Abstract

Tubulin glutamylation is a post-translational modification that occurs predominantly in the ciliary axoneme and has been suggested to be important for ciliary function1,2. However, its relationship to disorders of the primary cilium, termed ciliopathies, has not been explored. Here we mapped a new locus for Joubert syndrome (JBTS)3, which we have designated as JBTS15, and identified causative mutations in CEP41, which encodes a 41-kDa centrosomal protein4. We show that CEP41 is localized to the basal body and primary cilia, and regulates ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme5. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mice and results in glutamylation defects in the ciliary axoneme. Our data identify CEP41 mutations as a cause of JBTS and implicate tubulin post-translational modification in the pathogenesis of human ciliary dysfunction.

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Acknowledgements

We thank the Marshfield Clinic Research Foundation, Center for Inherited Disease Research (supported by the US National Institutes of Health and National Heart, Lung, and Blood Institute) for genotyping support. We thank the International JSRD Study Group, E. Bertini and the French Society of Foetal Pathology for subject referrals, J. Meerloo at the UCSD Microscopy Core (P30NS047101), T. Meerloo, Y. Jones, M. Farquhar and the Department of Cellular and Molecular Medicine (CMM) Electron Microscopy Core Facility at UCSD, S. Wirth and B. Willis for mutant mouse generation, C. Janke (Institute Curie Research Center) for GT335 antibody, TTLLs plasmids and technical advice, I. Drummond and N. Pathak (Massachusetts General Hospital) for ttll6 MO, M. Gorovsky (University of Rochester) for polyE and polyG antibodies, A.T. Look (Dana-Farber Cancer Institute) for the pCS2+ plasmid, S. Audollent for technical help, and B. Sotak, N. Akizu, A. Crawford, V. Cantagrel and E.-J. Choi for stimulating scientific discussion and comments. This work was supported by the US National Institutes of Health (R01NS048453 and R01NS052455 to J.G.G.; R01DK068306 to F.H.; and R01NS064077 to D.A.D.), the American Heart Association (09POST2250641 to J.E.L.), the Italian Ministry of Health (Ricerca Finalizzata Malattie Rare and Ricerca Corrente 2011), the Telethon Foundation Italy (GGP08145) and the Pierfranco and Luisa Mariani Foundation (to E.M.V.), Research was also supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Society for the Promotion of Science (JSPS), (23117517 and 23570209 to K.I.), the Newlife Charity, the Medical Research Council (G0700073), the Sir Jules Thorn Charitable Trust (09/JTA to C.A.J.), l'Agence National pour la Recherche (ANR) (07-MRAR-Fetalciliopathies to T.A.-B.), Simons Foundation Autism Research Initiative (to J.G.G.) and the Howard Hughes Medical Institute (to F.H. and J.G.G.).

Author information

Affiliations

  1. Department of Neurosciences, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California, USA.

    • Ji Eun Lee
    • , Jennifer L Silhavy
    • , Jana Schroth
    • , Stephanie L Bielas
    • , Sarah E Marsh
    • , Jesus Olvera
    • , Andrew M Schlossman
    • , Carrie M Louie
    • , Jeong Ho Lee
    •  & Joseph G Gleeson
  2. Neurogenetics Laboratory, Institute for Genomic Medicine, Department of Pediatrics, University of California, San Diego, La Jolla, California, USA.

    • Ji Eun Lee
    • , Jennifer L Silhavy
    • , Jana Schroth
    • , Stephanie L Bielas
    • , Sarah E Marsh
    • , Jesus Olvera
    • , Andrew M Schlossman
    • , Carrie M Louie
    • , Jeong Ho Lee
    •  & Joseph G Gleeson
  3. Clinical Genetics Department, Human Genetics and Genome Research Division, National Research Centre, Dokki, Egypt.

    • Maha S Zaki
  4. Casa Sollievo della Sofferenza (CSS) Hospital, CSS-Mendel Laboratory, San Giovanni Rotondo, Italy.

    • Francesco Brancati
    • , Miriam Iannicelli
    •  & Enza Maria Valente
  5. Department of Biopathology and Diagnostic Imaging, Medical Genetics Unit, Tor Vergata University, Rome, Italy.

    • Francesco Brancati
  6. Department of Cell Biology and Anatomy, Hamamatsu University School of Medicine, Hamamatsu, Japan.

    • Koji Ikegami
    •  & Mitsutoshi Setou
  7. Department of Human Genetics, Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.

    • Barry Merriman
    •  & Stanley F Nelson
  8. Département de Génétique, Institut National de la Santé et de la Recherche Médicale (INSERM) U781, Hôpital Necker–Enfants Malades, Université Paris Descartes, Paris, France.

