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 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 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 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

Sorting Nexin 17 Regulates ApoER2 Recycling and Reelin Signaling

<div><p>ApoER2 is a member of the low density-lipoprotein receptor (LDL-R) family. As a receptor for reelin, ApoER2 participates in neuronal migration during development as well as synaptic plasticity and survival in the adult brain. A previous yeast two-hybrid screen showed that ApoER2 is a binding partner of sorting nexin 17 (SNX17) - a cytosolic adaptor protein that regulates the trafficking of several membrane proteins in the endosomal pathway, including LRP1, P-selectin and integrins. However, no further studies have been performed to investigate the role of SNX17 in ApoER2 trafficking and function. In this study, we present evidence based on GST pull-down and inmunoprecipitation assays that the cytoplasmic NPxY endocytosis motif of ApoER2 interacts with the FERM domain of SNX17. SNX17 stimulates ApoER2 recycling in different cell lines including neurons without affecting its endocytic rate and also facilitates the transport of ApoER2 from the early endosomes to the recycling endosomes. The reduction of SNX17 was associated with accumulation of an ApoER2 carboxy-terminal fragment (CTF). In addition, in SNX17 knockdown cells, constitutive ApoER2 degradation was not modified, whereas reelin-induced ApoER2 degradation was increased, implying that SNX17 is a regulator of the receptor's half-life. Finally, in SNX17 silenced hippocampal and cortical neurons, we underscored a positive role of this endosomal protein in the development of the dendritic tree and reelin signaling. Overall, these results establish the role of SNX17 in ApoER2 trafficking and function and aid in identifying new links between endocytic trafficking and receptor signaling.</p></div

SNX17 knockdown decreases n-cofilin phosphorylation induced by reelin.

<p>(<b>A</b>) Dissociated mouse cortical neurons were transfected at DIV 5 with a GFP expression plasmid and the corresponding shRNA plasmid. At DIV 7, cells were treated with reelin, fixed, and analyzed by immunofluorescence to detect phospho-cofilin and βIII-tubulin. Images were captured, and the integrated fluorescence intensity of the soma was calculated using ImageJ software. Phosphorylation of n-cofilin was quantified in cells at each condition, and the intensity of βIII-tubulin was used for normalization. In (<b>B</b>), the quantification of the fluorescence intensity is shown of both cells transfected with pLKO plasmid (Control), and with shSNX17 in the presence of reelin (Reelin) or with DMSO (Mock). In (<b>C</b>), the same quantitative analysis of non-transfected cells present in each experimental condition is shown. **p<0.01; ***p<0.001.</p

The reelin signaling pathway is impaired in SNX17 knockdown cells.

<p>DIV 4 cortical mouse neurons were infected with the a lentiviral system expressing the parental plasmid pLKO (Control) or SNX17 shRNA (shSNX17) at MOI 1; three days after infection, the cells were incubated with reelin-containing medium. Next, cells were lysed and analyzed by western blot. Dab1 was immunoprecipitated (IP) with an anti-Dab1 antibody and analyzed by western blot using an anti-phosphotyrosine antibody. The Dab1 downstream targets AKT and GSK3β were also analyzed by western blot. For all of the proteins analyzed, the reelin-induced phosphorylation was reduced in the SNX17 knockdown cells.</p

SNX17 knockdown induces the retention of ApoER2 in early/recycling endosomes.

<p>(<b>A</b>) Control (pLKO) or SNX17 knockdown N2a cells expressing ApoER2 were subjected to subcellular fractionation to isolate endosomal compartments by a discontinuous sucrose gradient. Samples of the different fractions were resolved in Tris/Tricine gels and analyzed by western blot. The input corresponds to 5% of the lysate. EE/RE: early endosome/recycling endosome; LE: late endosome; fractions were free of endoplasmic reticulum (RAP) or Golgi (γ-Adaptin) contamination. (<b>B</b>) Quantification of ApoER2 in different compartments normalized by the Input. *p<0.05.</p

SNX17 interacts with the NPxY motif of ApoER2, and both proteins colocalize after receptor endocytosis.

<p>(<b>A</b>) HEK 293 cells were transfected with different myc-tagged SNX17 constructs, and their lysates were used for GST pull-down assays using GST or GST-ApoER2. The presence of SNX17 was evaluated by western blot with an anti-myc antibody. GST fusion proteins were detected by western blot using anti-GST antibody. F1, F2 and F3 indicates the three subdomains or modules of the FERM domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093672#pone.0093672-Ghai1" target="_blank">[35]</a>.(<b>B</b>) HEK 293 cells were transfected with HA-ApoER2 wild-type or mutated (NPxY/A) constructs. Cell lysates were used for a pull-down assay using GST or GST-SNX17. The receptor was evaluated by detecting the HA epitope. In both cases, Lys corresponds to 10% of the cell lysate used for the pull-down assay. (C) Cell extracts obtained from cells transiently transfected with myc-SNX17 and HA-ApoER2 were immunoprecipitated with anti-myc and probed for ApoER2 with the anti-HA antibody. Lys corresponds to 2% of the cell lysate used for the coinmunoprecipitation. (<b>D</b>) HeLa cells were transfected with HA-ApoER2, RAP, and myc-SNX17. Cells were incubated with anti-HA antibody for 1 h at 4°C, and receptor internalization was allowed for 10 min at 37°C. Cells were fixed and analyzed by immunofluorescence. Bar, 10 μm.</p