Figure 1. LRP1 minireceptor increases the uptake of Aβ42 in N2a-mLRP4 cells.

A, cell lysates from N2a cells stably expressing LRP1 minireceptor (N2a-mLRP4) or empty pcDNA3 vector (N2a-pcDNA3) were analyzed by 7.5% SDS-PAGE and Western blotted with anti-HA or anti-LRP1 antibodies. Both the endoplasmic reticulum precursor (open arrowhead) and the processed forms after furin cleavage (closed arrowhead) are readily detected with anti-HA antibodies. The level of LRP1-85 in N2a-pcDNA3 cells is negligible compared to that in N2a-mLRP4 cells. B, flow cytometric analyses of N2a cells stably transfected with pcDNA3 and mLRP4. Negative controls without primary antibody are indicated with light lines, whereas the signals from cell surface receptor staining are shown with dark lines. 84% of N2a-mLRP4 showed cell surface expression of the LRP1 minireceptor. C, LRP1 minireceptor expression increases the endocytosis of RAP in N2a cells. N2a-pcDNA3 and N2a-mLRP4 cells were incubated with 500 nM Alexa488-RAP at 37°C for 30 min, then fixed and analyzed by fluorescence microscopy. D, flow cytometric analysis from experiments as in C. N2a-pcDNA3 and N2a-mLRP4 cells were detached and additionally treated with pronase to remove cell surface-associated RAP. Quantification of FACS experiments indicates that N2a-mLRP4 cells internalize approximately 8 times more RAP than N2a-pcDNA3 cells. ** p<0.01, t-test. n = 3. E, LRP1 minireceptor expression increases the endocytosis of Aβ42 in N2a cells. N2a-pcDNA3 and N2a-mLRP4 cells were incubated with 5 µM FAM-Aβ42 for 1 h at 4°C and warmed to 37°C for 0.5, 4, 8 or 24 h, then fixed and examined by confocal microscopy. In N2a-mLRP4 cells, FAM-Aβ42 accumulation peaked 4 h after warming and decreased over time, suggesting that intracellular Aβ42 is eventually delivered for degradation.