Research ArticleParkinson’s Disease

A cullin-RING ubiquitin ligase targets exogenous α-synuclein and inhibits Lewy body–like pathology

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Science Translational Medicine  05 Jun 2019:
Vol. 11, Issue 495, eaau6722
DOI: 10.1126/scitranslmed.aau6722
  • Fig. 1 Internalization of insoluble fibrils leads to αSyn accumulation and proteome alterations.

    (A) Thioflavin T (ThT) fluorescence and WB analyses of αSyn species obtained by incubating recombinant M-αSyn for the indicated times. Representative M-αSyn, O- αSyn, F-αSyn, and iF-αSyn species are indicated. Number sign (#) indicates uncharacterized αSyn immunoreactive band. To show stacking gels (represented by black vertical dashed lines) at nonsaturating levels, a less exposed blot of the same membrane is shown (indicated by a curved arrow). a.u., arbitrary units. (B) Bar graph showing the amount of αSyn contained in cells treated with the species obtained in (A). (C) Cell viability of SH-SY5Y cells treated with the indicated αSyn using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. (D) Confocal microscopy and three-dimensional (3D) image reconstruction analyses of green fluorescent protein (GFP)–expressing SH-SY5Y cells treated with Alexa Fluor–labeled M-αSyn or iF-αSyn (both in red). GFP was used to delimitate cell morphology. Scale bars, 10 μm. (E) Volcano plots showing global protein quantities of SH-SY5Y cells treated with M-αSyn (black), O-αSyn (green), F-αSyn (blue), or iF-αSyn (red). Within dashed lines are shown differentially expressed proteins (DEPs), defined as those with fold change (FC) > 1.75 and P < 0.05 (unpaired, two-tailed distribution Student’s t test) compared to vehicle. Numbers of down- and up-regulated DEPs are indicated in left and right corners, respectively. For visualization purposes, only proteins with log10 P < 3 and log2 fold change < 3 and > −3, which represent >99% of the quantified proteins, are displayed. (F) Venn diagram of the overlap of 216 DEPs obtained from SH-SY5Y cells treated with the four species of αSyn. (G) Protein-protein interaction network analysis of DEPs from αSyn-treated cells using the STRING database. Functionally related DEPs are indicated by dashed ovals. In (A) and (C), the results are expressed as means + SD. *P < 0.05 compared to vehicle [one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test].

  • Fig. 2 An SCF E3 ubiquitin ligase targets internalized αSyn for ubiquitination and degradation.

    (A) WB of neuronal SH-SY5Y (left) and glial BV-2 (right) cells transfected with siGFP (control), siCul1, or siSKP1 and treated with iF-αSyn or vehicle. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Bar graph showing the amount of αSyn contained in Cos7 cells expressing DN-Cul1, DN-Cul2, DN-Cul3, and DN-Cul4A (see WB inset) and treated with iF-αSyn. Results show means + SD. *P < 0.01 compared to vehicle, **P < 0.05 compared to the rest of the treatments (one-way ANOVA followed by Tukey’s post hoc test). (C) WB showing the effects of DN-Cul1, Baf-A (bafilomycin-A), or MG132 (N-carbobenzyloxy-l-leucyl-l-leucyl-l-leucinal) on the degradation (but not uptake) of internalized αSyn. DN-Cul1 expression, as well as the Baf-A and MG132 treatments, was initiated in cells pretreated with iF-αSyn (+) (see also fig. S2H). (D) Immunofluorescence and confocal microscopy analyses of Cos7 cells transfected with siGFP, siCul1, or siSKP1 and treated with iF-αSyn. Quantification shows the percentage of cells with αSyn-positive foci (right) and the percentage of αSyn-positive foci that also contained Cul1 or SKP1 (left). Results show means + SEM. *P < 0.05 and **P < 0.01 compared to siGFP-transfected cells (unpaired, two-tailed distribution Student’s t test). Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (E and F) WB of Cos7 cells pretreated with (E) Baf-A or (F) stably expressing DN-Cul1 and treated with His-iF-αSyn (+). Cells were harvested under denaturing conditions, and whole-cell extracts (WCE) were subjected to metal-affinity purification (AP: Ni-NTA) to isolate His-iF-αSyn. ## and # indicate two αSyn immunoreactive bands of 15 and 60 kDa observed in all experiments with His-iF-αSyn. Quantification of the blots shows data expressed as means + SEM. *P < 0.05 and **P < 0.05 compared to vehicle and Baf-A (E) or DN-Cul1 (F), respectively (one-way ANOVA followed by Fisher’s post hoc test). (G) Quantification of ubiquitinated His-iF-αSyn purified from control cells (siGFP) or from cells depleted of Cul1 (siCul1). In this experiment, the cells were treated with Baf-A to avoid αSyn degradation. SRM traces of the αSyn peptides embedding K45, K58, and K60 bearing a GG-tag mass shift (indicated in red together with the modified lysine) resulting from trypsinized ubiquitinated proteins are shown. The unmodified αSyn peptide EGVVHGATVAEK and ENOA were analyzed as controls. Dashed lines indicate stacking gels.

