RESUMEN
The human Usher syndrome (USH) is a retinal ciliopathy, characterized by profound congenital deafness, variable vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. In the effected sensory cells, USH protein networks are assumed to function in ciliary transport processes. The USH1G protein SANS is a scaffold of the ciliary/periciliary USH protein network of photoreceptor cells. Moreover, SANS is associated with microtubules, the transport routes for protein delivery toward the cilium. To enlighten the role of SANS in ciliary transport processes, we aimed to identify transport related proteins associated with SANS. The intraflagellar transport (IFT) system is a conserved mechanism for bi-directional transport toward and through primary cilia. Thus, we tested the direct binding of SANS to IFT molecules, namely IFT20, IFT57, and IFT74 in 1:1 yeast-two-hybrid assay. The identified SANS-IFT interactions were validated in vitro via independent complementary interaction assays and in cells by applying membrane targeting assays. Quantitative immunofluorescence microscopy revealed the co-localization of SANS with IFT20, IFT52, and IFT57 particularly at ciliary base of wild type mouse photoreceptor cells. Analysis of photoreceptor cells of SANS knock out mice revealed the decrease of IFTs in the ciliary compartment indicating a role of SANS in the proper positioning of IFT-B molecules in primary cilia. Our study demonstrated direct binding of IFT complex B proteins IFT52 and IFT57 to the N-terminal ankyrin repeats and the central domain of SANS. Our data also indicate that pathologic mutations in the N-terminus of SANS lead to the loos of SANS binding to IFT-B molecules. Our findings provide direct evidence for a molecular link between the ciliary USH protein network and the IFT transport module in primary cilia.
RESUMEN
Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Degeneración Retiniana/genética , Síndromes de Usher/genética , Proteínas de Pez Cebra/genética , Pez Cebra , Animales , Apoptosis , Electrorretinografía , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/fisiología , Técnicas de Inactivación de Genes , Técnicas de Genotipaje , Proteínas de la Membrana/metabolismo , Microscopía Inmunoelectrónica , Mutación , Retina/fisiopatología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/fisiopatología , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/ultraestructura , Receptor de Retrovirus Xenotrópico y Politrópico , Proteínas de Pez Cebra/metabolismoRESUMEN
The Usher syndrome (USH) is the most common form of inherited deaf-blindness, accompanied by vestibular dysfunction. Due to the heterogeneous manifestation of the clinical symptoms, three USH types (USH1-3) and additional atypical forms are distinguished. USH1 and USH2 proteins have been shown to function together in multiprotein networks in photoreceptor cells and hair cells. Mutations in USH proteins are considered to disrupt distinct USH protein networks and finally lead to the development of USH.To get novel insights into the molecular pathomechanisms underlying USH, we further characterize the periciliary USH protein network in photoreceptor cells. We show the direct interaction between the scaffold protein SANS (USH1G) and the transmembrane adhesion protein ush2a and that both assemble into a ternary USH1/USH2 complex together with the PDZ-domain protein whirlin (USH2D) via mutual interactions. Immunohistochemistry and proximity ligation assays demonstrate co-localization of complex partners and complex formation, respectively, in the periciliary region, the inner segment and at the synapses of rodent and human photoreceptor cells. Protein-protein interaction assays and co-expression of complex partners reveal that pathogenic mutations in USH1G severely affect formation of the SANS/ush2a/whirlin complex. Translational read-through drug treatment, targeting the c.728C > A (p.S243X) nonsense mutation, restored SANS scaffold function. We conclude that USH1 and USH2 proteins function together in higher order protein complexes. The maintenance of USH1/USH2 protein complexes depends on multiple USH1/USH2 protein interactions, which are disrupted by pathogenic mutations in USH1G protein SANS.
Asunto(s)
Trastornos Sordoceguera/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Síndromes de Usher/genética , Trastornos Sordoceguera/patología , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patología , Humanos , Proteínas de la Membrana/química , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Unión Proteica , Mapas de Interacción de Proteínas/genética , Estructura Terciaria de Proteína , Síndromes de Usher/complicaciones , Síndromes de Usher/patologíaRESUMEN
BACKGROUND: Joubert syndrome (JBTS) and related disorders are defined by cerebellar malformation (molar tooth sign), together with neurological symptoms of variable expressivity. The ciliary basis of Joubert syndrome related disorders frequently extends the phenotype to tissues such as the eye, kidney, skeleton and craniofacial structures. RESULTS: Using autozygome and exome analyses, we identified a null mutation in KIAA0556 in a multiplex consanguineous family with hallmark features of mild Joubert syndrome. Patient-derived fibroblasts displayed reduced ciliogenesis potential and abnormally elongated cilia. Investigation of disease pathophysiology revealed that Kiaa0556 (-/-) null mice possess a Joubert syndrome-associated brain-restricted phenotype. Functional studies in Caenorhabditis elegans nematodes and cultured human cells support a conserved ciliary role for KIAA0556 linked to microtubule regulation. First, nematode KIAA0556 is expressed almost exclusively in ciliated cells, and the worm and human KIAA0556 proteins are enriched at the ciliary base. Second, C. elegans KIAA0056 regulates ciliary A-tubule number and genetically interacts with an ARL13B (JBTS8) orthologue to control cilium integrity. Third, human KIAA0556 binds to microtubules in vitro and appears to stabilise microtubule networks when overexpressed. Finally, human KIAA0556 biochemically interacts with ciliary proteins and p60/p80 katanins. The latter form a microtubule-severing enzyme complex that regulates microtubule dynamics as well as ciliary functions. CONCLUSIONS: We have identified KIAA0556 as a novel microtubule-associated ciliary base protein mutated in Joubert syndrome. Consistent with the mild patient phenotype, our nematode, mice and human cell data support the notion that KIAA0556 has a relatively subtle and variable cilia-related function, which we propose is related to microtubule regulation.
