Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice

The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins—myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans—do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner–outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients.

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Usher syndrome type 1–associated cadherins shape the photoreceptor outer segment

The Journal of Cell Biology ◽

10.1083/jcb.201612030 ◽

2017 ◽

Vol 216 (6) ◽

pp. 1849-1864 ◽

Cited By ~ 14

Author(s):

Cataldo Schietroma ◽

Karine Parain ◽

Amrit Estivalet ◽

Asadollah Aghaie ◽

Jacques Boutet de Monvel ◽

...

Keyword(s):

Outer Segment ◽

Usher Syndrome ◽

Retinal Dystrophy ◽

Syndrome Type ◽

Photoreceptor Outer Segment ◽

Outer Segments ◽

Protocadherin 15 ◽

Cadherin 23 ◽

Usher Syndrome Type

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15–containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal.

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Potential therapy for progressive vision loss due to PCDH15-associated Usher syndrome developed in an orthologous Usher mouse

10.1101/2021.06.08.447565 ◽

2021 ◽

Author(s):

Saumil Sethna ◽

Wadih M Zein ◽

Sehar Riaz ◽

Arnaud P.J. Giese ◽

Julie M Schultz ◽

...

Keyword(s):

Retinal Degeneration ◽

Vision Loss ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Usher Syndrome ◽

Visual Field Loss ◽

Type I ◽

Ashkenazi Jews ◽

Visual Deficits ◽

Protocadherin 15

Usher syndrome type I (USH1) is characterized by congenital deafness, vestibular areflexia, and progressive retinal degeneration with age. The protein-truncating p.Arg245* founder variant of PCDH15 has an ~2% carrier frequency among Ashkenazi Jews, accounting for nearly 60% of their USH1 cases. Here, longitudinal ocular phenotyping in thirteen USH1F individuals harboring the p.Arg245* variant revealed progressive retinal degeneration, leading to severe loss of vision with macular atrophy by the sixth decade. Half of the affected individuals met either the visual acuity or visual field loss definition for legal blindness by the middle of their fifth decade of life. Mice homozygous for p.Arg250* (Pcdh15R250X; equivalent to human p.Arg245*) also have early visual deficits evaluated using electroretinography. Light-dependent translocation of phototransduction cascade proteins, arrestin and transducin, was found to be impaired in Pcdh15R250X mice. Retinal pigment epithelium- (RPE) specific visual retinoid cycle proteins, RPE65 which converts all-trans retinoids to 11-cis retinoids and CRALBP that transports retinoids, and key retinoid levels were also reduced in Pcdh15R250X mice, suggesting a dual role for protocadherin-15 in photoreceptors and RPE. Administration of exogenous 9-cis retinal, an analog of the naturally occurring 11-cis retinal, improved ERG amplitudes in these mutant mice, suggesting a basis for a clinical trial of exogenous FDA approved retinoids to preserve vision in USH1F patients.

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Proposed therapy, developed in a Pcdh15-deficient mouse, for progressive loss of vision in human Usher Syndrome

eLife ◽

10.7554/elife.67361 ◽

2021 ◽

Vol 10 ◽

Author(s):

Saumil Sethna ◽

Wadih M Zein ◽

Sehar Riaz ◽

Arnaud PJ Giese ◽

Julie M Schultz ◽

...

Keyword(s):

Retinal Degeneration ◽

Vision Loss ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Usher Syndrome ◽