  • Fig. 3 An SCF inhibits the prion-like properties of extracellular αSyn.

    (A) Confocal microscopy images of Cos7 cells expressing GFP, GFP-αSyn, and/or DN-Cul1–Flag and treated with iF-αSyn. Arrows indicate GFP-αSyn–containing foci. Quantification shows means + SD. *P < 0.05 and **P < 0.05 compared to DN-Cul1 or GFP-αSyn, respectively (one-way ANOVA and the Tukey’s test). Scale bar, 10 μm. (B) WB analyses of GFP or GFP-αSyn–expressing SH-SY5Y cells transfected with siLuc, siCul1, or siSKP1 and treated with iF-αSyn. (C) Bar graph showing the amount of αSyn determined by SRM in Cos7 acceptor cells transfected with an empty vector, DN-Cul1 vector, or siCul1. In this experiment, exogenous αSyn was obtained from conditioned media (CM) from GFP- or GFP-αSyn–expressing SH-SY5Y cells (referred as donors). Data show means + SD. *P < 0.05 compared to vector (one-way ANOVA followed by Dunnett’s post hoc test). (D) Bar graph showing the amount of αSyn determined by SRM in HeLa cells transfected with siRNAs targeting 31 different F-box domain–containing proteins or GFP (siGFP; dashed horizontal line) and treated with iF-αSyn. Data show means + SD. *P < 0.05 by one-way ANOVA followed by Tukey’s post hoc test; #P < 0.05 by unpaired, two-tailed distribution Student’s t test compared to siGFP. (E) WB of whole-cell extracts (WCE) immunoprecipitates (IP: V5 and IP: αSyn) obtained from Cos7 cells expressing SKP1-V5 and Flag-FBXL5 or Flag-FBXL5-Δbox. In this experiment, the cells were pretreated with Baf-A and then exposed to iF-αSyn. (F) Flag-FBXL5 was expressed and immunoprecipitated from cells treated with iF-αSyn (+). A control immunoprecipitation (Ø) using an unrelated antibody is shown. #, heavy immunoglobulin chain. (G) Colocalization analyses between internalized iF-αSyn and Rab5A by immunofluorescence and confocal microscopy. Scale bar, 10 μm. Arrows show foci containing both αSyn and Rab5A. Quantification is shown on the right. (H and I) Colocalization analyses between internalized iF-αSyn and (H) galectin-3 (Gal3) and (I) lysosome-associated membrane protein-1 (Lamp1) by immunofluorescence and confocal microscopy. Scale bars, 10 μm. Arrows indicate foci positive for αSyn. Quantification is shown on the right. (G to I) Data show means + SEM. *P < 0.05 compared to vehicle (unpaired, two-tailed distribution Student’s t test). Dashed lines indicate stacking gels.

