Fig 1.
SCAMP5 is induced by autophagy regulator TFEB.
(A) Real-time PCR results showing increased SCAMP5 level in primary neuronal cultures treated with Rapamycin(100nM), MG132(3μM) or starvation. Values were normalized to control group as relative expression with respect to endogenous control gene actin (mean ±S.E.M.; n = 3; *p<0.05). (B) Dual-luciferase assay showing that TFEB activates from the SCAMP5 promoter. SH-SY5Y cells were transfected with either an empty vector pcDNA3 or Flag-TFEB and a firefly luciferase reporter directed by the human SCAMP5 promoter. Renilla-luciferase reporter was co-transfected as transfection efficiency control. The relative luminescence of Firefly/Renilla was examined 48h later (mean ±S.E.M.; n = 6; *p<0.05). (C) Immunoblots showing increased SCAMP5 level in SH-SY5Y cells transfected with Flag-TFEB (left panel). Right panel shows the quantification of SCAMP5/β-tubulin expression in the immunoblots of three independent experiments (mean ±S.E.M.; n = 3; *p<0.05). (D) The expression of SCAMP5 during autophagy response in TFEB-silenced cells. SH-SY5Y cells were transfected with siRNA against TFEB (TFEB) or scrambled non-specific siRNA as control (NC), and exposed to MG132 for 6 hour before harvest for analysis by real-time PCR. The silencing of the TFEB in SH-SY5Y cells was confirmed in (E) The expression of TFEB and SCAMP5 were normalized to actin and then to the control (mean ±S.E.M.; n = 3; *p<0.05, **p<0.01, ***p<0.001).
Fig 2.
SCAMP5 is ubiquitinated and can be degraded by the proteasome or autophagy pathway.
(A) Immunoblots showing quickly increased SCAMP5 in primary neuronal cultures treated with Rapamycin(100nM) or MG132(3μM). Representative results from four independent experiments with similar conclusions were shown. (B) Immunoblots and the quantification (C) showing that SCAMP5 degrades much quicker than β-actin. Primary neuronal cultures were treated with protein synthesis inhibitor Cycloheximide over a timecourse of 24 hours (mean ±S.E.M.; n = 3; **p<0.01). (D) Representative immunoblots showing that proteasome inhibitor MG132 causes a transient increase of exogenous SCAMP5. Primary neuronal cultures were infected with lentiviral vector expressing neuronal specific SCAMP5, and harvested 72 hours later. MG132 was added 2 to 12 hours before harvest. The results represent three independent experiments with similar results. (E) Representative immunoblots showing that autophagy inhibitor BafilomycinA1 causes a transient increase of exogenous SCAMP5 in primary neuronal cultures expressing lenti-viral delivered SCAMP5 as in (D). BafilomycinA1 was added 2 to 12 hours before harvest. The results represent three independent experiments with similar results. (F) SCAMP5 is ubiquitinated as shown by immunoprecipitation. HEK-293T cells were transfected with HA-Ubiquitin, Flag-SCAMP5, or both for 24 hours. One group was treated with MG132 for 6 hours before harvest. The cells were lysed with RIPA buffer and immunoprecipitated using anti-Flag antibody, then immunoblotted with anti-HA antibody.
Fig 3.
SCAMP5 blocks autophagy flux by inhibiting the fusion of autophagosomes and lysosomes.
