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

Abstract

Tubulin glutamylation is a post-translational modification that occurs predominantly in the ciliary axoneme and has been suggested to be important for ciliary function. 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), which we have designated as JBTS15, and identified causative mutations in CEP41, which encodes a 41-kDa centrosomal protein. We show that CEP41 is localized to the basal body and primary cilia, and regulates ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme. 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.

Figure 1

Identification of mutations in CEP41 in affected individuals linked to the JBTS15 locus. (a) Pedigree MTI-429 shows double first cousin marriage with five affected offspring. (b) Axial brain MRI images from patients with CEP41 mutations in each MTI-429, MTI-1491 (T1-weighted) and COR-98 (T2-weighted) family, showing the ‘molar tooth sign’ (red arrows). (c) JBTS15 is 5.5 Mbp located at Chr. 7q31.33–32.3 (red box) defined by rs766240 and rs4728251. Further narrowing of the interval to 2.8 Mbp based upon denser SNP scan to encompass rs17165226 and rs2971773 (black arrows). (d) CEP41 genomic organization, depicting locations of identified base changes including homozygous (red) splice mutations and heterozygous (blue) missense or 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 MTI-429 patient fibroblasts. Both CEP41 mutant cells failed to produce CEP41 mRNA, compared with WT. GAPDH is control. Green arrows: positions of primers, asterisk: region of splice mutation in MTI-429.

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 hpf), but at later stages, it is specifically expressed 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. Insets: co-localization of CEP41 with GT335, a marker of basal bodies and cilia. Scale bar 5 μm. (c) Knockdown of cep41 by injection of morpholino oligonucleotide (MO) causes heart asymmetry defects in Tg (myl7:egfp) zebrafish embryos. The cep41 morphants show either loss of asymmetry (midline) or inversion of V/A asymmetry (reversed) at 72hpf. *P < 0.01, **P < 0.001. Error bars: s.e.m. A, atrium; L, left; R, right; V, ventricle. (d) Murine Cep41 gene-trap shows altered embryonic morphogenesis. Range of phenotypes of Cep41^Gt/Gt embryos includes mild malformed hindbrain (arrowheads), exencephaly (brackets), hemorrhage in the head (asterisk), 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 (arrow), completely or partially.

Figure 3

CEP41 is required for tubulin glutamylation at the ciliary axoneme. (a) Absent CEP41 protein in CEP41 mutant patient cells. (b) Ciliary localization of endogenous CEP41 in human fibroblasts and loss of the protein in CEP41 mutant cilia. Scale bar 5 μm. (c) WT cells have both GT335-positive basal bodies (arrowheads) and cilia (arrows, marked by ARL13B), while mutant cells 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-stainined WT and cep41 morphant embryos were taken in anterior view (head is up and tail is down, D, dorsal; V, ventral), quantified below. (e) Exogenous CEP41 expression restores ciliary axoneme glutamylation in CEP41 mutant cells. Arrows: primary cilia stained for CEP41 and GT335. Insets: merged images at higher power, quantified below. Scale bar 5 μm. **P < 0.001; error bars = s.e.m. (f) Ultrastructural analysis of the pronephric ciliary axoneme at 72 hpf zebrafish embryos. Compared to WT, cep41 morphants have A-tubule specific defects in the outer doublet microtubules. Arrows: A-tubules and one of nine outer doublet microtubules is magnified in the red box. The numbers of cilia, categorized in normal and abnormal A-tubules according to the schematic, were counted in both WT embryos and cep41 morphants (n = 3 embryos, >20 cilia per animal), quantified below. Scale bar 100 nm. **P < 0.01; error bars = s.e.m.

Figure 4

CEP41 interacts with TTLL6 and is required for localizing TTLL6 to the cilium. (a) Morpholino knockdown of zebrafish ttll6 associates with ciliary phenotypes such as curved tail (arrows), abnormal number/orientation of ear otolith (boxes), cystic kidney (arrowheads) and peripheral cardiac edema (asterisks) at different dosages indicated and show A-tubule specific defects in the outer doublet microtubules. The numbers of defective cilia were counted in both WT embryos and ttll6 morphants (n = 3 embryos, >20 cilia per animal), quantified below. (b) GFP-CEP41 was immunoprecipitated with anti-Flag antibody recognizing Flag-tagged TTLL6 from whole-cell extract (WCE), compared with GFP-empty vector. In the reciprocal co-IP experiment with anti-GFP antibody, the interaction between CEP41 and TTLL6 was confirmed. (c) Disturbed localization of TTLL6 to the cilium following Cep41 siRNA co-transfection with GFP-TTLL6 into IMCD3 cells and immunostained with either GT335 or ARL13B antibody. Arrows: cilia; Arrowheads: basal bodies. Cells expressing ciliary localized TTLL6 were counted only in siRNA-transfected cells quantified in graph. Scale bar 5 μm. *P< 0.01, **P < 0.001. Error bars = s.e.m.