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 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 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 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

SNX17 knockdown diminishes surface levels of ApoER2 by decreasing its recycling.

<p>(<b>A</b>) HEK 293 cells or (<b>B</b>) N2a cells infected with a lentiviral vectors expressing shRNA against human or mouse SNX17 or empty pLKO vector were transfected with HA-ApoER2. Cells were lysed with 1% Triton X-100 in PBS and analyzed by western blot. (<b>C</b>) HEK 293 clones transfected with HA-ApoER2 and RAP were used to analyze the ratio of cell surface to total ApoER2 by FACS, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093672#s2" target="_blank">Methods</a> section. The graphic shows the ratio of the values of non-permeabilized versus permeabilized cells considering the control condition as 100%. (<b>D</b>) N2a clones expressing ApoER2 and SNX17 silenced or control were treated as in C. (<b>E</b>) Control or SNX17 knockdown HEK293 clones were transfected with a plasmid for mMeg4, a construct of megalin carrying the fourth ligand binding domain, the transmembrane domain, and the cytosolic domain. The receptor was determined in these cells by FACS. (<b>F</b>) SNX17 knockdown HEK293 clones expressing HA-ApoER2 were transfected with a shRNA-resistant mouse myc-SNX17. The presence of ApoER2 and SNX17 proteins were analyzed using a chicken anti-HA antibody and a mouse anti-Myc antibody respectively, in permeabilized and non-permeabilized conditions. (<b>G</b>) Control or SNX17 knockdown HEK293 clones expressing HA-ApoER2 were incubated with an Alexa 488-conjugated anti-HA antibody for 1 h at 4°C. The temperature was increased to 37°C for the indicated time period, and theremaining surface bound antibody was removed with an acid wash. The intracellular antibody was detected in the cells by flow cytometry. The total antibody bound to the cell was also determined. The endocytic rate was calculated by subtracting the value of the cells exposed to the acid wash at time 0 (A₀) from each time point, and dividing by A_0. (<b>H</b>) The same experimental procedure described in G was performed with control or SNX17 knockdown N2a cells expressing HA-ApoER2. (<b>I</b>) The same HEK293T cells used in G were utilized in a recycling experiment. The cells were labeled with an Alexa 488-conjugated anti-HA antibody for 30 min at 37°C. The recycled receptor was then chased at the surface with a quenching anti-Alexa 488 antibody. The cells were analyzed by flow cytometry, and the percentage of the initial fluorescence remaining at each time point was calculated as the difference in the fluorescence between time 0 and each chased time point. Every time (including time 0) was normalized to the non-chased value. The percentage of recycling efficiency was calculated by subtracting the percentage of internal fluorescence from 100. (<b>J</b>) Control or SNX17 knockdown N2a clones expressing HA-ApoER2 were treated as in C. *p<0.05, **p<0.01, ***p<0.001.</p

SNX17 knockdown diminishes the surface level of ApoER2 and reelin-induced dendritic development in neurons.

<p>(<b>A, B</b>) The cell surface level of ApoER2 was determined in DIV 5 mouse cortical neurons co-transfected with HA-ApoER2 and either SNX17 shRNA or pLKO. The cell surface receptor was labeled 48 h after transfection with a mouse anti-HA antibody. To control for the absence of permeabilization, cells were simultaneously incubated with an antibody against the cytoplasmic tail of ApoER2. The intracellular ApoER2 was detected thereafter in the fixed and permeabilized neurons with a chicken anti-HA antibody. Images of individual cells (n = 10 for each condition) were captured by confocal microscopy and analyzed using ImageJ software, selecting the threshold for each channel to avoid background. Total fluorescence was calculated by adding the fluorescence of the permeabilized and non-permeabilized channels. (<b>C, D</b>) Mouse dissociated hippocampal neurons were transfected with GFP and the corresponding shRNA, treated with reelin for 3 days, fixed and analyzed by immunofluorescence. Images were captured by confocal microscopy and used for Sholl analysis (n = 20 cells per condition). (<b>E</b>) The length of dendrites of reelin treated cells was significantly reduced in SNX17 knockdown neurons *p<0.05; **p<0.01; ***p<0.001. Bars, 20 μm.</p

SNX17 knockdown increases the ApoER2 CTF level.

