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A Druggable Genome Screen Identifies Modifiers of Α-Synuclein Levels Via a Tiered Cross-Species Validation Approach

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A Druggable Genome Screen Identifies Modifiers of Α-Synuclein Levels Via a Tiered Cross-Species Validation Approach This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. A link to any extended data will be provided when the final version is posted online. Research Articles: Neurobiology of Disease A druggable genome screen identifies modifiers of α-synuclein levels via a tiered cross-species validation approach Maxime W.C. Rousseaux1,2, Gabriel E. Vázquez-Vélez1,3,11, Ismael Al-Ramahi1,2, Hyun-Hwan Jeong1,2, Aleksandar Bajic1,4, Jean-Pierre Revelli1,2, Hui Ye1,2, Emily T. Phan1,2, Jennifer M. Deger1,2, Alma M. Perez1,2, Ji-Yoen Kim1,2, Laura A. Lavery1,2, Qikia Xu6, Mamie Z. Li6, Hyojin Kang1,2, Jean J. Kim12,13,14, Joshua M. Shulman1,2,5,7, Thomas F. Westbrook2,3,8,9, Stephen J. Elledge6, Zhandong Liu1,4, Juan Botas1,2 and Huda Y. Zoghbi1,2,3,4,10 1Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, TX, USA 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 3Program in Developmental Biology, Baylor College of Medicine, Houston, TX, USA 4Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA 5Department of Neurology, Baylor College of Medicine, Houston, TX, USA 6Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Division of Genetics, Brigham and Women's Hospital, Boston, MA, USA. 7Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA 8The Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, USA 9Therapeutic Innovation Center (THINC), Baylor College of Medicine, Houston, TX, USA 10Howard Hughes Medical Institute, Houston, TX, USA 11Medical Scientist Training Program, Baylor College of Medicine, Houston, TX, USA 12Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX, USA 13Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX, USA 14Human Stem Cell Core, Baylor College of Medicine, Houston, TX, USA DOI: 10.1523/JNEUROSCI.0254-18.2018 Received: 29 January 2018 Revised: 8 August 2018 Accepted: 13 August 2018 Published: 24 September 2018 Author contributions: M.W.R., G.E.V.-V., I.A.-R., J.-Y.K., L.A.L., T.F.W., S.J.E., J.B., and H.Y.Z. designed research; M.W.R., G.E.V.-V., I.A.-R., A.B., J.-P.R., H.Y., E.T.P., J.M.D., A.M.P., J.-Y.K., and L.A.L. performed research; M.W.R., G.E.V.-V., I.A.-R., H.-H.J., A.B., J.-P.R., and Z.L. analyzed data; M.W.R., G.E.V.-V., and H.Y.Z. wrote the first draft of the paper; M.W.R., G.E.V.-V., I.A.-R., H.-H.J., A.B., J.-P.R., H.Y., J.M.D., L.A.L., J.S., J.B., and H.Y.Z. edited the paper; M.W.R., G.E.V.-V., and H.Y.Z. wrote the paper; H.-H.J., Q.X., M.Z.L., H.K., J.J.K., J.S., T.F.W., S.J.E., and H.Y.Z. contributed unpublished reagents/analytic tools. Conflict of Interest: The authors declare no competing financial interests. Corresponding authors [email protected] and [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0254-18.2018 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 Title: A druggable genome screen identifies modifiers of α-synuclein levels via a 2 tiered cross-species validation approach 3 Abbreviated title: Pooled genetic screens for synucleinopathies 4 5 Author names and affiliations: 6 Maxime W.C. Rousseaux1,2,15,*,#, Gabriel E. Vázquez-Vélez1,3,11*, Ismael Al-Ramahi1,2, Hyun- 7 Hwan Jeong1,2, Aleksandar Bajic1,4, Jean-Pierre Revelli1,2, Hui Ye1,2, Emily T. Phan1,2, Jennifer 8 M. Deger1,2, Alma M. Perez1,2, Ji-Yoen Kim1,2, Laura A. Lavery1,2, Qikia Xu6, Mamie Z. Li6, 9 Hyojin Kang1,2,16, Jean J. Kim12,13,14, Joshua M. Shulman1,2,5,7, Thomas F. Westbrook2,3,8,9, 10 Stephen J. Elledge6, Zhandong Liu1,4, Juan Botas1,2, Huda Y. Zoghbi1-4,10,#. 