Disruption of intraflagellar protein transport in photoreceptor cilia causes Leber congenital amaurosis in humans and mice
Karsten Boldt, … , Ronald Roepman, Marius Ueffing
Karsten Boldt, … , Ronald Roepman, Marius Ueffing
Published May 23, 2011
Citation Information: J Clin Invest. 2011;121(6):2169-2180. https://doi.org/10.1172/JCI45627.
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Research Article

Disruption of intraflagellar protein transport in photoreceptor cilia causes Leber congenital amaurosis in humans and mice

Abstract

The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how mutations in LCA5 affect the connectivity of the encoded protein lebercilin at the interactome level. In photoreceptors, lebercilin is uniquely localized at the cilium that bridges the inner and outer segments. Using a generally applicable affinity proteomics approach, we showed that lebercilin specifically interacted with the intraflagellar transport (IFT) machinery in HEK293T cells. This interaction disappeared when 2 human LCA-associated lebercilin mutations were introduced, implicating a specific disruption of IFT-dependent protein transport, an evolutionarily conserved basic mechanism found in all cilia. Lca5 inactivation in mice led to partial displacement of opsins and light-induced translocation of arrestin from photoreceptor outer segments. This was consistent with a defect in IFT at the connecting cilium, leading to failure of proper outer segment formation and subsequent photoreceptor degeneration. These data suggest that lebercilin functions as an integral element of selective protein transport through photoreceptor cilia and provide a molecular demonstration that disrupted IFT can lead to LCA.

Authors

Karsten Boldt, Dorus A. Mans, Jungyeon Won, Jeroen van Reeuwijk, Andreas Vogt, Norbert Kinkl, Stef J.F. Letteboer, Wanda L. Hicks, Ron E. Hurd, Jürgen K. Naggert, Yves Texier, Anneke I. den Hollander, Robert K. Koenekoop, Jean Bennett, Frans P.M. Cremers, Christian J. Gloeckner, Patsy M. Nishina, Ronald Roepman, Marius Ueffing

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Figure 4

Clinical and morphological assessment of Lca5gt/gt mouse retina.

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Clinical and morphological assessment of Lca5gt/gt mouse retina. (A)...
(A) Fundus photography of Lca5gt/gt animals and littermate controls. At 2 months of age, depigmented patches were observed. (B) Scotopic and photopic ERG amplitudes of rod (left) and cone (right) waveforms at P25 (n = 3). Data are mean ± SEM. (C) Progressive retinal degeneration demonstrated in Lca5gt/gt mice. Retinal sections of Lca5gt/gt mice and wild-type controls, between P12 and 4 months of age, stained with hematoxylin and eosin. At P12, the OS/IS layers were remarkably thinner in Lca5gt/gt animals, whereas the ONL and INL were comparable to those of controls. At P28, the photoreceptor nuclei in the ONL were significantly reduced compared with controls. By 4 months of age, the photoreceptor layer was completely absent in Lca5gt/gt mice. (D) Scanning EM of control and Lca5gt/gt retinas at P10 and P15 from a scleral orientation (left) and fractured view (right). Control OSs at P10 were ovoid and regular in size. Vertically stacked OS discs (double asterisks) were rarely observed. OSs in Lca5gt/gt retinas were shorter and irregular in size and shape. By P15, OS discs were disorganized and did not reach the length observed in controls. Most OSs were rudimentary or abnormal in shape (asterisk). (E) Transmission EM of Lca5gt/gt retinas showed that the structure of the CC, including basal body, was preserved at P8. RPE, retinal pigment epithelium; NC, nascent cilia. Scale bars: 50 μm (C); 10 μm (D); 500 nm (E).