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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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α-Synuclein, Rab1a, and Atg9 affect omegasome and autophagosome formation. (A) The effect of Rab1a and Atg9 knockdown on omegasome formation in DFCP1-GFP HEK293, human embryonic kidney cells. (B) Quantification of A (*, P < 0.05; one-way ANOVA, Dunnett’s multiple comparison post hoc test; n = 10). (C) The effect of α-synuclein on omegasome formation in DFCP1-GFP HEK293 cells. httQ23-HA was used as a control for cells expressing α-synuclein–HA. (A and C) Arrows mark omegasomes. Images shown are single-plane images. (D) Quantification of C (*, P < 0.05; two-tailed Student’s t test; n = 10). (E) The effect of cells expressing α-synuclein–HA with empty CFP vector (control conditions) or Rab1a-CFP (rescue conditions) on omegasome formation. HA-tagged wild-type huntingtin exon-1 fragment with 23 polyglutamine repeats (httQ23) was used as a control for cells expressing α-synuclein–HA in both control and rescue conditions. Z-stack projections are shown. Arrows denote omegasomes. (F) Quantification of E (*, P < 0.05; two-tailed Student’s t test; n = 10). (B, D, and F) Error bars represent SEM. (G) Effect of Atg9 knockdown on LC3-II levels in SKNSH cell lysates. Actin was used to demonstrate equal loading. Bars: (A and C) 20 µm; (E) 10 µm.
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fig7: α-Synuclein, Rab1a, and Atg9 affect omegasome and autophagosome formation. (A) The effect of Rab1a and Atg9 knockdown on omegasome formation in DFCP1-GFP HEK293, human embryonic kidney cells. (B) Quantification of A (*, P < 0.05; one-way ANOVA, Dunnett’s multiple comparison post hoc test; n = 10). (C) The effect of α-synuclein on omegasome formation in DFCP1-GFP HEK293 cells. httQ23-HA was used as a control for cells expressing α-synuclein–HA. (A and C) Arrows mark omegasomes. Images shown are single-plane images. (D) Quantification of C (*, P < 0.05; two-tailed Student’s t test; n = 10). (E) The effect of cells expressing α-synuclein–HA with empty CFP vector (control conditions) or Rab1a-CFP (rescue conditions) on omegasome formation. HA-tagged wild-type huntingtin exon-1 fragment with 23 polyglutamine repeats (httQ23) was used as a control for cells expressing α-synuclein–HA in both control and rescue conditions. Z-stack projections are shown. Arrows denote omegasomes. (F) Quantification of E (*, P < 0.05; two-tailed Student’s t test; n = 10). (B, D, and F) Error bars represent SEM. (G) Effect of Atg9 knockdown on LC3-II levels in SKNSH cell lysates. Actin was used to demonstrate equal loading. Bars: (A and C) 20 µm; (E) 10 µm.

Mentions: Knockdown of Rab1a and overexpression of α-synuclein reduced omegasome vesicle count under basal conditions (Fig. 7, A–D). Overexpression of Rab1a was able to rescue the deficiency in omegasome formation caused by α-synuclein overexpression and increase omegasome levels to nearly that of the control condition (Fig. 7, E and F). Interestingly, Rab1a overexpression can increase omegasome count above that of the normal basal condition but is unable to increase LC3 vesicle formation above basel levels in α-synuclein–expressing cells. The transition of the omegasome to a functional LC3-positive autophagosome may be a potential rate-limiting step that cannot be overcome simply with an increase in omegasome formation.


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)

α-Synuclein, Rab1a, and Atg9 affect omegasome and autophagosome formation. (A) The effect of Rab1a and Atg9 knockdown on omegasome formation in DFCP1-GFP HEK293, human embryonic kidney cells. (B) Quantification of A (*, P < 0.05; one-way ANOVA, Dunnett’s multiple comparison post hoc test; n = 10). (C) The effect of α-synuclein on omegasome formation in DFCP1-GFP HEK293 cells. httQ23-HA was used as a control for cells expressing α-synuclein–HA. (A and C) Arrows mark omegasomes. Images shown are single-plane images. (D) Quantification of C (*, P < 0.05; two-tailed Student’s t test; n = 10). (E) The effect of cells expressing α-synuclein–HA with empty CFP vector (control conditions) or Rab1a-CFP (rescue conditions) on omegasome formation. HA-tagged wild-type huntingtin exon-1 fragment with 23 polyglutamine repeats (httQ23) was used as a control for cells expressing α-synuclein–HA in both control and rescue conditions. Z-stack projections are shown. Arrows denote omegasomes. (F) Quantification of E (*, P < 0.05; two-tailed Student’s t test; n = 10). (B, D, and F) Error bars represent SEM. (G) Effect of Atg9 knockdown on LC3-II levels in SKNSH cell lysates. Actin was used to demonstrate equal loading. Bars: (A and C) 20 µm; (E) 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3101586&req=5

fig7: α-Synuclein, Rab1a, and Atg9 affect omegasome and autophagosome formation. (A) The effect of Rab1a and Atg9 knockdown on omegasome formation in DFCP1-GFP HEK293, human embryonic kidney cells. (B) Quantification of A (*, P < 0.05; one-way ANOVA, Dunnett’s multiple comparison post hoc test; n = 10). (C) The effect of α-synuclein on omegasome formation in DFCP1-GFP HEK293 cells. httQ23-HA was used as a control for cells expressing α-synuclein–HA. (A and C) Arrows mark omegasomes. Images shown are single-plane images. (D) Quantification of C (*, P < 0.05; two-tailed Student’s t test; n = 10). (E) The effect of cells expressing α-synuclein–HA with empty CFP vector (control conditions) or Rab1a-CFP (rescue conditions) on omegasome formation. HA-tagged wild-type huntingtin exon-1 fragment with 23 polyglutamine repeats (httQ23) was used as a control for cells expressing α-synuclein–HA in both control and rescue conditions. Z-stack projections are shown. Arrows denote omegasomes. (F) Quantification of E (*, P < 0.05; two-tailed Student’s t test; n = 10). (B, D, and F) Error bars represent SEM. (G) Effect of Atg9 knockdown on LC3-II levels in SKNSH cell lysates. Actin was used to demonstrate equal loading. Bars: (A and C) 20 µm; (E) 10 µm.
Mentions: Knockdown of Rab1a and overexpression of α-synuclein reduced omegasome vesicle count under basal conditions (Fig. 7, A–D). Overexpression of Rab1a was able to rescue the deficiency in omegasome formation caused by α-synuclein overexpression and increase omegasome levels to nearly that of the control condition (Fig. 7, E and F). Interestingly, Rab1a overexpression can increase omegasome count above that of the normal basal condition but is unable to increase LC3 vesicle formation above basel levels in α-synuclein–expressing cells. The transition of the omegasome to a functional LC3-positive autophagosome may be a potential rate-limiting step that cannot be overcome simply with an increase in omegasome formation.

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