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α-Synuclein impairs macroautophagy: implications for Parkinson's disease.

Winslow AR, Chen CW, Corrochano S, Acevedo-Arozena A, Gordon DE, Peden AA, Lichtenberg M, Menzies FM, Ravikumar B, Imarisio S, Brown S, O'Kane CJ, Rubinsztein DC - J. Cell Biol. (2010)

Bottom Line: Our data show that α-synuclein compromises autophagy via Rab1a inhibition and Rab1a overexpression rescues the autophagy defect caused by α-synuclein.Inhibition of autophagy by α-synuclein overexpression or Rab1a knockdown causes mislocalization of the autophagy protein, Atg9, and decreases omegasome formation.Rab1a, α-synuclein, and Atg9 all regulate formation of the omegasome, which marks autophagosome precursors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, England, UK.

ABSTRACT
Parkinson's disease (PD) is characterized pathologically by intraneuronal inclusions called Lewy bodies, largely comprised of α-synuclein. Multiplication of the α-synuclein gene locus increases α-synuclein expression and causes PD. Thus, overexpression of wild-type α-synuclein is toxic. In this study, we demonstrate that α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Our data show that α-synuclein compromises autophagy via Rab1a inhibition and Rab1a overexpression rescues the autophagy defect caused by α-synuclein. Inhibition of autophagy by α-synuclein overexpression or Rab1a knockdown causes mislocalization of the autophagy protein, Atg9, and decreases omegasome formation. Rab1a, α-synuclein, and Atg9 all regulate formation of the omegasome, which marks autophagosome precursors.

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Autophagy is not dependent on all Rab proteins that regulate secretion. (A) To measure the effect of RNAi of Rab1a, Rab1b, Sar1A/B, Giantin, and VDP on constitutive secretion, HeLa-derived C1 cells were transfected with siRNA corresponding to these genes. The amount of GFP–growth hormone remaining in the cells 80 min after induction of secretion was determined using flow cytometry. As a positive control, the cells were transfected with STX5 (syntaxin5) siRNA, a known inhibitor of secretion (n = 3 independent experiments; ***, P < 0.001; multiple comparisons, one-way ANOVA, Dunnett’s multiple comparisons post hoc test). GH, growth hormone. (B) LC3-II levels were assessed by SDS-PAGE after knockdown of Sar1A and -B, Giantin, or VDP in HeLa cells. Actin levels demonstrate equal loading. (C) Bar graphs indicating the effect of knockdown, as in B, on httQ74 aggregation (**, P< 0.01; ***, P < 0.001; odds ratio; n = 9). (D) Tomato-p62 and DsRed were transfected into HeLa cells during the last 24 h of knockdown, as in B, and the effects were measured by SDS-PAGE. DsRed was used as a transfection control for tomato-p62. Quantification of tomato-p62 levels relative to DsRed is shown in the bar graph (*, P < 0.05; **, P < 0.01; Dunnett’s multiple comparison post hoc test; n = 3). Values were normalized to control values for separate experiments. (E) LC3-II levels were assessed by SDS-PAGE after knockdown of Rab1b (bands shown are from the same gel, and unrelated lanes have been omitted) or Rab2. Actin and tubulin levels demonstrate equal loading. (F) Bar graphs indicating the effect of knockdown, as in E, on the percentage of httQ74 aggregation in transfected cells (***, P < 0.001; odds ratio; n = 9). (G) Tomato-p62 and DsRed were transfected into HeLa cells during the last 24 h of knockdown, as in E, and the effects were measured by SDS-PAGE. DsRed was used as a transfection control for tomato-p62. Quantification of tomato-p62 levels relative to DsRed is shown in the bar graph (**, P < 0.01; Bonferroni’s multiple comparison post hoc test; n = 3). Values were normalized to control values for separate experiments. (A, C, D, F, and G) Error bars represent SEM.
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fig4: Autophagy is not dependent on all Rab proteins that regulate secretion. (A) To measure the effect of RNAi of Rab1a, Rab1b, Sar1A/B, Giantin, and VDP on constitutive secretion, HeLa-derived C1 cells were transfected with siRNA corresponding to these genes. The amount of GFP–growth hormone remaining in the cells 80 min after induction of secretion was determined using flow cytometry. As a positive control, the cells were transfected with STX5 (syntaxin5) siRNA, a known inhibitor of secretion (n = 3 independent experiments; ***, P < 0.001; multiple comparisons, one-way ANOVA, Dunnett’s multiple comparisons post hoc test). GH, growth hormone. (B) LC3-II levels were assessed by SDS-PAGE after knockdown of Sar1A and -B, Giantin, or VDP in HeLa cells. Actin levels demonstrate equal loading. (C) Bar graphs indicating the effect of knockdown, as in B, on httQ74 aggregation (**, P< 0.01; ***, P < 0.001; odds ratio; n = 9). (D) Tomato-p62 and DsRed were transfected into HeLa cells during the last 24 h of knockdown, as in B, and the effects were measured by SDS-PAGE. DsRed was used as a transfection control for tomato-p62. Quantification of tomato-p62 levels relative to DsRed is shown in the bar graph (*, P < 0.05; **, P < 0.01; Dunnett’s multiple comparison post hoc test; n = 3). Values were normalized to control values for separate experiments. (E) LC3-II levels were assessed by SDS-PAGE after knockdown of Rab1b (bands shown are from the same gel, and unrelated lanes have been omitted) or Rab2. Actin and tubulin levels demonstrate equal loading. (F) Bar graphs indicating the effect of knockdown, as in E, on the percentage of httQ74 aggregation in transfected cells (***, P < 0.001; odds ratio; n = 9). (G) Tomato-p62 and DsRed were transfected into HeLa cells during the last 24 h of knockdown, as in E, and the effects were measured by SDS-PAGE. DsRed was used as a transfection control for tomato-p62. Quantification of tomato-p62 levels relative to DsRed is shown in the bar graph (**, P < 0.01; Bonferroni’s multiple comparison post hoc test; n = 3). Values were normalized to control values for separate experiments. (A, C, D, F, and G) Error bars represent SEM.