(A) Immunoblots showing increased LC3-II in SCAMP5 over-expressed cells. HEK-293T were transfected with Flag-GFP, Flag-SCAMP1 or Flag-SCAMP5, and harvest 48 hours later. Autophagy inducer Rapamycin (100nM) was added to culture medium for 2 hours before harvest. In SDS-PAGE, the appeared molecular weight of SCAMP5 was about 25kD when the sample was not boiled. Large aggregated bands would form when the sample was boiled, probably due to its property as a membrane protein. (B) HEK-293T cells were transfected with Flag-GFP or Flag-SCAMP5 and harvest 48 hours later. Autophagy blocker Bafilomycin A1 (50nM) was added to culture medium for indicated time before harvest. The protein level of LC3 and p62 was examined by immunoblotting (upper panel). Lower panel shows the quantification of LC3II/actin in three independent experiments, BafilomycinA1 was applied for 3 hours (mean ±S.E.M.; n = 5; *p<0.05, ***p<0.001). (C) Immunoblots showing increased p62 in cells over-expressing SCAMP5. SH-SY5Y cells were transfected with Flag-GFP or Flag-SCAMP5. Lower panel showed the quantification of three independent experiments (mean ±S.E.M.; n = 3; *p<0.05). (D) Immunoblots (upper panel) and quantification (lower panel) of the effect of SCAMP5 knockdown on LC3II and p62 in the absence or the presence of Rapamycin (mean ±S.E.M.; n = 4 independent experiments; *p<0.05, **p<0.01). (E) Immunofluorescent results of mRFP-GFP-LC3 showing that SCAMP5 blocks the fusion of autophagosome and lysosome. HEK-293T cells were transfected with autophagy reporter mRFP-GFP-LC3 and either empty vector or Flag-SCAMP5, one group was treated with FBS free DMEM medium (starvation) for two hours before harvest. Immunofluorescent was performed 24 hours later using mouse anti-Flag primary antibody and donkey anti mouse Alexa Fluor 647 secondary antibody. Confocal microscopy examination confirmed that all the mRFP-GFP-LC3 transfected cells were Flag-SCAMP5 positive(data not show). The acid-sensitive green fluorescence is lost when autophagosomes fuse with lysosomes as shown in the representative image (left panel). The quantification of the number of RFP+GFP+ autophagosome and RFP+GFP- autolysosome was carried out double-blindly in more than 300 cells(mean ±S.E.M.; n = 3; *p≤0.05, **p<0.01) (right panel).
Fig 4.
SCAMP5 facilitates the secretion of α-synuclein and huntingtin protein.
(A) Immunofluorescent images of rat primary neuronal culture showing the co-localization of endogenous SCAMP5 (red) and α-synuclein (green) using confocal microscopy. (B) SCAMP5 increases the secretion of α-synuclein over time in cultured cells. EGFP-α-synuclein stable overexpressed SH-SY5Y cells were transfected with Flag-GFP or SCAMP5, and harvested 1 to 2 days after transfection. The amount of transfected EGFP-α-synuclein in cell lysate(CL), cell medium(CM), and SDS-insoluble pellet were analyzed by immunoblotting. (C) Quantification of secreted EGFP-α-synuclein in cell medium of SH-SY5Y cells overexpressed with SCAMP5 or control for 24 hours in three independent experiments. (mean ±S.E.M.; n = 3; *p<0.05). (D) MTS assay showing no significant difference in cell viability between control plasmid or SCAMP5 overexpressed cells. SH-SY5Y cells stably expressing EGFP-α-synuclein-WT were transfected with control plasmid or Flag-SCAMP5. 48 hours later, cells were digested, counted, and adjusted to the same concentration, then applied to MTS assay (mean ±S.E.M.; n = 3). (E) Knockdown of SCAMP5 decreases the secreted EGFP-α-synuclein in cell medium. SH-SY5Y cells stably expressing EGFP-α-synuclein were transfected with scrambled siRNA or SCAMP5 siRNA. The CL and CM were collected 72 hours later and examined by immunoblotting. Right panel shows the quantification of secreted EGFP-α-synuclein in cell medium of three independent experiments (mean ±S.E.M; n = 3; ***p<0.001). (F) SCAMP5 causes aggregation and secretion of HTT protein. Immunoblots showing HTT protein in cell lysates (cytoplasm and nucleus portion), RIPA-insoluble pellets (extracted with 8M Urea) and cell medium of HEK-293T cells transfected with an empty vector, SCAMP1 or SCAMP5.
