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 5

Localization study of phototransduction and IFT proteins in Lca5gt/gt retinas.

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Localization study of phototransduction and IFT proteins in Lca5gt/gt re...
(A) OS proteins rhodopsin (RHO) and cone opsin were mislocalized to ISs and ONL in Lca5gt/gt animals, while normally restricted to OSs in controls. All sections were obtained at P14. (B) Localization of transducin and arrestin in dark-adapted and light-stimulated animals (P14). In dark-adapted wild-type animals, transducin localized to OSs and arrestin to ISs, ONL, and outer plexiform layer (OPL). After light stimulation, transducin was mainly found in the ISs, with weaker staining in the ONL and outer plexiform layer, whereas arrestin translocated to the OSs. In Lca5gt/gt dark-adapted animals, the majority of transducin was found in the OSs, with weaker staining in other layers. Arrestin localization in Lca5gt/gt dark-adapted animals was similar to that in dark-adapted wild-type mice. In light-stimulated Lca5gt/gt mice, transducin not only was found in the ISs, but also showed diffuse staining in the entire ONL. Arrestin staining appeared diffusely throughout the photoreceptor layer, with less pronounced OS staining than in wild-type mice. (C) Localization of Ift20, Ift88, or Ift140 in connecting cilia of photoreceptors was not affected by inactivation of Lca5 compared with wild-type at P9. GT335 specifically stained connecting cilia. Scale bars: 50 μm (A and B); 10 μm (C). Enlarged views are shown in the insets (C, ×3).