Mentions: Given the role of Rab1a in secretion, we wanted to determine whether Rab1a regulates autophagy independently of general secretion or whether autophagy does in fact depend on proper functioning of the secretory pathway, as implicated in early yeast studies (Ishihara et al., 2001; Hamasaki et al., 2003; Reggiori et al., 2004). Understanding this difference may provide clues as to whether the autophagy defect caused by α-synuclein is a generic consequence of the secretory defect or whether Rab1a inhibition of autophagy can be dissociated from a general secretory defect, suggesting a specific role for Rab1a in autophagy regulation. To begin to address this issue, we used RNAi to decrease protein levels of Rab1a effectors (Giantin and VDP [vesicle-docking protein 115]), early secretory proteins critical for Rab1a secretion (Sar1A and -B), and Rab1b, an alternative isoform of Rab1, also involved in early secretion. After knockdown of these proteins, we analyzed the effects of their depletion on both secretion and autophagy to more closely look at the interplay of these two pathways. Knockdown of Rab1a, VDP, Giantin, and Sar1A and -B all partially inhibited constitutive secretion (Fig. 4 A), as measured by the percentage of protein secreted 80 min after secretion induction. Rab1b (which appears to traffic different subsets of vesicles to Rab1a; Allan et al., 2000; Alvarez et al., 2003) inhibited secretion to a similar extent as Rab1a, Giantin, and VDP. Like Rab1a, knockdown of known Rab1a effectors Giantin and VDP and Sar1A and -B decreased LC3-II levels (Fig. 4 B), increased the percentage of cells with huntingtin aggregates (Fig. 4 C), and increased p62 accumulation (Fig. 4 D). Unexpectedly, knockdown of Rab1b had the opposite effect on autophagy. Rab1b RNAi increased LC3-II (Fig. 4 E) levels, decreased the percentage of cells with huntingtin aggregates (Fig. 4 F), and decreased accumulation of p62 (Fig. 4 G).


