LIPID DROPLET TARGETING AND FUNCTION OF APOLIPOPROTEIN E

APOE is the strongest genetic risk factor for late-onset Alzheimer’s Disease (AD). Individuals with one or two copies of the APOE4 variant are at an increased risk of developing AD compared to those homozygous for the APOE3 variant. In the central nervous system, apolipoprotein E (APOE) is highly expressed in astrocytes. Astrocytes secrete APOE-coated lipoprotein particles, which mediate bidirectional lipid transport between astrocytes and neurons. However, the role of APOE in intracellular lipid metabolism is not understood, nor is the mechanism by which the E4 variant predisposes individuals to AD. We demonstrate that in response to neutral lipid synthesis, APOE can divert from translocation into the endoplasmic reticulum (ER) lumen and traffic to the cytoplasmic surface of lipid droplets (LDs) during LD biogenesis in astrocytes. This raises the possibility that APOE plays previously unrecognized roles in cellular lipid metabolism by acting directly on LDs. Using immunogold electron microscopy and live-cell imaging, we found that APOE translocates onto LDs via membrane bridges at ER-LD contact sites. Expression of APOE truncation mutants indicated that the C-terminal amphipathic alpha-helical domain is required for LD targeting. APOE knockdown promotes more unsaturated triglycerides and a smaller number of larger LDs upon OA loading. This LD size distribution phenotype was rescued by expressing chimeric APOE that targets only LDs. Similar to APOE knockdown, APOE4-expressing astrocytes also form a small number of large LDs that additionally exhibit impaired turnover and increased sensitivity to peroxidation. Lipidomics studies revealed that E4 cells also contain more unsaturated triglyceride as well as less unsaturated phospholipid. Finally, using affinity purification-mass spectrometry, we identified interacting partners of APOE on LDs. Our data indicate that APOE plays a previously unrecognized role as an LD surface protein that regulates LD size and composition. We hypothesize that APOE affects LD composition and size via modulating the activity of interacting partners on the LD surface. We propose that APOE4 astrocytes with large, unsaturated LDs are sensitized to lipid peroxidation or lipotoxicity, which could contribute to Alzheimer’s disease risk.