Gene augmentation of LCA5-associated Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme
Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman
Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman
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Research Article Cell biology Genetics

Gene augmentation of LCA5-associated Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme

Abstract

Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy, we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localized at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient in lebercilin exhibited early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, adeno-associated virus–based LCA5 gene augmentation partially restored the bulge region, preserved OS axoneme structure and membrane disc formation, and resulted in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.

Authors

Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman

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Figure 3

Effect of lebercilin loss on bulge formation, distal axoneme organization, and CC length.

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Effect of lebercilin loss on bulge formation, distal axoneme organizatio...
(A) Widefield (original magnification, 63×) images of expanded photoreceptors stained for POC5 (green) and tubulin (magenta) from P10 to P22 in Lca5gt/gt (HOM) mice and in P22 Lca5+/gt (HET) mice. Lines in images illustrate the measurements used in C of tubulin width at 3 locations: +500 nm, 0 nm, −500 nm. The distal end of CC inner scaffold marker POC5 was used to set the 0 location. Scale bars: 500 nm. (B) Distal axoneme (above CC) conformations of HET versus HOM photoreceptors at P18 and P22 indicated in percentages. Photoreceptor distal axoneme conformations: normal (HET: 99.6%; HOM: 50.2%), open/broken (HET: 0.4%; HOM: 40.8%), and bent/curled (HET: 0%; HOM: 9%). n = 247 (HET), n = 217 (HOM). (C) Tubulin width measurements of the photoreceptor at the 3 locations depicted in A from P10 to P22. Average tubulin width at each location is indicated by a gray or red dot for HET and HOM, respectively. P28 not included, since most of the distal axonemes are lost at this time point. Only photoreceptors that were stained for tubulin and POC5 were used for the measurements. Three animals per time point. Data presented as mean ± SD; n = 16–29. *P < 0.05, **P < 0.01, ***P < 0.001 by F test. Significance represents the tubulin width dispersion between HET and HOM. (D–F) Widefield (original magnification, 63×) images of expanded photoreceptors stained for tubulin (magenta) and CEP290 (cyan, D), POC5 (green, E), or RP1 (white/gray, F) from P10 to P28 in HOM mice. Scale bars: 500 nm. (G–I) Impact of lebercilin loss on CEP290 length (G), CC inner scaffold length (POC5, H), or RP1-normalized intensity at the bulge region (I) from P10 to P28. Three animals per time point. Data presented as mean ± SD; n = 39–65 (G), n = 19–60 (H), n = 39–72 (I). **P < 0.01, ****P < 0.0001 by Mann-Whitney test.