α-Synuclein impairs macroautophagy: implications for Parkinson's disease.

Winslow AR, Chen CW, Corrochano S, Acevedo-Arozena A, Gordon DE, Peden AA, Lichtenberg M, Menzies FM, Ravikumar B, Imarisio S, Brown S, O'Kane CJ, Rubinsztein DC - J. Cell Biol. (2010)

Autophagy is not dependent on all Rab proteins that regulate secretion. (A) To measure the effect of RNAi of Rab1a, Rab1b, Sar1A/B, Giantin, and VDP on constitutive secretion, HeLa-derived C1 cells were transfected with siRNA corresponding to these genes. The amount of GFP–growth hormone remaining in the cells 80 min after induction of secretion was determined using flow cytometry. As a positive control, the cells were transfected with STX5 (syntaxin5) siRNA, a known inhibitor of secretion (n = 3 independent experiments; ***, P < 0.001; multiple comparisons, one-way ANOVA, Dunnett’s multiple comparisons post hoc test). GH, growth hormone. (B) LC3-II levels were assessed by SDS-PAGE after knockdown of Sar1A and -B, Giantin, or VDP in HeLa cells. Actin levels demonstrate equal loading. (C) Bar graphs indicating the effect of knockdown, as in B, on httQ74 aggregation (**, P< 0.01; ***, P < 0.001; odds ratio; n = 9). (D) Tomato-p62 and DsRed were transfected into HeLa cells during the last 24 h of knockdown, as in B, and the effects were measured by SDS-PAGE. DsRed was used as a transfection control for tomato-p62. Quantification of tomato-p62 levels relative to DsRed is shown in the bar graph (*, P < 0.05; **, P < 0.01; Dunnett’s multiple comparison post hoc test; n = 3). Values were normalized to control values for separate experiments. (E) LC3-II levels were assessed by SDS-PAGE after knockdown of Rab1b (bands shown are from the same gel, and unrelated lanes have been omitted) or Rab2. Actin and tubulin levels demonstrate equal loading. (F) Bar graphs indicating the effect of knockdown, as in E, on the percentage of httQ74 aggregation in transfected cells (***, P < 0.001; odds ratio; n = 9). (G) Tomato-p62 and DsRed were transfected into HeLa cells during the last 24 h of knockdown, as in E, and the effects were measured by SDS-PAGE. DsRed was used as a transfection control for tomato-p62. Quantification of tomato-p62 levels relative to DsRed is shown in the bar graph (**, P < 0.01; Bonferroni’s multiple comparison post hoc test; n = 3). Values were normalized to control values for separate experiments. (A, C, D, F, and G) Error bars represent SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3101586&req=5

