Fig. 2.

Reduced surface expression of LRP1 receptor in ApoE4 astrocytes. (A) Four independent approaches to evaluate ApoE isotype-specific surface expression of LRP1. (B) Surface biotinylation (Left) and quantification (Right) of biological triplicates showing that plasma membrane levels of LRP1 are depressed by ∼50% in ApoE4 astrocytes, relative to ApoE3 (**P = 0.0022, Student’s t test; n = 3). Plasma membrane protein Na⁺/K⁺ ATPase is used as a loading control. (C) Fraction of LRP1-positive cells quantified following surface antibody labeling and fluorescence-activated cell sorting (FACS) analysis of 10,000 live, nonpermeabilized cells in biological triplicates. Unstained cells were used as a control. Note the ∼55% lower surface LRP1 positivity in ApoE4 relative to ApoE3 (****P = 4.8 × 10⁻⁻⁵, Student’s t test; n = 3). (D) Representative surface immunofluorescence micrographs (Left) and quantification (Right) showing prominent LRP1 staining on the cell surface and processes and faint, ∼49% lower, labeling on ApoE4 cells (****P = 2.4 × 10⁻¹⁶, Student’s t test; n = 75 per condition). (E) Plasma membrane level of LRP1 receptor was monitored by a ligand (fluorescent Aβ)-binding assay performed on ice that only allows Aβ to bind surface receptors. Representative images are shown (Left), and mean fluorescence ± SD was plotted (Right). Surface-bound Aβ was 66% lower in ApoE4 astrocytes, relative to ApoE3 (****P = 2.2 × 10⁻²⁴, Student’s t test; n = 75 per condition). (F, Left) Confocal micrographs revealing a striking loss of LRP1 from astrocyte processes (white arrow) and increased perinuclear accumulation in ApoE4 cells, relative to ApoE3. (F, Right) Zoomed-in images of the boxed regions. Increased perinuclear accumulation of LRP1 in ApoE4 cells is also evident in orthogonal slices (Z) (black arrows). (G) Confocal micrographs revealing prominent colocalization of LRP1 (green) with TFN (red) in DAPI (blue)-stained ApoE4 astrocytes. Colocalization is evident in the merge and orthogonal slices (Z) as yellow puncta (SI Appendix, Figs. S2–S4). DIC, differential interference contrast. (Scale bars, 10 μm.)