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. 2008 Mar 21;4(3):e1000027.
doi: 10.1371/journal.pgen.1000027.

C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging

Tjakko J van Ham  1 , Karen L Thijssen, Rainer Breitling, Robert M W Hofstra, Ronald H A Plasterk, Ellen A A Nollen
Affiliations

C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging

Tjakko J van Ham et al. PLoS Genet. .

Abstract

Inclusions in the brain containing alpha-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a C. elegans model that makes it possible to monitor, in living animals, the formation of alpha-synuclein inclusions. In worms of old age, inclusions contain aggregated alpha- synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in alpha-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between alpha-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson's disease and other alpha-synuclein related disorders.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Α-synuclein-YFP in Transgenic Animals Relocalizes to Discrete Inclusions during Aging.
(A) Confocal laser scanning images showing α-synuclein-YFP expression in the head region of transgenic C. elegans during aging. (B) Immunoblotting analysis of SDS/PAGE separated protein extracts from α-synuclein-YFP, N2 (wt) and YFP animals using α-synuclein (LB509) and YFP (anti-GFP) antibodies. Loading control is α-actin. (C) Immunoblotting analysis of protein extracts from 3-, 5-, 11- and 17-day old α-synuclein-YFP synchronized animals using anti-α- synuclein antibody.
Figure 2
Figure 2. Fluorescent Recovery after Photo Bleaching Reveals α-Synuclein Inclusions Contain Mobile as well as Immobilized Protein Material.
(A,F,K,P) Images of YFP, Q40-YFP and α-synuclein-YFP transgenic animals. (B-E,G-J, L-O,Q-T) High magnification images of the area indicated (red box) before photo bleaching and during recovery. (U-X) Graphical representation of fluorescence recovery after photo bleaching in (B-E, G-J, L-O, Q-T). Relative fluorescence intensity (RFI) value on y-axis represents percentage fluorescence corrected for background bleaching. (Y) Average number of inclusions larger than ∼2 µm2 per animal between tip of the nose and pharyngeal bulb during aging (n = 9 for day 11, n = 10 for days 9, 13, 15 and 17). (Z) Percentage of foci containing immobile material during aging. Bar in d-g represents 50 µm (overview) and 5 µm in higher magnification images. Error bars in (Y) indicate standard deviation.
Figure 3
Figure 3. Suppressors of Inclusion Formation Identified by RNAi.
(A) Confocal images showing head region of α-synuclein-YFP transgenic animals fed on OP50 bacteria, bacteria containing L4440 (empty vector) and expressing double stranded RNA targeting two representative genes (F26H11.4 and Y48G1A.6) found to increase inclusion formation. Phenotypes of increased inclusion formation were analyzed in liquid culture by observing at least five out of the first ten animals screened to show an increased presence of inclusions compared to wild type. Scale bar represents 50 µm. (B) Quantification of the number of inclusions present in worms as shown in (A) (n = 2).
Figure 4
Figure 4. Sir-2.1, ymel-1 and lagr-1 Are Suppressors of α-Synuclein Inclusion Formation.
(A,B) Confocal images showing α-synuclein-YFP transgenic animals and the transgenic strains containing a deletion in the sir-2.1gene (sir-2.1(ok434)) on day 9. (C) Average number of inclusions between tip of the head and pharyngeal bulb of the worm (n = 8). *P≤0.025 (Student's t test). (D, E, F) Confocal images showing α-synuclein-YFP transgenic worms and the transgenic strains containing a deletion in the ymel-1and lagr-1 gene (ymel-1(tm1920) and lagr-1(gk331)), on day 9. (G) Average number of inclusions between tip and pharyngeal bulb of the worm (n = 8 (wt and ymel-1), n = 7 (lagr-1)). *P≤0.05, **P≤0.05 (Student's t test).
Figure 5
Figure 5. RNAi and Deletion of grk-1 and grk-2 Decreases Inclusion Formation.
(A) Confocal images showing head region of α-synuclein-YFP transgenic animals fed on OP50 bacteria, bacteria containing empty RNAi vector (L4440), and expressing double stranded RNA targeting grk-1 and grk-2. (B) Confocal images of α-synuclein-YFP C. elegans, in wild type background, grk-1(ok1239) and grk-2(gk268) background. Scale bar represents 25 μm. (C) Quantification of the number of worms within the population (n = 20) with the same amount (wt), fewer (<wt) or no inclusion (none). (D) Western blot analysis of protein extracts from 20 staged transgenic C. elegans with a grk-1 or grk-2 genetic deletion showing similar levels of fusion protein expressed and actin (loading control).
Figure 6
Figure 6. RNAi of hsp-70 Does Not Affect Inclusion Formation.
(A) Confocal images showing Q24-YFP, Q35-YFP and α-synuclein-YFP transgenic animals fed on bacteria containing empty RNAi vector (L4440), and expressing double stranded RNA targeting hsp-70.
Figure 7
Figure 7. Subcellular Distribution α-Synuclein Modifiers.
(A) and (B) bar graphs showing the percentage of genes within each subcellular location class for the genes identified in the α-synuclein modifier screen (A), the polyglutamine modifier screen (B) versus random lists of genes. Random genes were obtained from UniProt (Universal Protein Resource). The subcellular quantities were calculated by adding up the subcellular annotation for 20 sets of 55 randomly chosen proteins (black bars). Values shown are average (n = 20, error bars indicate standard deviation). (A) Overrepresentation of genes in ER/Golgi class and vesicular class in the α-synuclein screen (P-values for the differences between the number of observed genes in ER/Golgi and vesicular class and the number expected: P<0.005 and P<0.001). (B) Overrepresentation of genes in cytoplasm and nucleus in the polyglutamine screen (P-values for the differences between the percentage of observed genes in cytoplasm and nucleus and the number expected: P<0.001 and P<0.02).
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