    • Tania Attié-Bitach
  9. Section of Ophthalmology and Neurosciences, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, UK.

    • Clare V Logan
    •  & Colin A Johnson
  10. Department of Pediatrics, Division of Developmental Medicine, University of Washington, Seattle Children's Hospital, Seattle, Washington, USA.

    • Ian A Glass
    •  & Daniel A Doherty
  11. Division of Genetic Medicine, University of Washington, Seattle Children's Hospital, Seattle, Washington, USA.

    • Ian A Glass
    •  & Daniel A Doherty
  12. Department of Pediatrics and Communicable Diseases, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan, USA.

    • Andrew Cluckey
    •  & Friedhelm Hildebrandt
  13. Saul R. Korey Department of Neurology, Albert Einstein College of Medicine, New York, New York, USA.

    • Hilary R Raynes
    •  & Isabelle Rapin
  14. Department of Pediatrics, Albert Einstein College of Medicine, New York, New York, USA.

    • Hilary R Raynes
    •  & Isabelle Rapin
  15. Rose F. Kennedy Intellectual and Developmental Disabilities Research Center, Albert Einstein College of Medicine, New York, New York, USA.

    • Hilary R Raynes
    •  & Isabelle Rapin
  16. Pediatric Neurology Service, University Hospital La Paz, Madrid, Spain.

    • Ignacio P Castroviejo
  17. Serviço de Neuropediatria, Hospital de Crianças Maria Pia, Porto, Portugal.

    • Clara Barbot
  18. Department of Pediatric Neurology, University Children's Hospital of Zürich, Zurich, Switzerland.

    • Eugen Boltshauser
  19. Department of Medical and Surgical Pediatric Sciences, University of Messina, Messina, Italy.

    • Enza Maria Valente

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Contributions

J.E.L., M.S.Z. and J.G.G. designed the study and experiments with substantial contributions from B.M. S.F.N. helped with fine mapping. J.L.S., S.L.B., J.O., F.B., M.I., A.M.S., T.A.-B., C.V.L., I.A.G., A.C., F.H., C.A.J., D.A.D. and E.M.V. performed genetic screening. J.E.L., J.L.S., J.S., J.O., F.B., M.I., T.A.-B., I.A.G., D.A.D., C.M.L. and J.H.L. performed mutation analysis. M.S.Z., S.E.M., H.R.R., I.R., I.P.C., E.B., C.B. and E.M.V. identified and recruited subjects. K.I. and M.S. shared critical reagents. J.S. helped with genotyping of mutant mice. J.E.L. performed microscopy, biochemical assays and zebrafish and mouse experiments. J.E.L. and J.G.G. interpreted the data and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Joseph G Gleeson.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–14 and Supplementary Tables 1–4

Videos

  1. 1.

    Supplementary Video 1

    A small piece of debris in Kupffer's vesicle of a 12 hpf (6 somite) WT embryo is caught moving ciliary currents. The debris (marked by arrow) follows a counter-clockwise circular path around Kupffer's vesicle. nc, notochord; A, anterior; P, posterior; L, left; R, right directions.

  2. 2.

    Supplementary Video 2

    Small pieces of debris in Kupffer's vesicle of a 12 hpf (6 somite) cep41 morphant are caught moving ciliary currents. The debris (within a circle) shows no directional movement, but rather bounces around or stalls. nc, notochord; A, anterior; P, posterior; L, left; R, right directions.

  3. 3.

    Supplementary Video 3

    Movement of cilia at the junction area of the pronephric duct and tubule in a 2.5 dpf WT zebrafish embryo. The observed cilium (an arrow) shows rhythmic undulations. Dashed lines demarcate the outline of the pronephric duct lumen and an arrow point out the observed cilium. A, anterior; D, dorsal; P, posterior; V, ventral directions.

  4. 4.

    Supplementary Video 4

    Movement of cilia at the junction area of the pronephric duct and tubule in a 2.5 dpf cep41 MO-injected embryo. Motile cilium, observed in the WT embryo, is not found. Dashed lines demarcate the outline of the pronephric duct lumen. A, anterior; D, dorsal; P, posterior; V, ventral directions.

  5. 5.

    Supplementary Video 5

    Movement of cilia at the pronephric duct in a 2.5 dpf WT zebrafish embryo. The cilium (an arrow) shows rhythmic undulations. Dashed lines demarcate the outline of the pronephric duct lumen and an arrow point out the observed cilium. A, anterior; D, dorsal; P, posterior; V, ventral directions.

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DOI

https://doi.org/10.1038/ng.1078

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