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

Data_Sheet_1_Nanoparticles With Affinity for α-Synuclein Sequester α-Synuclein to Form Toxic Aggregates in Neurons With Endolysosomal Impairment.pdf

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. It is characterized pathologically by the aggregation of α-synuclein (αS) in the form of Lewy bodies and Lewy neurites. A major challenge in PD therapy is poor efficiency of drug delivery to the brain due to the blood–brain barrier (BBB). For this reason, nanomaterials, with significant advantages in drug delivery, have gained attention. On the other hand, recent studies have shown that nanoparticles can promote αS aggregation in salt solution. Therefore, we tested if nanoparticles could have the same effect in cell models. We found that nanoparticle can induce cells to form αS inclusions as shown in immunocytochemistry, and detergent-resistant αS aggregates as shown in biochemical analysis; and nanoparticles of smaller size can induce more αS inclusions. Moreover, the induction of αS inclusions is in part dependent on endolysosomal impairment and the affinity of αS to nanoparticles. More importantly, we found that the abnormally high level of endogenous lysosomotropic biomolecules (e.g., sphingosine), due to impairing the integrity of endolysosomes could be a determinant factor for the susceptibility of cells to nanoparticle-induced αS aggregation; and deletion of GBA1 gene to increase the level of intracellular sphingosine can render cultured cells more susceptible to the formation of αS inclusions in response to nanoparticle treatment. Ultrastructural examination of nanoparticle-treated cells revealed that the induced inclusions contained αS-immunopositive membranous structures, which were also observed in inclusions seeded by αS fibrils. These results suggest caution in the use of nanoparticles in PD therapy. Moreover, this study further supports the role of endolysosomal impairment in PD pathogenesis and suggests a possible mechanism underlying the formation of membrane-associated αS pathology.</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 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