CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium

Journal name:
Nature Genetics
Volume:
44,
Pages:
193–199
Year published:
DOI:
doi:10.1038/ng.1078
Received
Accepted
Published online

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.

At a glance

Figures

  1. Identification of mutations in CEP41 in affected persons linked to the JBTS15 locus.
    Figure 1: Identification of mutations in CEP41 in affected persons linked to the JBTS15 locus.

    (a) The pedigree of MTI-429 shows two first cousin marriages with a total of five affected offspring (filled symbols). The symbol with a slash indicates an individual who is deceased. (b) Axial brain MRI images from individuals with CEP41 mutations in the MTI-429, MTI-1491 (T1-weighted) and COR-98 (T2-weighted) families, showing the molar tooth sign (red arrows). (c) The JBTS15 locus spans 5.5 Mb at chromosome 7q31.33-32.3 (red box) defined by rs766240 and rs4728251. A denser SNP scan further narrowed the JBTS15 interval to a 2.8-Mb region encompassing rs17165226 and rs2971773 (black arrows). (d) CEP41 genomic organization, depicting locations of identified base changes, including homozygous (red) splice-site mutations and heterozygous (blue) missense and nonsense mutations. Capital letters, exon sequences; small letters, intron sequences; asterisks, point mutations; underline, deletion. (e) RT-PCR confirmation of the splicing defect of CEP41 in fibroblasts from MTI-429 subjects. Both CEP41 mutant cell lines failed to produce CEP41 mRNA compared with cells isolated from a control subject (WT, wild type). GAPDH was used as a control. Green arrows, positions of primers; asterisk, region of splice-site mutation in MTI-429.

  2. CEP41 is expressed in ciliated tissues and its loss recapitulates ciliopathy-related phenotypes in zebrafish and mouse.
    Figure 2: CEP41 is expressed in ciliated tissues and its loss recapitulates ciliopathy-related phenotypes in zebrafish and mouse.

    (a) Zebrafish cep41 mRNA is expressed ubiquitously at gastrulation stages (6 h post-fertilization (h.p.f.)), but at later stages, it is expressed specifically in ciliated organs: Kupffer's vesicle (red box), inner ear (white boxes), brain (brackets), eyes (arrows), pronephric duct (black box) and heart (asterisks). A, anterior; D, dorsal; P, posterior; V, ventral. (b) CEP41 is predominantly localized to the basal body (arrowheads) and primary cilium (arrows) in ciliated IMCD3 and hTERT-RPE1 cells. Scale bars, 5 μm. Insets, co-localization of CEP41 with GT335, a marker of basal bodies and cilia. (c) Knockdown of cep41 by injection with cep41 MO causes heart laterality defects in Tg (myl7:egfp) zebrafish embryos. The cep41 morphants show either loss of asymmetry (midline) or inversion of ventricle-atrium asymmetry (reversed) at 72 h.p.f. Error bars, s.e.m. *P < 0.01, **P < 0.001. A, atrium; V, ventricle. (d) Cep41 gene-trap mice show altered embryonic morphogenesis. The range of phenotypes for Cep41Gt/Gt embryos includes mild malformed hindbrain (arrowheads), exencephaly (brackets), hemorrhage in the head (asterisks), dilated pericardial sac (arrows), failure to rotate and lethality at E10–12. (e) Injection of human CEP41 RNA into cep41 morphants rescued ciliary phenotypes of pericardial edema (arrowhead), hydrocephalus (asterisk) and curved tail (arrows), completely or partially.

  3. CEP41 is required for tubulin glutamylation at the ciliary axoneme.
    Figure 3: CEP41 is required for tubulin glutamylation at the ciliary axoneme.

    (a) Absence of CEP41 protein in CEP41 mutant cells from human subjects. (b) Ciliary localization of endogenous CEP41 in human fibroblasts and loss of the protein in CEP41 mutant cilia. Scale bar, 5 μm. (c) Cells with wild-type CEP41 have both GT335-positive basal bodies (arrowheads) and cilia (arrows, marked by ARL13B), whereas cells with mutant CEP41 show staining of GT335 only at the basal bodies. Scale bar, 5 μm. (d) Depletion of cep41 causes glutamylation defects in zebrafish olfactory placode cilia (red arrows). Images of GT335-stained wild-type and cep41 morphant embryos were taken in anterior view (head is up, tail is down), with quantification shown below. D, dorsal; V, ventral. (e) Exogenous CEP41 expression restores ciliary axoneme glutamylation in CEP41 mutant cells. Arrows, primary cilia stained for CEP41 and GT335. Scale bar, 5 μm. Insets, merged images at higher magnification, with quantification shown below. Error bars, s.e.m. **P < 0.001. (f) Ultrastructural analysis of the pronephric ciliary axoneme at 72 h.p.f. in zebrafish embryos. Compared to wild-type embryos, cep41 morphants have A-tubule–specific defects in the outer doublet microtubules. Arrows, A-tubules. Scale bars, 100 nm. One of nine outer doublet microtubules is magnified in the red box. The numbers of cilia, categorized as having normal and abnormal A-tubules according to the schematic were counted in both wild-type embryos and cep41 morphants (n = 3 embryos each, >20 cilia per animal), with quantification shown below. Error bars, s.e.m. *P < 0.01.

  4. CEP41 interacts with TTLL6 and is required for TTLL6 localization to the cilium.
    Figure 4: CEP41 interacts with TTLL6 and is required for TTLL6 localization to the cilium.

    (a) MO-mediated knockdown of zebrafish ttll6 results in ciliary phenotypes, such as abnormal number and/or orientation of ear otolith (boxes), cystic kidney (arrowheads) and peripheral cardiac edema (asterisks) as well as curved tail (arrows) at the different dosages indicated and causes A-tubule–specific defects in the outer doublet microtubules. The numbers of defective cilia were counted in both wild-type embryos and ttll6 morphants (n = 3 embryos each, >20 cilia per animal), with quantification shown below. (b) GFP-CEP41 and its associated proteins were immunoprecipitated with antibody to the Flag epitope recognizing Flag-tagged TTLL6 from the whole-cell extract (WCE) of cells transfected with this expression construct, with immunoprecipitation in WCE from cells transfected with empty GFP vector serving as a control. In the reciprocal co-immunoprecipitation experiment with antibody to GFP, the interaction between CEP41 and TTLL6 was confirmed. (c) Disturbed localization of TTLL6 to the cilium following the co-transfection of Cep41 siRNA with GFP-TTLL6 into IMCD3 cells as determined by immunostaining with either GT335 or ARL13B antibody. Arrows, cilia; arrowheads, basal bodies. Scale bar, 5 μm. Cells expressing cilium-localized TTLL6 were counted only in siRNA-transfected cells and are quantified in the graph. Error bars, s.e.m. *P < 0.01, **P < 0.001.

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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

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 financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (8M)

    Supplementary Figures 1–14 and Supplementary Tables 1–4

Movies

  1. Supplementary Video 1 (13M)

    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. Supplementary Video 2 (4M)

    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. Supplementary Video 3 (2M)

    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. Supplementary Video 4 (2M)

    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. Supplementary Video 5 (11M)

    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.

Additional data