Type I ◽

Ashkenazi Jews ◽

Visual Deficits ◽

Severe Vision Loss ◽

Protocadherin 15

Usher syndrome type I (USH1) is characterized by deafness, vestibular areflexia and progressive retinal degeneration. The protein-truncating p.Arg245* founder variant of PCDH15 (USH1F) has an ~2% carrier frequency amongst Ashkenazi Jews accountings for ~60% of their USH1 cases. Here, longitudinal phenotyping in thirteen USH1F individuals revealed progressive retinal degeneration, leading to severe vision loss with macular atrophy by the sixth decade. Half of the affected individuals were legally blind by their mid-fifties. The mouse Pcdh15R250X variant is equivalent to human p.Arg245*. Homozygous Pcdh15R250X mice also have visual deficits and aberrant light-dependent translocation of the phototransduction cascade proteins, arrestin and transducin. Retinal pigment epithelium- (RPE) specific retinoid cycle proteins, RPE65 and CRALBP, were also reduced in Pcdh15R250X mice, indicating a dual role for protocadherin-15 in photoreceptors and RPE. Exogenous 9-cis retinal improved ERG amplitudes in Pcdh15R250X mice, suggesting a basis for a clinical trial of FDA approved retinoids to preserve vision in USH1F patients.

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PCARE and WASF3 regulate ciliary F-actin assembly that is required for the initiation of photoreceptor outer segment disk formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903125117 ◽

2020 ◽

Vol 117 (18) ◽

pp. 9922-9931 ◽

Cited By ~ 5

Author(s):

Julio C. Corral-Serrano ◽

Ideke J. C. Lamers ◽

Jeroen van Reeuwijk ◽

Lonneke Duijkers ◽

Anita D. M. Hoogendoorn ◽

...

Keyword(s):

Outer Segment ◽

Actin Polymerization ◽

Retinal Dystrophy ◽

Photoreceptor Cells ◽

Actin Dynamics ◽

Mouse Retina ◽

Specific Gene ◽

Common Denominator ◽

Photoreceptor Outer Segment ◽

Disk Formation

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare−/− mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.

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Differential requirement of NPHP1 for compartmentalized protein localization during photoreceptor outer segment development and maintenance

10.1101/2021.01.20.427412 ◽

2021 ◽

Author(s):

Poppy Datta ◽

J. Thomas Cribbs ◽

Seongjin Seo

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Retinal Degeneration ◽

Outer Segment ◽

Protein Localization ◽

Retinal Dystrophy ◽

Gene Trap ◽

Mutant Mice ◽

Plasma Membrane Proteins ◽

Photoreceptor Outer Segment

AbstractNephrocystin (NPHP1) is a ciliary transition zone protein and its ablation causes nephronophthisis (NPHP) with partially penetrant retinal dystrophy. However, the precise requirements of NPHP1 in photoreceptors are not well understood. Here, we characterize retinal degeneration in a mouse model of NPHP1 and show that NPHP1 is required to prevent infiltration of inner segment plasma membrane proteins into the outer segment during the photoreceptor maturation. We demonstrate that Nphp1 gene-trap mutant mice, which were previously described as null, are in fact hypomorphs due to the production of a small quantity of functional mRNAs derived from nonsense-associated altered splicing and skipping of two exons including the one harboring the gene-trap. In homozygous mutant animals, inner segment plasma membrane proteins such as syntaxin-3 (STX3), synaptosomal-associated protein 25 (SNAP25), and interphotoreceptor matrix proteoglycan 2 (IMPG2) accumulate in the outer segment when outer segments are actively elongating. This phenotype, however, is spontaneously ameliorated after the outer segment elongation is completed. Retinal degeneration also occurs temporarily during the photoreceptor maturation but stops afterward. We further show that Nphp1 genetically interacts with Cep290, another NPHP gene, and that a reduction of Cep290 gene dose results in retinal degeneration that continues until adulthood in Nphp1 mutant mice. These findings demonstrate that NPHP1 is required for the confinement of inner segment plasma membrane proteins during the outer segment development, but its requirement diminishes as photoreceptors mature. Our study also suggests that additional mutations in other NPHP genes may influence the penetrance of retinopathy in human NPHP1 patients.

