Fig. 4.

NHE6 restores defective Aβ clearance in ApoE4 astrocytes. (A) Endosomal pH is precisely tuned by a balance of proton pumping (acidification) through the V-ATPase and proton leak (alkalization) via NHE6. (B) Box plots of NHE6 transcript levels in postmortem brains extracted from a large microarray dataset (GSE15222; n = 363) showing significant down-regulation of NHE6 expression in ApoE4⁺ AD brains (****P = 1.2 × 10⁻¹⁰, Student’s t test). (C, Top) Western blot analysis of NHE6 protein revealed that it was ∼45% lower in ApoE4 astrocytes, relative to ApoE3. (C, Bottom) Densitometric quantification of the monomeric and dimeric forms of NHE6 relative to β-actin (***P = 0.0009, Student’s t test; n = 4). (D) Aβ levels were significantly higher in the brains of the NHE6-null mouse model (NHE6^KO), relative to WT (**P = 0.0013, Student’s t test; n = 5 per condition), consistent with our hypothesis. Aβ was measured using ELISA and normalized to total protein concentration in the brain homogenate. (E) Fluorescence-activated cell sorting (FACS) scatter plots (Left) and quantification (Right) demonstrating Aβ internalization by an empty vector expressing ApoE3 (orange) and ApoE4 (gray) astrocytes and ApoE4 astrocytes with restored NHE6 expression (green). Note the remarkable correction of Aβ clearance in ApoE4 astrocytes with NHE6 expression to ApoE3 levels (****P = 4.9 × 10⁻⁶, Student’s t test; n = 3). (F) Representative surface immunofluorescence images (Left) and quantification (Right) of nonpermeabilized cells showing an ∼2.5-fold increase in plasma membrane LRP1 expression in ApoE4 cells expressing exogenous NHE6 (****P = 6.02 × 10⁻¹², Student’s t test; n = 40 per condition) (SI Appendix, Figs. S2 and S5–S7). DIC, differential interference contrast. (Scale bars, 2.5 μm.)