Introduction Sporadic as well as familial Parkinson's disease are characterized by protein inclusions in the brain containing α-synuclein [1]. Similar inclusions are also present in other neurodegenerative diseases, including dementia with Lewy bodies [2]. The α-synuclein gene is causatively related to Parkinson's disease, since mutations in the gene, and duplication or triplication of the α-synuclein locus cause familial forms of Parkinson's disease in humans [3]–[5]. Sporadic Parkinson's disease, seen in 1–4% of the population over 65 years of age, appears to be unrelated to mutations or multiplications of the α-synuclein locus. How α-synuclein inclusions are produced is unknown, but identifying cellular factors and processes involved in the formation of these inclusions may provide some understanding of the molecular cause of Parkinson's disease and of the link between aging and the sporadic form of the disease. To study pathological α-synuclein accumulation, we used a genetic model organism, the nematode Caenorhabditis elegans. We chose C. elegans for its thoroughly characterized aging properties, its amenability to genome-wide RNAi screening, and its transparency throughout its lifetime, which allows visualization of inclusions in living animals during aging. We expressed human α-synuclein fused to yellow fluorescent protein in the body wall muscle of C. elegans, where it, age-dependently, accumulated into inclusions. In old age these inclusions contained aggregated material, similar to human pathological inclusions. We used a genome-wide RNAi screen to identify genes and cellular processes involved in age-related α-synuclein accumulation in inclusions. Results/Discussion To visually trace expression of α-synuclein, we expressed human α-synuclein fused to yellow fluorescent protein (YFP) in C. elegans under control of the unc-54 promoter, which drives expression to the body wall muscle cells. Muscle expression rather than neuronal expression was chosen for several reasons. The unc-54 promoter is strong and muscle cells are large, allowing for visual detection of α-synuclein expression and its subcellular localization. Furthermore, RNAi by feeding seems to work more efficiently in muscles than in neurons, which better allows for genome-wide RNAi screening. Finally, muscle expression has been used successfully to model protein-misfolding diseases and to identify modifier genes in previous studies [6]–[8]. The α-synuclein-YFP chimaeric protein is recognized by an antibody specific for human α-synuclein and an antibody for YFP (Figure 1B). YFP fused to human α-synuclein relocates to inclusions (Figure 1A), which are visible as early as day 2 after hatching and increase in number and size during the animals' aging up to late adulthood. As YFP alone remains diffusely localized throughout aging, this indicates that relocation of α-synuclein-YFP into foci is caused by intrinsic properties of the α-synuclein protein. 10.1371/journal.pgen.1000027.g001 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. One of the characteristics of late inclusions in the brains of Parkinson's patients is the presence of electron-dense filamentous and granular protein material, which is typical for aggregated protein [9]. To address whether α-synuclein was aggregated within the inclusions in our C. elegans model, we measured the mobility of the α-synuclein-YFP chimaera by fluorescence recovery after photo bleaching (FRAP) [10]. We observed two types of inclusions. One type contained mostly mobile material (Figure 2P–2T, 2W; ∼80% fluorescence recovery), whereas the other type contained immobilized material (Figure 2K-2O, 2X; ∼40% fluorescence recovery), similar to Q40- YFP aggregates (Figure 2F-2J, V; ∼30% fluorescence recovery), indicating aggregated protein, a characteristic of α-synuclein deposits in Parkinson's disease. 