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Differential requirement of NPHP1 for compartmentalized protein localization during photoreceptor outer segment development and maintenance

PLoS ONE ◽

10.1371/journal.pone.0246358 ◽

2021 ◽

Vol 16 (5) ◽

pp. e0246358

Author(s):

Poppy Datta ◽

J. Thomas Cribbs ◽

Seongjin Seo

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Retinal Degeneration ◽

Outer Segment ◽

Protein Localization ◽

Retinal Dystrophy ◽

Gene Trap ◽

Mutant Mice ◽

Plasma Membrane Proteins ◽

Photoreceptor Outer Segment

Nephrocystin (NPHP1) is a ciliary transition zone protein and its ablation causes nephronophthisis (NPHP) with partially penetrant retinal dystrophy. However, the precise requirements of NPHP1 in photoreceptors are not well understood. Here, we characterize retinal degeneration in a mouse model of NPHP1 and show that NPHP1 is required to prevent infiltration of inner segment plasma membrane proteins into the outer segment during the photoreceptor maturation. We demonstrate that Nphp1 gene-trap mutant mice, which were previously described as null, are likely hypomorphs due to the production of a small quantity of functional mRNAs derived from nonsense-associated altered splicing and skipping of two exons including the one harboring the gene-trap. In homozygous mutant animals, inner segment plasma membrane proteins such as syntaxin-3 (STX3), synaptosomal-associated protein 25 (SNAP25), and interphotoreceptor matrix proteoglycan 2 (IMPG2) accumulate in the outer segment when outer segments are actively elongating. This phenotype, however, is spontaneously ameliorated after the outer segment elongation is completed. Consistent with this, some photoreceptor cell loss (~30%) occurs during the photoreceptor maturation period but it stops afterward. We further show that Nphp1 genetically interacts with Cep290, another NPHP gene, and that a reduction of Cep290 gene dose results in retinal degeneration that continues until adulthood in Nphp1 mutant mice. These findings demonstrate that NPHP1 is required for the confinement of inner segment plasma membrane proteins during the outer segment development, but its requirement diminishes as photoreceptors mature. Our study also suggests that additional mutations in other NPHP genes may influence the penetrance of retinopathy in human NPHP1 patients.

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Loss of CEP162 function at the primary cilium delays ciliogenesis and causes retinal ciliopathy in humans

10.1101/2021.11.23.469779 ◽

2021 ◽

Author(s):

Nafisa Nuzhat ◽

Kristof Van Schil ◽

Sandra Liakopoulos ◽

Miriam Bauwens ◽

Alfredo Dueñas Rey ◽

...

Keyword(s):

Transition Zone ◽

Primary Cilium ◽

Outer Segment ◽

Retinal Dystrophy ◽

Mrna Levels ◽

Loss Of Function ◽

Photoreceptor Outer Segment ◽

Zone Formation ◽

Mother Centriole

Ciliopathies often comprise retinal degeneration since the photoreceptor outer segment is an adapted primary cilium. CEP162 is a distal end centriolar protein required for proper transition zone assembly during ciliogenesis and whose loss causes ciliopathy in zebrafish. CEP162 has so far not been implicated in human disease. Here, we identified a homozygous CEP162 frameshift variant, c.1935dupA (p.(E646R*5)), in retinitis pigmentosa patients from two unrelated Moroccan families, likely representing a founder allele. We found that even though mRNA levels were reduced, the truncated CEP162-E646R*5 protein was expressed and localized to the mitotic spindle during mitosis, but not at the basal body of the cilium. In CEP162 knockdown cells, expression of the truncated CEP162-E646R*5 protein is unable to restore ciliation indicating its loss of function at the cilium. In patient fibroblasts, cilia overcome the absence of CEP162 from the primary cilium by delaying ciliogenesis through the persistence of CP110 at the mother centriole. The patient fibroblasts are ultimately able to extend some abnormally long cilia that are missing key transition zone components. Defective transition zone formation likely disproportionately affects the long-living ciliary outer segment of photoreceptors resulting in retinal dystrophy. CEP162 is expressed in human retina, and we show that wild-type CEP162, but not truncated CEP162-E646R*5, specifically localizes to the distal end of centrioles of mouse photoreceptor cilia. Together, our genetic, cell-based, and in vivo modeling establish that CEP162 deficiency causes retinal ciliopathy in humans.

