C. elegans Model Identifies Genetic Modifiers of a- Synuclein Inclusion Formation During Aging Tjakko J. van Ham 1 , Karen L. Thijssen 1 , Rainer Breitling 2 , Robert M. W. Hofstra 1 , Ronald H. A. Plasterk 3 , Ellen A. A. Nollen 1 * 1 Department of Genetics, University Medical Centre Groningen and University of Groningen, Groningen, The Netherlands, 2 Groningen Bioinformatics Centre, University of Groningen, Haren, The Netherlands, 3 Hubrecht Laboratory, Netherlands Institute of Developmental Biology, Utrecht, The Netherlands Abstract Inclusions in the brain containing a-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 a-synuclein inclusions. In worms of old age, inclusions contain aggregated a- 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 a-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 a-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 a-synuclein related disorders. Citation: van Ham TJ, Thijssen KL, Breitling R, Hofstra RMW, Plasterk RHA, et al. (2008) C. elegans Model Identifies Genetic Modifiers of a-Synuclein Inclusion Formation During Aging. PLoS Genet 4(3): e1000027. doi:10.1371/journal.pgen.1000027 Editor: Stuart K. Kim, Stanford University Medical Center, United States of America Received October 2, 2007; Accepted February 8, 2008; Published March 21, 2008 Copyright: ß 2008 van Ham et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was funded by a ZonMW Research Institute of the Elderly grant (TvH, EAAN and RHAP) and an NWO VENI grant (EAAN). Competing Interests: The authors have declared that no competing interests exist. * E-mail: e.a.a.nollen@medgen.umcg.nl Introduction Sporadic as well as familial Parkinson’s disease are characterized by protein inclusions in the brain containing a-synuclein [1]. Similar inclusions are also present in other neurodegenerative diseases, including dementia with Lewy bodies [2]. The a-synuclein gene is causatively related to Parkinson’s disease, since mutations in the gene, and duplication or triplication of the a-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 a- synuclein locus. How a-synuclein inclusions are produced is unknown, but identifying cellular factors and processes involved in the formation of these inclusions may provide some understand- ing of the molecular cause of Parkinson’s disease and of the link between aging and the sporadic form of the disease. To study pathological a-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 a-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 a- synuclein accumulation in inclusions. Results/Discussion To visually trace expression of a-synuclein, we expressed human a-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 a- 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 a-synuclein-YFP chimaeric protein is recognized by an antibody specific for human a-synuclein and an antibody for YFP (Figure 1B). YFP fused to human a-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 a-synuclein-YFP into foci is caused by intrinsic properties of the a-synuclein protein. 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 a-synuclein was aggregated within the inclusions in our C. elegans model, we measured the mobility of the a-synuclein-YFP chimaera by fluorescence recovery after PLoS Genetics | www.plosgenetics.org 1 2008 | Volume 4 | Issue 3 | e1000027