LRP1 mediates internalization and accumulation of Aβ42 in GT1-7 and MEF cells.

<p><i>A,</i> LRP1 knockdown decreases Aβ42 accumulation in GT1-7 cells. GT1-7 cells were transiently transfected with LRP1 siRNA or with control, scrambled siRNA. After 72 h, cells were treated with 500 nM FAM-Aβ42 for 4 h and intracellular Aβ42 was determined by flow cytometric analyses of pronase-treated cells as described in the <i>Experimental Procedures</i>. <i>B,</i> decreased Aβ42 accumulation in mouse embryonic fibroblasts from LRP1 knockout mice. Wild type (MEF1) and LRP1 knockout (MEF2) fibroblasts were treated with 500 nM FAM-Aβ42 for 4 h, and intracellular Aβ42 was determined by flow cytometric analyses. ** p<0.01, <i>t</i>-test. n = 3. <i>C,</i> cell lysates from GT1-7 and MEF cells treated as in <i>A</i> and <i>B</i>, analyzed by 7.5% SDS-PAGE, and Western blotted with anti-LRP1 antibodies. Levels of LRP1 were efficiently decreased in both cell types.</p

Increased susceptibility to Aβ42-mediated cell death in LRP1 minireceptor-expressing cells.

<p>N2a-pcDNA3 and N2a-mLRP4 cells were incubated with increasing concentrations of Aβ42 for 24, 48 and 72 h and cell viability was assessed by the reduction of the MTS dye. Decreased viability was detected only after 72 h incubation with a high concentration of Aβ42 in LRP1 minireceptor expressing cells. * p<0.05, <i>t</i>-test. n = 3.</p

Increased accumulation of intracellular Aβ42 within lysosomes in LRP1 minireceptor-expressing cells.

<p><i>A,</i> increased Aβ42 accumulation in LRP1 minireceptor-expressing cells. N2a-mLRP4 and N2a-pcDNA3 cells were treated with 500 nM of FAM-Aβ42 at 37°C for 24, 48 and 72 h, and steady-state levels of intracellular Aβ42 were determined by flow cytometric analyses of pronase-treated cells as described in the <i>Experimental Procedures</i>. N2a-mLRP4 cells showed increased Aβ42 accumulation compared to N2a-pcDNA3 cells starting at 48 h of Aβ42 incubation. ** p<0.01, *** p<0.001, <i>t</i>-test. n = 3. <i>B,</i> increased co-localization of intracellular Aβ42 and lysosomes in N2a-mLRP4 cells. N2a-pcDNA3 and N2a-mLRP4 cells were grown in glass chamber slides and treated with 500 nM of FAM-Aβ42 at 37°C for 24, 48 and 72 h. Lysosomes were labeled with LysoTracker 30 min before the end of each incubation. Cells were then fixed and analyzed by confocal microscopy. Intracellular accumulated Aβ42 was highly co-localized with LysoTracker and increased over time in N2a-mLRP4 cells.</p

LRP1 endocytosis is required for Aβ42 uptake and accumulation in N2a cells.

<p><i>A,</i> clathrin heavy chain (CHC) knockdown increases cell surface levels of mLRP4. N2a-mLRP4 cells were infected with CHC shRNA lentivirus or pLKO, control lentivirus. The levels of cell surface and total pools of mLRP4 were determined by flow cytometric analyses with anti-HA antibody in non-permeabilized and saponin-treated cells, respectively. The surface-to-total ratio were calculated and plotted as fold-change to control-infected cells. <i>Right panel</i>, decreased CHC levels and normal mLRP4 levels in transduced N2a-mLRP4 cells were verified by Western blot from sister cultures. <i>B,</i> CHC knockdown decreases accumulation of Aβ42 in N2a-mLRP4 cells. N2a-mLRP4 cells infected with clathrin heavy chain lentivirus as in <i>A</i>) were treated with 500 nM of FAM-Aβ42 or the corresponding control, scrambled peptide for 48 h. The intracellular level of FAM-Aβ42 was determined by flow cytometric analyses of pronase-treated cells. <i>C,</i> deletion of LRP1 tail increases cell surface levels of mLRP4. The cell surface and total pools of the LRP1 minireceptor were determined by flow cytometric analyses with anti-HA antibody as in <i>A</i> in N2a-mLRP4 cells and in N2a cells stably transfected with a deletion variant lacking the cytoplasmic tail of mLRP4 (mLRP4-Tless). The surface-to-total ratios were then calculated and plotted as fold-change to N2a-mLRP4 cells. <i>Right panel</i>, HA blot showing the expression level of minireceptors in N2a stable cell lines. <i>D,</i> deletion of LRP1 tail decreased the mLRP4 endocytosis rate in N2a cells. N2a-mLRP4 and N2a-mLRP4-Tless cells were incubated with 5 nM ¹²⁵I-RAP at 4°C for 60 min, and then shifted to 37°C for the indicated times. At each time point, the amounts of ligand that is either internalized or that remains at the cell surface were determined and the ratios of internalized to total cell-associated ligand were plotted against time. Values are the average of triple determinations with the S.D. indicated by <i>error bars</i>. <i>E</i>, impaired LRP1 endocytosis rate decreases accumulation of Aβ42 in N2a-mLRP4 cells. N2a-pcDNA3, N2a-mLRP4 and N2a-mLRP4-Tless cells were treated with 500 nM of FAM-Aβ42 or the corresponding control, scrambled peptide for 48 h and the cell-associated (without pronase) and intracellular (with pronase) levels of Aβ42 were determined by flow cytometric analyses.</p