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PRCD is Concentrated at the Base of Photoreceptor Outer Segments and is Involved in Outer Segment Disc Formation

Human Molecular Genetics ◽

10.1093/hmg/ddz248 ◽

2019 ◽

Cited By ~ 4

Author(s):

Gilad Allon ◽

Irit Mann ◽

Lital Remez ◽

Elisabeth Sehn ◽

Leah Rizel ◽

...

Keyword(s):

Retinal Degeneration ◽

Knockout Mice ◽

Outer Segment ◽

Mouse Retina ◽

Cone Photoreceptors ◽

Photoreceptor Outer Segment ◽

Photoreceptor Outer Segments ◽

Outer Segments ◽

Rod And Cone Photoreceptors ◽

Disc Formation

Abstract Mutations of the PRCD gene are associated with rod-cone degeneration in both dogs and humans. Prcd is expressed in the mouse eye as early as embryonic day 14. In the adult mouse retina PRCD is expressed in the outer segments of both rod and cone photoreceptors. Immunoelectron microscopy revealed that PRCD is located at the outer segment rim, and that it is highly concentrated at the base of the outer segment. Prcd-knockout mice present with progressive retinal degeneration, starting at 20 weeks of age and onwards. This process is reflected by a significant and progressive reduction of both scotopic and photopic electroretinographic responses, and by thinning of the retina, and specifically of the outer nuclear layer, indicating photoreceptor loss. Electron microscopy revealed severe damage to photoreceptor outer segments, which is associated with immigration of microglia cells to the Prcd-knockout retina, and accumulation of vesicles in the inter-photoreceptor space. Phagocytosis of photoreceptor outer segment discs by the retinal pigmented epithelium is severely reduced. Our data show that Prcd-knockout mice serve as a good model for retinal degeneration caused by PRCD mutations in humans. Our findings in these mice support the involvement of PRCD in outer segment disc formation of both rod and cone photoreceptors. Furthermore, they suggest a feedback mechanism which coordinates the rate of photoreceptor outer segment disc formation, shedding and phagocytosis. This study has important implications for understanding the function of PRCD in the retina, as well as for future development of treatment modalities for PRCD-deficiency in humans.

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A Computational Pipeline for Functional Gene Discovery

10.21203/rs.3.rs-770297/v1 ◽

2021 ◽

Author(s):

Aolani Colon ◽

Rishabh Hirday ◽

Ami Patel ◽

Amrita Poddar ◽

Emma Tuberty-Vaughan ◽

...

Keyword(s):

Candidate Genes ◽

Outer Segment ◽

Gene Discovery ◽

Immunohistochemical Analysis ◽

Transcriptome Profiling ◽

Photoreceptor Cells ◽

Functional Gene ◽

Mouse Retina ◽

Computational Pipeline ◽

Photoreceptor Outer Segment

Abstract Many computational pipelines exist for the detection of differentially expressed genes. However, computational pipelines for functional gene detection are rarely exist. We developed a new computational pipeline for functional gene identification from transcriptome profiling data. Key features of the pipeline include clustering optimization by gap statistics, gene ontology analysis for each cluster, and literature analysis for functional gene discovery. By leveraging this pipeline on RNA-seq datasets of mouse retinal development studies, we identified 14 candidate genes involved in the formation of the photoreceptor outer segment. The expression of top three candidate genes (Pde8b, Laptm4b, and Nr1h4) in the outer segment of the developing mouse retina were experimentally validated by immunohistochemical analysis. This computational pipeline can accurately predict novel functional gene for a specific biological process, e.g., the outer segment development of the photoreceptor cells in the mouse retina. This pipeline is also applicable to functional gene discovery for any other biological processes and in any other organs and tissues.

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