Low-density lipoprotein receptor-related protein 1 (LRP1) mediates neuronal Abeta42 uptake and lysosomal trafficking

Abstract

Background: Alzheimer's disease (AD) is characterized by the presence of early intraneuronal deposits of amyloid-beta 42 (Abeta42) that precede extracellular amyloid deposition in vulnerable brain regions. It has been hypothesized that endosomal/lysosomal dysfunction might be associated with the pathological accumulation of intracellular Abeta42 in the brain. Our previous findings suggest that the LDL receptor-related protein 1 (LRP1), a major receptor for apolipoprotein E, facilitates intraneuronal Abeta42 accumulation in mouse brain. However, direct evidence of neuronal endocytosis of Abeta42 through LRP1 is lacking.

Methodology/principal findings: Here we show that LRP1 endocytic function is required for neuronal Abeta42 uptake. Overexpression of a functional LRP1 minireceptor, mLRP4, increases Abeta42 uptake and accumulation in neuronal lysosomes. Conversely, knockdown of LRP1 expression significantly decreases neuronal Abeta42 uptake. Disruptions of LRP1 endocytic function by either clathrin knockdown or by removal of its cytoplasmic tail decreased both uptake and accumulation of Abeta42 in neurons. Finally, we show that LRP1-mediated neuronal accumulation of Abeta42 is associated with increased cellular toxicity.

Conclusions/significance: These results demonstrate that LRP1 endocytic function plays an important role in the uptake and accumulation of Abeta42 in neuronal lysosomes. These findings emphasize the central function of LRP1 in neuronal Abeta metabolism.

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.

Figure 2. Increased accumulation of intracellular Aβ42 within lysosomes in LRP1 minireceptor-expressing cells.

A, 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 Experimental Procedures. 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, t-test. n = 3. B, 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.

Figure 3. LRP1 mediates internalization and accumulation of Aβ42 in GT1-7 and MEF cells.

A, 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 Experimental Procedures. B, 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, t-test. n = 3. C, cell lysates from GT1-7 and MEF cells treated as in A and B, analyzed by 7.5% SDS-PAGE, and Western blotted with anti-LRP1 antibodies. Levels of LRP1 were efficiently decreased in both cell types.

Figure 4. LRP1 endocytosis is required for Aβ42 uptake and accumulation in N2a cells.

A, 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. Right panel, decreased CHC levels and normal mLRP4 levels in transduced N2a-mLRP4 cells were verified by Western blot from sister cultures. B, CHC knockdown decreases accumulation of Aβ42 in N2a-mLRP4 cells. N2a-mLRP4 cells infected with clathrin heavy chain lentivirus as in A) 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. C, 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 A 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. Right panel, HA blot showing the expression level of minireceptors in N2a stable cell lines. D, 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 error bars. E, 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.

Figure 5. Increased susceptibility to Aβ42-mediated cell death in LRP1 minireceptor-expressing cells.

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, t-test. n = 3.