrying individuals, decades before the appearance of Aβ fibrilization. Therefore, we asked if cholesterol and endo‐lysosomal trafficking dysregulation in APOE4 carriers were due to differential lipidation of APOE3 and APOE4.MethodLocalization of ATP Binding Cassette Subfamily A Member 1 (ABCA1) in early, late, and recycling endosomal compartments were analyzed in human isogenic induced pluripotent stem cell (iPSC)‐derived neural cells and in cell lines treated with recombinant APOE in microscopic images. Membrane and cytoplasmic ABCA1 were compared using Western blots. Secreted high‐density lipoprotein (HDL) particles containing fluorescently tagged APOE3 or APOE4 were analyzed using ion‐mobility spectrometry. CS‐6253, an ABCA1 agonist peptide, was used to increase APOE lipidation.ResultThe protein levels of ABCA1, which mediates cholesterol and phospholipid efflux to the nascent apolipoprotein particles, were lower in APOE4/4 iPSC‐derived astrocytes compared to astrocytes derived from isogenic APOE3/3 iPSCs. Advanced image analysis revealed lower ABCA1 protein levels in Rab11+ recycling endosomes and less colocalization between ABCA1 and Rab7, a late endosome marker, in APOE4/4 astrocytes than APOE3/3 astrocytes. Consequently, less ABCA1 was detected at the cell membrane. Combined with previous data, these findings suggest APOE4 lipidation was reduced due to reduced ABCA1 activity. Additionally, BHK cells treated with recombinant APOE3 or APOE4 showed differences in ABCA1 localization that were altered by increasing APOE lipidation by activating ABCA1 via CS‐6253. Finally, fluorescently tagged APOE3 and APOE4 were incorporated in HDL particles and were secreted whose sizes were altered after CS‐6253 treatment, suggesting differences in lipidation levels.ConclusionWe demonstrated that diminished APOE4 lipidation is, at least in part, due to dysregulation of endo‐lysosomal trafficking of ABCA1 and that increasing APOE4 lipidation could alleviate cellular defects.