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eSymposia | Autophagy: Mechanisms and Disease


Investigation of mechanisms involved in Apolipoprotein E autophagic degradation and endocytosis


Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Description

Investigation of mechanisms involved in Apolipoprotein E autophagic degradation and endocytosis Gianna M. Fote1, Nicolette R. Geller2, Nikolaos Efstathiou3, Demetrios G. Vavvas3, Jack C. Reidling4, Leslie M. Thompson1,2,4,5, Joan S. Steffan*2,4 1UC Irvine Department of Biological Chemistry, 2UC Irvine Department of Psychiatry and Human Behavior, 3Harvard Medical School Department of Opthalmology, 4UC Irvine MIND Institute, 5UC Irvine Department of Neurobiology and Behavior The Apolipoprotein E4 allele (APOE4) is the greatest genetic risk factor for late-onset Alzheimer’s disease (AD). Compared to the more common APOE3, APOE4 expression results in enlarged endosomes and reduced autophagic flux. Impaired endolysosomal trafficking has been proposed as one potential mechanism contributing to AD. Levels of APOE4 are reduced relative to APOE3 in human tissue, and APOE4 is believed to be degraded rapidly, but the mechanism of degradation is unknown. We have found that APOE is degraded by the lysosome, and have investigated autophagic mechanisms of APOE degradation. Lysosomal de-acidification with bafilomycin A1 (Baf) causes APOE accumulation in transiently transfected immortalized neuronal cells (St14a). Both Baf and Brefeldin A (BFA), which disrupts the golgi apparatus, prevent pH-sensitive dual-tagged APOE-mCherry-SepHluorin from entering acidic compartments and fluorescing red. To investigate the mechanism of APOE lysosomal degradation, we knocked down autophagy proteins Lamp2, Atg7, Stx17, and non-canonical autophagy protein Rubicon. In mouse brain tissue, Lamp2 knockout results in accumulation of mouse APOE in vivo. In transfected St14a cells, dual-tagged APOE3 and APOE4 significantly accumulate with Lamp2A knockdown, but APOE2, which is protective for AD, does not. Imaging of APOE3-mCherry shows accumulation around the periphery of Lamp2A knockdown lysosomes, and more APOE3 was found to immunoprecipitate with lysosomes in Lamp2A knockdown cells. In human hepatic HepG2 cells, knockdown of Lamp2A, Atg7 or Stx17 all significantly increase endogenous APOE3 levels. Atg7 knockdown also impairs endocytosis of APOE, as does knockdown of Rubicon. Endocytosis of APOE3 significantly increases LC3 lipidation in St14a and HepG2 cells, suggesting that APOE internalization may be LC3-dependent. GABARAPL1, another member of the Atg8 family, co-localizes with endocytosed APOE3 following treatment with the fusion inhibitor chloroquine. Imaging of the endocytosis of fluorescent APOE suggests that APOE is endocytosed in an isoform-dependent manner, with APOE4 endocytosed more robustly than APOE3, and APOE2 endocytosed the least. In conclusion, our data suggest that APOE is degraded by the lysosome through autophagy that may require Lamp2A, Atg7, and Stx17, and may enter the cell through LC3-dependent endocytosis. APOE4, the AD risk allele, appears to be degraded by the lysosome through autophagy and to be endocytosed more robustly than APOE2, suggesting a possible mechanism for rapid APOE4 degradation observed in tissue, which may contribute to AD pathogenesis. Understanding how APOE traffics through the endolysosomal system may lead to insight into mechanisms that contribute to AD progression, and may be targeted to develop novel therapies for AD. Funding sources: NIH AG016573, F30 AG060704

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