Fig 5.
SCAMP5 facilitates the secretion of α-synuclein through Golgi-independent secretion pathway, and causes Golgi fragmentation.
(A) Inhibitors of Golgi-dependent conventional secretion did not block SCAMP5-mediated secretion of α-synuclein. SH-SY5Y cells stably expressing EGFP-α-synuclein-WT or EGFP-α-synuclein-A53T were transfected with Flag-GFP or Flag-SCAMP5. Cell medium and cell lysate were harvested 48 hours later. Six hours before harvested, the exocytosis inhibitor Tetanus toxic (TeNT, 2nM) or BrefeldinA (BFA, 2μM), or DMSO were applied with a medium change. (B) Representative immunofluorescent images (z-projection) showing Golgi fragmentation in SCAMP5 over-expressed SH-SY5Y cells. Cells were transfected with Flag-SCAMP5, and treated with or without Bafilomycin A1 for 6 hours. 48 hours later, cells were fixed and immuno-stained with antibodies against Flag (green) and Golgi marker GM130 (red) or Giantin (red), and examined with confocal microscopy. Arrows indicate fragmented Golgi apparatus. (C) Quantificantion of Golgi fragmentation. The Golgi apparatus were immuno-stained with GM130 antibody and categorized into three types according to the integrity of its structure: normally stacked Golgi (green arrow), slightly collapsed compact Golgi (yellow arrow), and fragmented Golgi (red arrow). The percentages of cells with indicated Golgi morphology in SCAMP5-/+ cells were determined in three independent experiments (total cell number≥300; mean ±S.E.M.; *p<0.05, **p<0.01, ***p<0.001). (D) Immunofluorescent images showing impaired ER-Golgi transport in SCAMP5 over-expressed SH-SY5Y cells. Cells were transfected with Flag-SCAMP5 and immuno-stained with antibodies against Flag (green) and ERGIC53 (red). Representative morphology of accumulated (arrow) ER-Golgi intermediate compartment was as showed. (E) Statistics shows the percentage of cells with impaired ER-Golgi transport in SCAMP5- or SCAMP5+ cells treated with BafilomycinA1 in four independent experiments (total cell number≥300; mean ±S.E.M.; **p<0.01).
Fig 6.
SCAMP5 facilitates the secretion of α-synuclein through exosome pathway.
(A) Flowchart showing the exosome extraction procedure. (B) Quality control of exosome extraction. Cell lysates(CL), Cell medium(CM), exosome(EXO), Flow through(FT), and RIPA insoluable pellets of EGFP-α-synuclein stable overexpressed SH-SY5Y cells were collected according to the flowchart in 6A. The difference in apparent molecular weight was mainly due to different buffer between samples. Immunoblotting of exosome marker CD63 is conducted using native lysis buffer and native PAGE. Other proteins such as α-synuclein and ER marker Calnexin were examined with SDS-PAGE. (C) Electron microscopy images of exosomes isolated from the cell medium of SH-SY5Y cells stably expressing EGFP-α-synuclein. (D) SCAMP5 is abundant in exosomes, and it increases α-synuclein secretion via exosome. SH-SY5Y cells stably expressing EGFP-α-synuclein were transfected with Flag-GFP or Flag-SCAMP5, and harvested 48 hour later. All the samples were processed identically. The loading/total volumes of CL, CM, EXO, and FT were 2/1,200μL, 30/6,000μL, 30/200μL, 30/6,000μL respectively. (E) Quantification of secreted EGFP-α-synuclein in exosome of SH-SY5Y cells overexpressed with SCAMP5 or control in three independent experiments. (mean ±S.E.M.; n = 3; *p<0.05). (F) SH-SY5Y cells stably expressing EGFP-α-synuclein were transfected with Flag-GFP or Flag-SCAMP5, and harvested 48 hour later. BrefeldinA was added 6 hours before harvest with a change of medium.