Super-resolution microscopy reveals photoreceptor-specific subciliary location and function of ciliopathy-associated protein CEP290

Potter, Valencia; Moye, Abigail R.; Robichaux, Michael A.; Wensel, Theodore G.

doi:10.1172/jci.insight.145256

Cited by 21 publications

(27 citation statements)

References 83 publications

(129 reference statements)

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“…Moreover, we demonstrated that CEP290 setup and maintenance in the CC are independent of the CC inner scaffold, revealing that different modules can be discriminated inside the CC. Consistently, Potter and colleagues showed that Cep290 -knockout photoreceptors still possess the CC inner scaffold protein CENTRIN, suggesting that CEP290 and the CC inner scaffold are probably independent [ 20 ]. These results suggest that the CC might differ from a long canonical TZ, as previously suggested.…”

Section: Discussionmentioning

confidence: 94%

“…We next investigated the precise localization of the inner scaffold components POC5, CENTRIN, and FAM161A in adult mouse photoreceptors, with the exception of POC1B owing to the lack of appropriate antibodies. We also inspected the distribution of CEP290, known to localize along the CC from super-resolution microscopy [ 20 ], and Lebercilin (LCA5), a proposed FAM161A interactor [ 10 ], mutations in which have been linked to Leber congenital amaurosis, a retinopathy causing severe visual deficiency from the first year after birth [ 21 , 22 ]. Consistent with our previous work, all 3 inner scaffold proteins were found in the central core region of centrioles, where the inner scaffold structure lies [ 16 ] ( Fig 1C–1E ).…”

Section: Resultsmentioning

confidence: 99%

“…Since then, different TZ proteins have been described as decorating the CC in photoreceptor cells, corroborating this observation. For example, the protein CEP290 has been proposed to be part of the Y-links structure, even if this is still a matter of debate in the literature [ 20 , 27 , 35 ]. Here, we showed for the first time the 9-fold symmetry of the CEP290 signal all along the CC, and positioned it at the level of the Y-links structure.…”

Section: Discussionmentioning

confidence: 99%

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The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration

et al. 2022

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Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. These cells possess a photosensitive outer segment linked to the cell body through the connecting cilium (CC). While structural defects of the CC have been associated with retinal degeneration, its nanoscale molecular composition, assembly, and function are barely known. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC inner scaffold containing POC5, CENTRIN, and FAM161A. Dissecting CC inner scaffold assembly during photoreceptor development in mouse revealed that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Furthermore, we show that Fam161a disruption in mouse leads to specific CC inner scaffold loss and triggers microtubule doublet spreading, prior to outer segment collapse and photoreceptor degeneration, suggesting a molecular mechanism for a subtype of retinitis pigmentosa.

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Section: Discussionmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration

et al. 2022

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“…The ciliary protein CEP290 localises to the TZ in photoreceptor cilia and is known to interact with RPGR [ 31 , 32 ]. In both control 30-week-old iPSC-ROs, there was strong co-localisation of CEP290 and RPGR at the photoreceptor TZ ( Figure 5 D(i) and Supplementary Figure S4A , white arrows, yellow co-stain in merged images).…”

Section: Resultsmentioning

confidence: 99%

Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic RPGR Variant Assessment for Therapy Suitability

Karam

Loi

et al. 2022

JPM

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The RPGR gene encodes Retinitis Pigmentosa GTPase Regulator, a known interactor with ciliary proteins, which is involved in maintaining healthy photoreceptor cells. Variants in RPGR are the main contributor to X-linked rod-cone dystrophy (RCD), and RPGR gene therapy approaches are in clinical trials. Hence, elucidation of the pathogenicity of novel RPGR variants is important for a patient therapy opportunity. Here, we describe a novel intronic RPGR variant, c.1415 − 9A>G, in a patient with RCD, which was classified as a variant of uncertain significance according to current clinical diagnostic criteria. The variant lay several base pairs intronic to the canonical splice acceptor site, raising suspicion of an RPGR RNA splicing abnormality and consequent protein dysfunction. To investigate disease causation in an appropriate disease model, induced pluripotent stem cells were generated from patient fibroblasts and differentiated to retinal pigment epithelium (iPSC-RPE) and retinal organoids (iPSC-RO). Abnormal RNA splicing of RPGR was demonstrated in patient fibroblasts, iPSC-RPE and iPSC-ROs, leading to a predicted frameshift and premature stop codon. Decreased RPGR expression was demonstrated in these cell types, with a striking loss of RPGR localization at the ciliary transitional zone, critically in the photoreceptor cilium of the patient iPSC-ROs. Mislocalisation of rhodopsin staining was present in the patient’s iPSC-RO rod photoreceptor cells, along with an abnormality of L/M opsin staining affecting cone photoreceptor cells and increased photoreceptor apoptosis. Additionally, patient iPSC-ROs displayed an increase in F-actin expression that was consistent with an abnormal actin regulation phenotype. Collectively, these studies indicate that the splicing abnormality caused by the c.1415 − 9A>G variant has an impact on RPGR function. This work has enabled the reclassification of this variant to pathogenic, allowing the consideration of patients with this variant having access to gene therapy clinical trials. In addition, we have identified biomarkers of disease suitable for the interrogation of other RPGR variants of uncertain significance.

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“…To gain a better understanding of CEP290-LCA, many aspects of CEP290 cell biology have been extensively studied. Combined, these studies suggest that CEP290 influences both ciliogenesis 4,5 and ciliary protein content by acting as a gatekeeper in protein trafficking through the connecting cilium that normally bridges the inner and outer segment of photoreceptors 6–8 . CEP290 mutations can preclude the ability for normal outer segments to form and cause an accumulation of proteins typically in the outer segment, such as rhodopsin, within the inner segment 6 .…”

Section: Introductionmentioning

confidence: 94%

Genetic modifiers of Cep290-mediated retinal degeneration

Meyer

Whitmore

Burnight

et al. 2022

Preprint

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Mutations in CEP290 cause up to 30% of cases of Leber congenital amaurosis (LCA), a severe childhood blindness resulting from abnormalities in the photoreceptor connecting cilia that lead to rapid retinal degeneration. Like many genetic diseases, CEP290-LCA has considerable variable expressivity, indicating the presence of other factors that influence phenotypic outcome. Here, we have undertaken a phenotype-driven approach in mice to identify genetic modifiers of CEP290-mediated retinal degeneration through backcrosses and intercrosses between BXD24-Cep290rd16 mice and the genetically distinct inbred CAST strain to introduce genetic variation. Optical coherence tomography was used to quantitatively measure retinal thickness as a surrogate indication of photoreceptor degeneration in the resulting rd16-mutant mice. It was readily apparent that CEP290-mediated retinal degeneration in the resulting mice is sensitive to genetic background, with some mice exhibiting relatively thick laminated retinas and others having thin retinas with advanced disease. Quantitative trait locus (QTL) analysis identified multiple genomic loci capable of influencing the retinal degeneration phenotype in Cep290-mutant mice that together account for 71.7% of the phenotypic variation in retinal thickness observed in our population. Following the QTL analysis, two suppressor loci were studied in detail through a combination of physical and molecular approaches to narrow the critical region for each QTL and identify the probable causative genetic variations.

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Super-resolution microscopy reveals photoreceptor-specific subciliary location and function of ciliopathy-associated protein CEP290

Cited by 21 publications

References 83 publications

The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration

The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration

Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic RPGR Variant Assessment for Therapy Suitability

Genetic modifiers of Cep290-mediated retinal degeneration

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