Mechanisms of endosomal dysfunction in APOE4.

Abstract

Lipid poor apolipoprotein E (ApoE) aggregates are central to the pathogenesis of Alzheimer's disease (AD), promoting cerebral amyloid-β plaques formation, and altering endosomal recycling of apoE complexed proteins. Activating the ATP binding cassette A1 (ABCA1) to lipidate ApoE reduces its aggregation, promotes ApoE recycling and presents an attractive mechanism to rescue AD pathology. ABCA1 was immunoprecipitated using anti-GFP beads (Chromotek) from ApoE3/ApoE4 treated Hela-ABCA1GFP cell lysates. A unbiased proteomics (LC-MS) screen was employed to indentify ABCA1 binding proteins from the immunoprecipitated samples. E3/E4 AD (n=9, cogdx=4) and E3/E3 AD (n=11, cogdx=4) patient inferior frontal cortex from the Religious Order Study and the Memory and Aging Project (ROSMAP) were lysed and analyzed by Western blot for targets from proteomics screen. ApoE4 was associated with lower astrocytic ABCA1 activity. We identified caveolin-1 (Cav-1), an integral membrane protein in plasma membrane lipid rafts, as one of the ABCA1-binding proteins that regulate ABCA1 cell surface level. Using multiple models, we observed elevated Cav-1 in primary astrocytes and brains of ApoE4- targeted replacement (TR) mice. Knockdown Cav-1 through siRNA treatment restored ABCA1 surface expression level, underscoring the importance of Cav-1 in ABCA1 trafficking. In AD patient samples from ROSMAP, Cav-1 protein expression in E3/E4 AD inferior frontal cortex (n=9) demonstrated non-significant increase from 1.2 to 2.6 (normalized to β-actin) compared to E3/E3 AD (n=11). Ongoing efforts are planned to expand the human study sample size. ApoE4 is associated with elevated Cav-1 level and knockdown of Cav-1 can restore ABCA1 surface expression. Our work provides a deeper understanding of the critical mechanisms that define the pathogenicity of ApoE4 and can ultimately identify key pathways that regulate endosomal dysregulation in ApoE4.