fig4: Autophagy is not dependent on all Rab proteins that regulate secretion. (A) To measure the effect of RNAi of Rab1a, Rab1b, Sar1A/B, Giantin, and VDP on constitutive secretion, HeLa-derived C1 cells were transfected with siRNA corresponding to these genes. The amount of GFP–growth hormone remaining in the cells 80 min after induction of secretion was determined using flow cytometry. As a positive control, the cells were transfected with STX5 (syntaxin5) siRNA, a known inhibitor of secretion (n = 3 independent experiments; ***, P < 0.001; multiple comparisons, one-way ANOVA, Dunnett’s multiple comparisons post hoc test). GH, growth hormone. (B) LC3-II levels were assessed by SDS-PAGE after knockdown of Sar1A and -B, Giantin, or VDP in HeLa cells. Actin levels demonstrate equal loading. (C) Bar graphs indicating the effect of knockdown, as in B, on httQ74 aggregation (**, P< 0.01; ***, P < 0.001; odds ratio; n = 9). (D) Tomato-p62 and DsRed were transfected into HeLa cells during the last 24 h of knockdown, as in B, and the effects were measured by SDS-PAGE. DsRed was used as a transfection control for tomato-p62. Quantification of tomato-p62 levels relative to DsRed is shown in the bar graph (*, P < 0.05; **, P < 0.01; Dunnett’s multiple comparison post hoc test; n = 3). Values were normalized to control values for separate experiments. (E) LC3-II levels were assessed by SDS-PAGE after knockdown of Rab1b (bands shown are from the same gel, and unrelated lanes have been omitted) or Rab2. Actin and tubulin levels demonstrate equal loading. (F) Bar graphs indicating the effect of knockdown, as in E, on the percentage of httQ74 aggregation in transfected cells (***, P < 0.001; odds ratio; n = 9). (G) Tomato-p62 and DsRed were transfected into HeLa cells during the last 24 h of knockdown, as in E, and the effects were measured by SDS-PAGE. DsRed was used as a transfection control for tomato-p62. Quantification of tomato-p62 levels relative to DsRed is shown in the bar graph (**, P < 0.01; Bonferroni’s multiple comparison post hoc test; n = 3). Values were normalized to control values for separate experiments. (A, C, D, F, and G) Error bars represent SEM.
Mentions: Given the role of Rab1a in secretion, we wanted to determine whether Rab1a regulates autophagy independently of general secretion or whether autophagy does in fact depend on proper functioning of the secretory pathway, as implicated in early yeast studies (Ishihara et al., 2001; Hamasaki et al., 2003; Reggiori et al., 2004). Understanding this difference may provide clues as to whether the autophagy defect caused by α-synuclein is a generic consequence of the secretory defect or whether Rab1a inhibition of autophagy can be dissociated from a general secretory defect, suggesting a specific role for Rab1a in autophagy regulation. To begin to address this issue, we used RNAi to decrease protein levels of Rab1a effectors (Giantin and VDP [vesicle-docking protein 115]), early secretory proteins critical for Rab1a secretion (Sar1A and -B), and Rab1b, an alternative isoform of Rab1, also involved in early secretion. After knockdown of these proteins, we analyzed the effects of their depletion on both secretion and autophagy to more closely look at the interplay of these two pathways. Knockdown of Rab1a, VDP, Giantin, and Sar1A and -B all partially inhibited constitutive secretion (Fig. 4 A), as measured by the percentage of protein secreted 80 min after secretion induction. Rab1b (which appears to traffic different subsets of vesicles to Rab1a; Allan et al., 2000; Alvarez et al., 2003) inhibited secretion to a similar extent as Rab1a, Giantin, and VDP. Like Rab1a, knockdown of known Rab1a effectors Giantin and VDP and Sar1A and -B decreased LC3-II levels (Fig. 4 B), increased the percentage of cells with huntingtin aggregates (Fig. 4 C), and increased p62 accumulation (Fig. 4 D). Unexpectedly, knockdown of Rab1b had the opposite effect on autophagy. Rab1b RNAi increased LC3-II (Fig. 4 E) levels, decreased the percentage of cells with huntingtin aggregates (Fig. 4 F), and decreased accumulation of p62 (Fig. 4 G).

Bottom Line: Our data show that α-synuclein compromises autophagy via Rab1a inhibition and Rab1a overexpression rescues the autophagy defect caused by α-synuclein.Inhibition of autophagy by α-synuclein overexpression or Rab1a knockdown causes mislocalization of the autophagy protein, Atg9, and decreases omegasome formation.Rab1a, α-synuclein, and Atg9 all regulate formation of the omegasome, which marks autophagosome precursors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, England, UK.

ABSTRACT
Parkinson's disease (PD) is characterized pathologically by intraneuronal inclusions called Lewy bodies, largely comprised of α-synuclein. Multiplication of the α-synuclein gene locus increases α-synuclein expression and causes PD. Thus, overexpression of wild-type α-synuclein is toxic. In this study, we demonstrate that α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Our data show that α-synuclein compromises autophagy via Rab1a inhibition and Rab1a overexpression rescues the autophagy defect caused by α-synuclein. Inhibition of autophagy by α-synuclein overexpression or Rab1a knockdown causes mislocalization of the autophagy protein, Atg9, and decreases omegasome formation. Rab1a, α-synuclein, and Atg9 all regulate formation of the omegasome, which marks autophagosome precursors.

Show MeSH
Related in: MedlinePlus