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

Additional file 3: Figure S3. of α-synuclein interacts with SOD1 and promotes its oligomerization

Co-IP studies confirm the α-synuclein-SOD1 binding. (A) α-Synuclein immunoprecipitation of co-transfected H4 cells using α-synuclein antibody co-immunoprecipitated SOD1-myc and endogenous SOD1. Input: 5 μg. (B) Immunoprecipitation using myc antibody co-immunoprecipitated α-synuclein. After α-synuclein detection, membrane was incubated with stripping buffer, blocked and incubated with an anti-tau antibody and secondary antibody. (C) α-Synuclein immunoprecipitation of wt mouse brain homogenate without the usage of the DSP crosslinking reagent detects co-immunoprecipitated SOD1. Input: 8 μg. (TIF 4670 kb

Additional file 2: Figure S2. of α-synuclein interacts with SOD1 and promotes its oligomerization

Luciferase signal of S1/SOD1-2 and S2/SOD1-1 is not based on a nonspecific binding of luciferase halves. (A) Luciferase activity measurement of living H4 cells and (B) conditioned medium 24 h post transfection with luciferase halves alone (L1 + L2) or luciferase halves tagged to α-synuclein and SOD1. Figure shows pooled data from 3 independent experiments after normalization to the respective mean of luciferase activity of co-transfected cells with L1 + L2 (two tailed, unpaired student’s t-test, n = 12, * p < 0,05, ** p < 0,005, *** p < 0,0005). (TIF 158 kb

Additional file 1: Figure S1. of α-synuclein interacts with SOD1 and promotes its oligomerization

Concept of the extracellular complementation assay: Cells expressing proteins with n-terminal halve of hGluc or proteins with c-terminal halve of hGluc were lysed, followed by determination the concentrations α-synuclein and SOD1. Lysates were adjusted to the same molarity of α-synuclein or SOD1 and combined. After incubation, luciferase activity was measured. (TIF 2014 kb

Additional file 4: Figure S4. of α-synuclein interacts with SOD1 and promotes its oligomerization

Co-localization of α-synuclein and SOD1 in wt mouse brain. Representative images of C57Bl/6 wt mouse brain sections co-immunostained for α-synuclein (Alexa 488, green) and SOD1 (Alexa 546, red). Nuclei were stained with DAPI. As control, sections were stained either with α-synuclein or SOD1 primary antibody but with both fluorophores (Alexa 488, Alexa 546) conjugated secondary antibodies. (TIF 7729 kb

Additional file 1: Figure S1. of Neonatal AAV delivery of alpha-synuclein induces pathology in the adult mouse brain

Representative intensity of Human αsyn immunostaining (a) Photomicrographs representative of the variability of expression observed in the different group of animals at 1, 3 and 6 months of age (b) Level of expression of the transgene was assessed by western blot in AAV-αsyn at 3 months of age and compared to transgenic mice overexpressing αsyn under Thy1 promoter (line 61) at the same age. Antibody recognizing human and mouse αsyn was used (clone 42). (c) Quantification of the western blot shows αsyn level increase of 2.93 ± 0.33 fold in the AAV-αsyn animals and 3.23 ± 0.12 fold in the line 61. .The data are expressed as the amount of total level of αsyn normalized to actin (*, p < 0.05) and are from 3 repeated experiments. (PDF 1678 kb

Additional file 2: Figure S2. of Neonatal AAV delivery of alpha-synuclein induces pathology in the adult mouse brain

Neither ThioS positive structures nor neurodegeneration are observed in AAV-αsyn animals. (a-i) Sagittal brain sections were incubated with anti human asyn antibody followed by 5 min in 1% thioS solution. Thalamus (a-c) and cortex (d-f) of AAV-asyn animal show strong asyn immunoreactivity (a, d) that is not thioS- positive (b, e). As a control, human DLBD brain was co-stained in parallel. Cortical Lewy bodies positive for human asyn (g) are reactive to thioS (h, i). Representative images of NeuN-labeled cells in the cortex of AAV-asyn (n = 9) and AAV-venus (n = 7) at 6 months of age (k). Quantification of NeuN-positive cells in the whole cortex (area delineated in blue). Data are presented as as mean ± S.E.M means. Scale bars in i = 40 μm and applied to a-h; Scale bars in k = 2 mm. Abbreviation: DLBD; Diffuse Lewy Body Disease. (PDF 1541 kb