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 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-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

MOESM4 of Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

Additional file 4: Figure S4. (a) Representative cropped western blot of lysates from dissected substantia nigra/midbrain (SN) of three rats injected unilaterally with control virus, AAV8-Hgluc (gaussia luciferase) demonstrates no change in SIRT3 protein levels when contralateral (C) uninjected SN is compared to ipsilateral (I) injected SN (b) Representative cropped western blot of AMPKα, p-AMPKα (Thr 172), CREB, and p-CREB (Ser 133) in SN lysate of AAV8-SL1&SL2 injected rat. αSyn expression leads to decreased p-AMPK and p-CREB protein levels in ipsilateral (I) injected SN compared to contralateral (C) uninjected SN, n = 5 rats total. Error bars represent the mean ± SD. *p < 0.05, **p < 0.01

MOESM2 of Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

Additional file 2: Figure S2. (a) Representative cropped western blots showing αsyn in cytosolic and mitochondrial fractions from H4 WT-αsyn overexpressing cells. αSyn, GAPDH, and COXIV are all from same samples and immunoblot. (b) Western blot and quantification for SIRT3 in primary embryonic mouse neurons treated at DIV7 for 5 days with either AAV2/8 WT-αsyn or AAV2/8 venus control. Untagged αsyn overexpression shows a significant decrease in SIRT3 level compared to control. Error bars represent the mean ± SD. **p < 0.01 (n = 4)

MOESM1 of Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

Additional file 1: Figure S1. (a) Luciferase activity in cytosolic fractions of H4 SL1&SL2 cells over time, n = 5 (b) Representative cropped western blots of denaturing SDS-page (left) and native-page (right) performed at 72 h to confirm the purity of the mitochondrial-enriched fractions, the lysates were probed for the golgi marker (GM130), inner mitochondrial membrane marker (COXIV), GAPDH and αsyn in cytosol and mitochondria from H4 SL1&SL2 cells. (c) Representative image of dot-blot assay of the mitochondrial fractions at 72 h probed for amyloid-specific A11 and αsyn disease-associated (5G4) antibodies. (d) SIRT3 expression was detected by immune-fluorescence in mitochondria. SIRT3 expression is decreased at 72 h in cells overexpressing αsyn. Representative image from 3 experiments. DAPI (nucleus; blue); αsyn (green); SIRT3 (mitochondria; orange); merged images (yellow). Scale bar = 10 μm. Error bars represent the mean ± SD (n = 3–5). **p < 0.01

MOESM3 of Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

Additional file 3: Figure S3. (a) Representative cropped western blot showing DRP1 and p-DRP1 (Ser 616) in whole cells lysates from H4 SL1&SL2 cells over time (n = 4). Removal of tet leads to significantly increased p-DRP1 by 72 h. (b) Quantification of p-DRP1/DRP1 protein ratio in whole cell lysates. Error bars represent the mean ± SD. **p < 0.01

Additional file 5: Figure S5. of α-synuclein interacts with SOD1 and promotes its oligomerization

Increase in luciferase activity in presence of α-synuclein and reduced luciferase activity in absence of α-synuclein does not derive from unequal expression levels. A Densitometry of western blots from H4 cell co-transfected with SOD1-1 /-2 complementation pair and with myc, wt, A30P or A53T α-synuclein. B Expression level of S1/S2 in the presence of myc, wt and mutated SOD1-myc relative to β-actin (two tailed, unpaired student’s t-test, n = 3). (C) Expression level of SOD1-1 and SOD1-2 of lysates from scrambled-shRNA stabile H4 cell line and two α-synuclein-shRNA stabile H4 cell lines (α-syn I, α-syn II) relative to β-actin (two tailed, unpaired student’s t-test, n = 4). (D) Densitometry of western blots from SOD1-1 and SOD1-2 co-transfected H4 cells treated with 7 μM recombinant α-synuclein or solvent PBS (two tailed, unpaired student’s t-test, n = 3). (n.s. = not significant). (TIF 964 kb