Asunto(s)
Cuerpos Basales/metabolismo , Cerebelo/anomalías , Proteínas Asociadas a Microtúbulos/genética , Mutación , Retina/anomalías , Factores de Ribosilacion-ADP/metabolismo , Anomalías Múltiples/genética , Anomalías Múltiples/patología , Adenosina Trifosfatasas/metabolismo , Adulto , Animales , Cuerpos Basales/patología , Encéfalo/metabolismo , Encéfalo/patología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Cerebelo/patología , Niño , Preescolar , Cilios/genética , Cilios/patología , Exoma , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Femenino , Humanos , Katanina , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Linaje , Unión Proteica , Retina/patologíaRESUMEN
Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.
Asunto(s)
Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Marcadores Genéticos , Pruebas Genéticas/métodos , Genómica/métodos , Células Fotorreceptoras , Interferencia de ARN , Anomalías Múltiples , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestructura , Enfermedades Cerebelosas/genética , Cerebelo/anomalías , Cilios/metabolismo , Cilios/patología , Trastornos de la Motilidad Ciliar/metabolismo , Trastornos de la Motilidad Ciliar/patología , Proteínas del Citoesqueleto , Bases de Datos Genéticas , Síndrome de Ellis-Van Creveld/genética , Anomalías del Ojo/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Enfermedades Renales Quísticas/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestructura , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Proteínas/genética , Proteínas/metabolismo , Reproducibilidad de los Resultados , Retina/anomalías , Factores Supresores Inmunológicos/genética , Factores Supresores Inmunológicos/metabolismo , Transfección , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Mutations in the FAM161A gene were previously identified as the cause for autosomal-recessive retinitis pigmentosa 28. To study the effects of Fam161a dysfunction in vivo, we generated gene-trapped Fam161a(GT/GT) mice with a disruption of its C-terminal domain essential for protein-protein interactions. We confirmed the absence of the full-length Fam161a protein in the retina of Fam161a(GT/GT) mice using western blots and showed weak expression of a truncated Fam161a protein by immunohistochemistry. Histological analyses demonstrated that photoreceptor segments were disorganized in young Fam161a(GT/GT) mice and that the outer retina was completely lost at 6 months of age. Reactive microglia appeared in the outer retina and electroretinography showed an early loss of photoreceptor function in 4-month-old Fam161a(GT/GT) animals. Light and electron microscopy revealed a remarkable phenotype of a significantly shortened connecting cilium, spread ciliary microtubule doublets and disturbed disk organization in Fam161a(GT/GT) photoreceptor cells. Co-immunolabeling experiments demonstrated reduced expression and mislocalization of centrin 3 and disturbed targeting of the Fam161a interactors lebercilin and Cep290, which were restricted to the basal body and proximal connecting cilium in Fam161a(GT/GT) retinas. Moreover, we identified misrouting of the outer segment cargo proteins opsin and rds/peripherin 2 in Fam161a(GT/GT) mice. In conclusion, our results suggest a critical role for the C-terminal domain of Fam161a for molecular interactions and integrity of the connecting cilium. Fam161a is required for the molecular delivery into the outer segment cilium, a function which is essential for outer segment disk formation and ultimately visual function.
