LRP1 regulates GluA1-mediated neurite outgrowth and filopodia formation.

<p>Mouse primary neurons were infected with lentivirus carrying control vector or GluA1 cDNA and lentivirus carrying NT-shRNA or LRP1-shRNA. Control and LRP1-suppressed neurons with or without forced GluA1 expression were stained with anti-MAP2 antibody and their neurite outgrowth (<b><i>A</i></b>; scale bar  = 25 µm) and filopodia formation (<b><i>B</i></b>; scale bar  = 15 µm) were observed using confocal microscopy. Total outgrowth (<b><i>C</i></b>), mean process length (<b><i>D</i></b>) and Filopodia density (<b><i>E</i></b>) were quantified by MetaMorph software. The data are plotted as mean ± SEM. N.S., Not significant; *, p<0.05; **, p<0.01.</p

LRP1 interacts with GluA1 and regulates its turnover in neurons.

<p>(<b><i>A</i></b>) Brain lysates from wild-type mice were immune-precipitated using specific antibodies against LRP1, GluA1, GluA2/3 or PSD95, and their interactions were examined by Western blot (<b><i>B–E</i></b>). After infection with control NT-shRNA or LRP1-shRNA, control and LRP1-knockdown neurons were treated with cycloheximide (CHX), and the levels of GluA1 (<b><i>C</i></b>), GluA2/3 (<b><i>D</i></b>) and PSD95 (<b><i>E</i></b>) were analyzed by Western blot at different time points. (<b><i>F</i></b>) LRP1-knockdown neurons were treated with DMSO (control), proteasomal inhibitor lactacystin (Lac; 10 µM) or lysosomal inhibitor bafilomycin A1 (BA1; 5 nM) in addition to CHX. (<b><i>G</i></b>) GluA1 and PSD95 levels were analyzed by Western blot, and densitometrically quantified. The data are plotted as mean ± SD (n = 3). *, p<0.05; **, p<0.01.</p

LRP1-knockdown suppresses GluA1-mediated calcium influx in neurons.

<p>Primary mouse neurons were first infected with lentivirus carrying control vector or GluA1 plasmid, and then with lentivirus carrying NT-shRNA or LRP1-shRNA (<b><i>A</i></b>). Expression levels of LRP1 (<b><i>B</i></b>) and GluA1 (<b><i>C</i></b>) were detected by Western blot. (<b><i>D</i></b>) Calcium influx detected with the fluorescence microplate reader using Fluo-4 AM as a fluorescent indicator of intracellular calcium concentration in neurons after stimulation of AMPA in the presence of NMDAR antagonist. The scale bar represents 200 µm. (<b><i>E</i></b>) Calcium fluorescence intensities were measured with the excitation and emission wavelengths set at 494 and 535 nm, respectively. The data are plotted as mean ± SD (n = 3). N.S., Not significant; **, p<0.01.</p

LRP1-knockdown disturbs the trafficking of GluA1 to the cell surface and suppresses GluA1 phosphorylation in neurons.

<p>Primary mouse cortical neurons were infected with lentivirus carrying LRP1-shRNA or NT-shRNA for 4 days. Cell surface proteins were labeled with biotin in live neurons, and the cell lysates were precipitated with streptavidin beads. (<b><i>A, B</i></b>) The precipitates and total cell lysates were examined by Western blot to detect cell surface GluA1 and total GluA1, respectively. The ratio of surface GluA1 versus total GluA1 was quantified (<b><i>A</i></b>). Similarly, ratio of surface GluA2/3 versus total GluA2/3 was analyzed (<b><i>B</i></b>). (<b><i>C</i></b>) In control and LRP1-knockdown neurons, the expression of total GluA1 and phosphorylated GluA1 (pSer-845 and pSer-831) were analyzed by Western blot. The phosphorylation at Ser-845 (<b><i>D</i></b>) and Ser-831(<b><i>E</i></b>) sites of GluA1 versus total GluA1 were quantified. The data are plotted as mean ± SD (n = 3). N.S., not significant; *, p<0.05; **, p<0.01.</p

LRP1 knockdown decreases the expression levels of GluA1 in neurons.

<p>Primary cortical neurons cultured from C57Bl/6 mice were infected with lentivirus carrying LRP1-shRNA or control NT-shRNA on day 8 <i>in vitro</i> (DIV) and then harvested after 2 or 4 days of infection. The expression level of LRP1 in neurons was detected by Western blot (<b><i>A</i></b>), and densitometrically quantified (<b><i>B</i></b>). (<b><i>C</i></b>) The cell viability of neurons was assessed by MTT assay at 2 or 4 days following infection. In LRP1-knockdown neurons, the expression levels of PSD95 (<b><i>D</i></b>, <b><i>E</i></b>), GluA1 (<b><i>D</i></b>, <b><i>F</i></b>), and GluA2/3 (<b><i>D</i></b>, <b><i>G</i></b>) at 4 days post-infection were detected by Western blot and densitometrically quantified. In addition, the mRNA levels of PSD95 (<b><i>H</i></b>) and GluA1 (<b><i>I</i></b>) were also analyzed by quantitative real-time PCR. The data are plotted as mean ± SD (n = 3). N.S., Not significant; **, p<0.01.</p

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

Additional file 1 of A mutant methionyl-tRNA synthetase-based toolkit to assess induced-mesenchymal stromal cell secretome in mixed-culture disease models

Additional file 1: Fig. S1. Uncropped agarose gel from Figure 1B