  • Fig. 4 SCFFBXL5inhibits LB-like pathology induced by extracellular αSyn fibrils.

    (A) Representative confocal microscopy images of brain slices from healthy subjects (n = 5) and from DLB (n = 5) and PD (n = 5) patients. Immunofluorescence shows αSyn in red and Cul1 and SKP1 in green. Data show means + SEM. *P < 0.05 and **P < 0.01 (unpaired, two-tailed distribution Student’s t test). n.d., nondetected. Scale bars, 10 μm. (B) Representative images of paraffin-embedded sections of striatum, cortex, and hippocampus (CA2) of αSyn transgenic mice injected with LV-shGFP (n = 6) or LV-shSKP1 (n = 6) and then with iF-αSyn. Immunohistochemistry was carried out with αSyn (clone LB509) or pSer129-αSyn antibodies. Scale bars, 20 μm. (C) Quantification of αSyn- and pSer129-αSyn–positive inclusions of the αSyn mice of (B). (D and E) Representative images of paraffin-embedded sections of striatum, cortex, and CA2 from wild-type nontransgenic mice injected with LV-shGFP (n = 6) and LV-shFBXL5 (n = 6) (left and right hemispheres, respectively) and subsequently with iF-αSyn. Animals were sacrificed at (D) 5 or (E) 10 weeks after iF-αSyn injection. Immunohistochemistry was carried out with αSyn (clone 509) and pSer129-αSyn antibodies. Arrows indicate neuronal projections. Scale bars, 25 μm. (F) Schematic representation of three brain sections of wild-type mice of (D) and (E). αSyn-positive inclusions are indicated by red dots, and quantification is indicated in the upper corner. (G) Quantification of αSyn- and pSer129-αSyn–positive inclusions of the mice of (E). (C and G) Data show means + SEM. *P < 0.05 and **P < 0.01 compared to LV-shGFP–injected mice (C) or hemispheres (G) (paired, two-tailed distribution Student’s t test).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/495/eaau6722/DC1

    Materials and Methods

    Fig. S1. Internalization of insoluble fibrils leads to αSyn accumulation and proteome alterations.

    Fig. S2. An SCF E3 ubiquitin ligase targets internalized αSyn for ubiquitination and degradation.

    Fig. S3. An SCF inhibits the prion-like properties of extracellular αSyn.

    Fig. S4. SCFFBXL5 inhibits LB-like pathology induced by extracellular αSyn fibrils.

    Table S1. Label-free quantification of proteins from SH-SY5Y cells treated with the αSyn species.

    Table S2. Label-free quantification of peptides from SH-SY5Y cells treated with the αSyn species.

    Table S3. DEPs from SH-SY5Y cells treated with the αSyn species.

    Table S4. Gene ontology enrichment analysis according to the Panther database.

    Table S5. KEEG pathway enrichment analysis according to the STRING database.

    Data file S1. Source data (Excel file).

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Internalization of insoluble fibrils leads to αSyn accumulation and proteome alterations.
    • Fig. S2. An SCF E3 ubiquitin ligase targets internalized αSyn for ubiquitination and degradation.
    • Fig. S3. An SCF inhibits the prion-like properties of extracellular αSyn.
    • Fig. S4. SCFFBXL5 inhibits LB-like pathology induced by extracellular αSyn fibrils.
    • Table S4. Gene ontology enrichment analysis according to the Panther database.
    • Table S5. KEEG pathway enrichment analysis according to the STRING database.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Label-free quantification of proteins from SH-SY5Y cells treated with the αSyn species.
    • Table S2 (Microsoft Excel format). Label-free quantification of peptides from SH-SY5Y cells treated with the αSyn species.
    • Table S3 (Microsoft Excel format). DEPs from SH-SY5Y cells treated with the αSyn species.
    • Data file S1. Source data (Excel file).

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