Asunto(s)
Proteínas del Ojo/genética , Mutación , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Degeneración Retiniana/genética , Potenciales de Acción , Animales , Proteínas Portadoras/metabolismo , Femenino , Expresión Génica , Marcación de Gen , Sitios Genéticos , Genotipo , Humanos , Masculino , Ratones , Ratones Transgénicos , Microglía/metabolismo , Células Fotorreceptoras/ultraestructura , Unión Proteica , Transporte de Proteínas , Retina/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Trastornos de la Visión/genética , Trastornos de la Visión/patología , Trastornos de la Visión/fisiopatologíaRESUMEN
The human Usher syndrome (USH) is the most frequent cause of combined hereditary deaf-blindness. USH is genetically and clinically heterogeneous: 15 chromosomal loci assigned to 3 clinical types, USH1-3. All USH1 and 2 proteins are organized into protein networks by the scaffold proteins harmonin (USH1C), whirlin (USH2D) and SANS (USH1G). This has contributed essentially to our current understanding of the USH protein function in the eye and the ear and explains why defects in proteins of different families cause very similar phenotypes. Ongoing in depth analyses of USH protein networks in the eye indicated cytoskeletal functions as well as roles in molecular transport processes and ciliary cargo delivery in photoreceptor cells. The analysis of USH protein networks revealed molecular links of USH to other ciliopathies, including non-syndromic inner ear defects and isolated retinal dystrophies but also to kidney diseases and syndromes like the Bardet-Biedl syndrome. These findings provide emerging evidence that USH is a ciliopathy molecularly related to other ciliopathies, which opens an avenue for common therapy strategies to treat these diseases.
Asunto(s)
Retina/patología , Retina/fisiopatología , Síndromes de Usher/patología , Síndromes de Usher/fisiopatología , Cilios/patología , Cilios/fisiología , Trastornos de la Motilidad Ciliar/patología , Trastornos de la Motilidad Ciliar/fisiopatología , Encefalocele/patología , Encefalocele/fisiopatología , Humanos , Amaurosis Congénita de Leber/patología , Amaurosis Congénita de Leber/fisiopatología , Enfermedades Renales Poliquísticas/patología , Enfermedades Renales Poliquísticas/fisiopatología , Retinitis Pigmentosa/patología , Retinitis Pigmentosa/fisiopatologíaRESUMEN
Mutations in the myosin VIIa gene cause Usher syndrome type IB (USH1B), characterized by deaf-blindness. A delay of opsin trafficking has been observed in the retinal photoreceptor cells of myosin VIIa-deficient mice. We identified spectrin ßV, the mammalian ß-heavy spectrin, as a myosin VIIa- and rhodopsin-interacting partner in photoreceptor cells. Spectrin ßV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other ß spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin ßV-rhodopsin complex could be detected in the differentiating photoreceptors as soon as their outer segment emerges. A failure of the spectrin ßV-mediated coupling between myosin VIIa and opsin molecules thus probably accounts for the opsin transport delay in myosin VIIa-deficient mice. We showed that spectrin ßV also associates with two USH1 proteins, sans (USH1G) and harmonin (USH1C). Spectrins are supposed to function as heteromers of α and ß subunits, but fluorescence resonance energy transfer and in vitro binding experiments indicated that spectrin ßV can also form homodimers, which likely supports its αII-independent ßV functions. Finally, consistent with its distribution along the connecting cilia axonemes, spectrin ßV binds to several subunits of the microtubule-based motor proteins, kinesin II and the dynein complex. We therefore suggest that spectrin ßV homomers couple some USH1 proteins, opsin and other phototransduction proteins to both actin- and microtubule-based motors, thereby contributing to their transport towards the photoreceptor outer disks.
Asunto(s)
Miosinas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Espectrina/genética , Espectrina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Células HeLa , Humanos , Fototransducción , Ratones , Proteínas de Microtúbulos/metabolismo , Miosina VIIa , Retina/metabolismo , Rodopsina/metabolismo , Síndromes de Usher/metabolismoRESUMEN
Loss-of-function mutations in the gene encoding FAM161A were recently discovered as the cause for RP28, an autosomal recessive form of retinitis pigmentosa. To initiate the characterization of the cellular role of FAM161A in the retina, we focused on its subcellular localization and conducted in vitro studies to identify FAM161A-interacting proteins and associated cellular structures. Immunohistochemistry revealed the presence of mouse FAM161A in the photoreceptor inner segments, the synaptic regions of the outer and inner plexiform layers and the ganglion cells. In mouse and human retinal sections from unfixed eyes, FAM161A localized to the ciliary region linking photoreceptor outer and inner segments. High-resolution immunofluorescence and immunoelectron microscopy mapped FAM161A to the connecting cilium, the basal body region and the adjacent centriole. Ectopic FAM161A was found in the centrosome and concentrated at the base of primary cilia in cultured cells. In addition, overexpressed FAM161A was clearly associated with microtubules during interphase and mitosis. The presence of FAM161A increased microtubule acetylation and stabilization. We further show that the evolutionarily conserved UPF0564 domain of FAM161A is crucial for its binding to microtubules and mediates homo- and heterotypic FAM161A and FAM161B interaction. In conclusion, our study shows that FAM161A is a microtubule-associated ciliary protein presumably involved in microtubule stabilization to maintain the microtubule tracks and/or in transport processes along microtubules in photoreceptors and other retinal cell types.