11 1Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, 12 TX, USA 13 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 14 USA 15 3Program in Developmental Biology, Baylor College of Medicine, Houston, TX, USA 16 4Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA 17 5Department of Neurology, Baylor College of Medicine, Houston, TX, USA 18 6Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Division 19 of Genetics, Brigham and Women’s Hospital, Boston, MA, USA. 20 7Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA 21 8The Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor 22 College of Medicine, Houston, USA 23 9Therapeutic Innovation Center (THINC), Baylor College of Medicine, Houston, TX, USA 24 10Howard Hughes Medical Institute, Houston, TX, USA 25 11Medical Scientist Training Program, Baylor College of Medicine, Houston, TX, USA 26 12Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX, USA 27 13Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX, 28 USA 29 14Human Stem Cell Core, Baylor College of Medicine, Houston, TX, USA 30 15Present address: University of Ottawa Brain and Mind Research Institute, Department of 31 Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada. 32 16Present address: HPC-enabled Convergence Technology Research Division, Korea Institute of 33 Science and Technology Information, Daejeon, South Korea 34 *Authors contributed equally 35 #Corresponding authors [email protected] and [email protected] 36 Submitting author: [email protected] 37 Number of Pages: 40 38 Number of Figures: 11 (7 main, 4 extended data) 39 Number of Tables: 3 (1 main, 2 extended data) 40 Multimedia: 1 (video) 1 41 Number of words for Abstract: 197 42 Number of words for Introduction: 609 43 Number of words for Discussion: 1,496 44 Conflict of Interest: This study was partially funded by UCB pharmaceuticals (H.Y.Z.). 45 2 46 Acknowledgements: 47 The authors thank members of the Zoghbi lab for important discussions and critical feedback on the 48 manuscript, Nan Lu (BCM) for helping with the initial screen optimization and library construction, 49 Christof Fellmann (U.C. Berkeley) for providing the shRNA scores (SplashRNA scores) and important 50 discussions, the Viral Vector Core at the University of Texas MD Anderson and the Gene Vector and the 51 Genome and RNA Profiling (GARP) cores at Baylor College of Medicine. Neural progenitor cells used in 52 this study were derived from the WA09 human ESC line (H9 ESC) derived by Dr. James Thomson and 53 distributed by WiCell Research Institute under SLA agreement. This study was funded in part by grants 54 from the Huffington Foundation (H.Y.Z. and J.B.), the Robert A. and Renée E. Belfer Family Foundation 55 (H.Y.Z. and J.B.), and the Howard Hughes Medical Institute (H.Y.Z. and S.J.E.). H.Y.Z. also received 56 support for this study from UCB Pharma and The Hamill Foundation. S.J.E. was funded by an NIH grant 57 AG11083. M.W.C.R. received funding from the Canadian Institutes of Health Research (Fellowship 58 #201210MFE-290072-173743) and the Parkinson’s Disease Foundation Stanley Fahn Junior Faculty 59 Award (Grant No. PF-JFA-1762). I.A. was supported by R21NS096395 grant from the NIH and by the 60 Darrell K Royal Research Fund for Alzheimer’s Disease. J.M.S. was additionally supported by NIH 61 grants (R21NS089854) and a Career Award for Medical Scientists from the Burroughs Welcome Fund. 62 The project was supported in part by Baylor College of Medicine IDDRC Grant Number U54HD083092 63 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The 64 IDDRC Microscopy Core was used for this project. The content is solely the responsibility of the authors 65 and does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of 66 Child Health and Human Development or the National Institutes of Health. 67 3 68 Abstract 69 Accumulation of α-Synuclein (α-Syn) causes Parkinson’s disease (PD) as well as other synucleopathies. 70 α-Syn is the major component of Lewy bodies and Lewy neurites, the proteinaceous aggregates that are a 71 hallmark of sporadic PD. In familial forms of PD, mutations or copy number variations in SNCA (the α- 72 Syn gene) result in a net increase of its protein levels. Furthermore, common risk variants tied to PD are 73 associated with small increases of wild-type α-Syn levels. These findings are further bolstered by animal 74 studies which show that overexpression of α-Syn is sufficient to cause PD-like features. Thus, increased 75 α-Syn levels are intrinsically tied to PD pathogenesis and underscore the importance of identifying the 76 factors that regulate its levels. In this study, we establish a pooled RNAi screening approach and 77 validation pipeline to probe the druggable genome for modifiers of α-Syn levels and identify 60 78 promising targets. Using a cross-species, tiered validation approach, we validate six strong candidates that 79 modulate α-Syn levels and toxicity in cell lines, Drosophila, human neurons and mouse brain of both 80 sexes.
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