<p>Control or SNX17 knockdown (<b>A</b>) HEK293 cells and (<b>B</b>) N2a cells expressing HA-ApoER2 were lysed in PBS-T. The samples were separated on Tris-Tricine gels and analyzed by western blot. (<b>C</b>) DIV 4 cortical mouse neurons were infected with a lentiviral vector expressing SNX17 shRNA or empty pLKO at an MOI of 1. Three days after the infection, the cells were lysed and the lysates were resolved on Tris-Tricine gels. (<b>D</b>) Control or SNX17 knockdown HEK293 cell clones were transfected with the ApoER2 plasmid and either the mouse myc-SNX17 plasmid or pcDNA3 as a control. The resultant cell lysates were analyzed by western blot. (<b>E</b>) The band intensity was quantified to determine the ApoER2 CTF level normalized to actin. In all conditions, the bands were normalized to the control condition (empty pLKO and pcDNA3). The figure shows the average of three independent experiments. *p<0.05, **p<0.01, ns p>0.05. (<b>F</b>) Control or SNX17 knockdown N2a cells expressing ApoER2 were treated with DAPT or DMSO for 16 h, and then the presence of ApoER2 and its CTF were determined by western blot. (<b>G</b>) Quantification was performed as previously described, using pLKO cells treated with DMSO as the control condition. **p<0.001 and ns p>0.05.</p

Mesd Is a Universal Inhibitor of Wnt Coreceptors LRP5 and LRP6 and Blocks Wnt/β-Catenin Signaling in Cancer Cells

Mesd is a specialized chaperone for low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6. In our previous studies, we found that Mesd binds to mature LRP6 on the cell surface and blocks the binding of Wnt antagonist Dickkopf-1 (Dkk1) to LRP6. Herein, we demonstrate that Mesd also binds to LRP5 with a high affinity and is a universal inhibitor of LRP5 and LRP6 ligands. Mesd not only blocks binding of Wnt antagonists Dkk1 and Sclerostin to LRP5 and LRP6 but also inhibits Wnt3A and Rspondin1-induced Wnt/β-catenin signaling in LRP5- and LRP6-expressing cells. We also found that Mesd, Dkk1, and Sclerostin compete with one another for binding to LRP5 and LRP6 at the cell surface. More importantly, we demonstrated that Mesd is able to suppress LRP6 phosphorylation and Wnt/β-catenin signaling in prostate cancer PC-3 cells and inhibits PC-3 cell proliferation. Our results indicate that recombinant Mesd protein is a useful tool for studying Wnt/β-catenin signaling on the cell surface and has a potential therapeutic role in Wnt-dependent cancers

SNX17 does not regulate the half-life of ApoER2 under basal conditions.

<p>(<b>A</b>) HEK 293 cells treated with shRNA against SNX17 and pLKO control clones were transfected with HA-ApoER2 and RAP, incubated with medium containing [35S]Met/Cys for 90 min, and chased with medium without [35S]Met/Cys for 1, 3, 6, 9, and 20 h, followed by immunoprecipitation with anti-HA antibody. The samples were separated by SDS-PAGE and subjected to autoradiography. (<b>B</b>) The radioactive intensity was plotted against time. In both conditions, ApoER2 is degraded with similar kinetics. (<b>C</b>) Western blot of equal amounts of cell lysate of N2a cells (pLKO and SNX17 KD) expressing ApoER2, which was detected with anti-HA antibody. The ER precursor and the mature fully glycosylated forms are shown. (<b>D</b>) There is no difference in the amount of mature form with respect to the precursor in SNX17 knockdown cells indicating that ApoER2 degradation was not affected by the lack of SNX17 (n = 3).</p