10.1371/journal.pgen.1000027.g002 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. Notably, there was an increase in the number of “immobile” inclusions relative to “mobile” inclusions during aging (Figure 2Y and 2Z), which appeared unrelated to the expression level of α- synuclein-YFP (Figure 1C). Interestingly, immobile inclusions were not observed before late adulthood, which is consistent with age-related aggregation in Parkinson's disease patients [11]. We noted that the motility of the mobile material contained in inclusions was similar at all ages, suggesting that aggregation was not a gradual process but caused by a sharp transition from mobile to immobile material (data not shown). Taken together, the time-dependent accumulation and immobilization of α-synuclein in inclusions verify the suitability of our α-synuclein-YFP C. elegans system as a model for human Parkinson's disease. To identify processes involved in inclusion formation we searched for genes that, when inactivated, increased the amount of inclusions using a genome-wide RNAi screen. Worms were screened by visual inspection using fluorescence microscopy at days 4 and 5 after transfer of synchronized, first stage (L1) larvae to RNAi clones. Clones were scored positive when at least five out of the first ten worms screened showed an increase in the amount of inclusions (Figure 3). Genes for which RNAi results were confirmed in duplicate, in liquid culture and on agar plates, were considered suppressors of inclusion formation. 10.1371/journal.pgen.1000027.g003 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). In this screen we found 80 suppressors of inclusion formation (Table 1). Forty-nine of these genes have an established human ortholog (Blast E-value ≤2.5e-5), indicating involvement of these genes in molecular pathways conserved between humans and nematodes (Table S1 online). The effect of RNAi was confirmed in genetic deletion strains for three genes: sir-2.1, an NAD+-dependent protein deacetylase, lagr-1, a sphingolipid synthase, and ymel-1, a mitochondrial protease, which is an ortholog of the human presenilin associated metalloprotease (PAMP) (Figure 4). 10.1371/journal.pgen.1000027.g004 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). 10.1371/journal.pgen.1000027.t001 Table 1 Suppressors of α-Synuclein Inclusion Formation. Function Cosmid no. Gene Description Strength CS R05H10.6 cdh-7 FAT tumor suppressor homolog 3 + CS Y54E2A.2 Similar to Ca2+-binding actin-bundling protein (spectrin) + DR E03A3.2 rcq-5 DEAD/DEAH box helicase ++ DR F32D1.10 mcm-7 DNA-replication licensing factor + DR W02D9.1 pri-2 Eukaryotic-type DNA primase, large subunit ++ ECM F41F3.4 col-139 Structural component of the cuticle, phosphate transport + EM C07A9.8 Bestrophin, anion channel ++ EM C28H8.11 Tryptophan 2,3-dioxygenase ++ EM R03E1.2 Renin-precursor, lysosomal ATPase H+ transporter ++ EM T14F9.1 vha-15 (phi-52) ATPase coupled proton transport, Vacuolar ATP synthase sub + EM Y37H9A.6 ndx-4 NUDIX hydrolase + ET B0213.12 cyp-34A7 Cytochrome P450 2B6 + ET W01B11.2 sulp-6 STAS domain, Sulfate transporter family ++ ET W01B11.6 Thioredoxin, Yeast: required for ER-Golgi transport, stress protection ++ GM F29F11.2 UDP-glucuronosyltransferase 1–8 precursor ++ GM K07A3.1 fbp-1(K07A3.b) Fructose-1-6-bisphosphatase + LM C28C12.7 spp-10 sphingolipid metabolism/lysosomal ++ LM F28H1.4 Membrane-associating domain, Plasmolipin ++ LM F54F11.1 Lipolytic enzyme, putative phospholipase ++ LM K09H9.6 lpd-6 RNA-binding protein required for 60S ribosomal subunit biogenesis + LM T08H10.1 Aldo-keto reductase family 1 member B10, oxido-reductase + LM W03G9.6 paf-1 acetylhydrolase/phospholipase A2 ++ LM Y48G9A.10 Carnitine O-palmitoyltransferase I ++ LM Y6B3B.10 lagr-1 LAG1, (dihydro)ceramide synthase, spingholipid synthesis, ER ++ PD C46F9.3 math-24 MATH domain, Ubiquitin carboxyl-terminal hydrolase 7 ++ PD C47B2.1 F-box domain + PD F32H2.7 E3 ubiquitin-protein ligase HECTD1 + PD F49B2.6 Peptidase family M1 + PD M03C11.5 ymel-1 Peptidase M41, FtsH, metallo protease: unfolded mito-proteins ++ PD R09B3.4 ubc-12 Ub-conj. enzyme (E2), NEDD8-conj. enzyme NCE2 + PD R151.6 Human Derlin-2, protein degradation in ER + PD T06A4.1 Peptidase M14, carboxypeptidase A, Zinc-metalloprotease + PD Y63D3A.9 fbxb-93 F-box + PF F52C12.2 Uncharacterized conserved protein + PF R151.7 Hsp90 protein ++ PS F42A10.4 efk-1 Ca/calmodulin-dep. kinase, Elongation factor 2 kinase + PS F56H6.9 Protein of unknown function, DUF288 + PS Y67D8A_370.a puf-4 Translational repression, Splice Isoform 2 of Pumilio homolog 2 ++ RSP C04F5.5 srab-2 7TM chemoreceptor, GPCR activity, DNA biding/Zn-finger + RSP C28H8.5 ShTK domain ++ RSP D2089.1 rsp-7 Splicing factor, arginine/serine-rich ++ RSP F26H11.4 Neurofilament triplet H protein ++ RSP T12A2.3 Protein AF-9 ++ RSP Y113G7B.18 mdt-17 Transcriptional cofactor + RSP Y116A8C.35 uaf-2 Splicing factor U2AF 35 kDa subunit + RSP Y48G1A.6 Y48G1A_53.a Polycomb group protein SCM/L(3)MBT ++ SG C24B9.8 str-13 7TM chemoreceptor, GPCR activity ++ SG F10E9.3 Splice Isoform 1 of Death domain-associated protein 6 ++ SG F12A10.6 Serine/threonine kinase (haspin family) ++ SG F19H8.2 ∼to Rho kinase + SG F21F3.3 Isoprenylcysteine carboxyl methyltransferase family, yeast: ER ++ SG R09E12.1 srbc-59 7TM chemoreceptor, srbc family ++ SG R52.4 Chemoreceptor + SG T25B9.2 protein phosphatase 1, catalytic subunit, alpha isoform 3 ++ SG T28F2.2 Splice Isoform 1 of COMM domain-containing protein 4 ++ SG W05B5.2 GPCR, membrane ++ SG Y71G12B.20 mab-20 Semaphorin-4G precursor ++ SG ZK355.1 ZK355.h Tyr-kinase Receptor ++ U B0238.11 HMG box-containing protein, transcriptional + U C02E7.6 Splice Isoform 1 of AMME syndrome candidate gene 1 protein ++ U C29F9.1 Unknown ++ U D1022.5 Dienelactone hydrolase family + U F01G12.5a let-2 Unknown ++ U F47F6.5 Similarity to C-type lectin ++ U F54E7.6 Unknown ++ U F56C4.1 Unknown + U H23L24.e Unknown ++ U K01G12.3 HIV TAT specific factor 1 + U R09H3.3 Unknown ++ U R10H10.2 spe-26 kelch-like 20 ++ U R11A8.4 sir-2.1 NAD-dependent deacetylase sirtuin-1 ++ U T05A10.4 Similarity to cysteine-rich secretory protein 2 precursor + U T06G6.8 Unknown ++ U XE249 Chondroitin 6-sulfotransferase ++ U Y19D10A.j C-type lectin + U ZC334.9 ins-28 Insulin-like peptide ++ U ZK1290.11 Unknown ++ VT C07G1.5 hgrs-1 (pqn-9) HGF-reg tyr-kinase substrate, membrane trafficking/protein sorting + VT C34C12.2 Role in preribosome assembly or transport/t-snare domain ++ VT M151.3 Girdin, Yeast: ER-Golgi transport, SNARE assembly ++ VT T05G5.9 Ran-binding protein 2-like 4/yeast: ER-Golgi/SNARE assembly ++ VT W02A11.2 ESCRT-II complex subunit, Yeast: vacuolar protein sorting protein 25 + U: Unknown, RSP: RNA synthesis and processing, PT: Protein Transport, PS: Protein synthesis, PF: Protein Folding, DR: DNA replication, ECM: Extracellular matrix, CS: Cytoskeleton, EM: Energy metabolism, GM: Glucose Metabolism, SG: Signaling, VT: vesicle transport, ET: electron transport. +: up to a two-fold increase, ++: more than a two-fold increase in the amount of inclusions. The modifier genes function in a variety of biological processes, some of which have previously been suggested to be involved in Parkinson's disease, such as vesicular transport and lipid metabolism. Lipid metabolism, lipid membranes, and vesicle-mediated transport have previously been linked to α-synuclein pathology in a yeast model [12],[13]. In Parkinson's patients, lipids and membrane material have been found to be directly associated with α-synuclein in lipid droplets and Lewy bodies [11], [14]–[16]. Although we did identify a C. elegans ortholog of the recently discovered modifier of neuronal alpha-synuclein toxicity SIRT2, we did not pick up two other modifiers of neuronal alpha-synuclein pathology: the G-protein coupled receptor kinase 2 and the molecular chaperone Hsp70 [17]–[19]. In Drosophila, overexpression of G-protein coupled receptor kinase 2 (Gprk2) increases neuronal toxicity of α-synuclein [18]. In addition, expression of an S129A mutant of α-synuclein, which cannot be phosphorylated by Grpk2, is less toxic, while forming more aggregates [18]. Based on these observations, one might expect that knockdown of Gprk2 would result in an increase in the amount of inclusions as well. Two orthologs of the Gprk2 gene are present in C. elegans, grk-1 and grk-2. We tested the effect of knockdown of these two genes by RNAi on the formation of inclusions. Unexpectedly, RNAi knockdown of grk-1 or grk-2, and not RNAi of random tyrosine or serine kinases, resulted in a decrease in the amount of inclusions (Figure 5A and data not shown), indicating that we could not have recovered these genes in our screen for more inclusions. There was no obvious difference in the level of α-synuclein-YFP expression at day 5 after synchronization, indicating that formation of the inclusions themselves is affected (Figure 5D). The effect of knockdown of grk-1 and grk-2 by RNAi was confirmed by crossing in deletion alleles of both genes, grk-1(ok1239) and grk-2(gk268), into the α-synuclein-YFP strain (Figure 5B). Note that an RNAi screen for a reduction in the amount of inclusions yielded only one other kinase, which supports the idea that GRKs act specifically (data not shown). In all, C. elegans orthologs of Gprk2 act as modifiers of α-synuclein inclusion formation. Knockdown of Gprk2 in Drosophila or overexpression of grk-1 or grk-2 in C. elegans will be required to establish whether their specific role in alpha-synuclein pathology is comparable between the two species. 10.1371/journal.pgen.1000027.g005 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 (
