RESUMEN
Leber hereditary optic neuropathy (LHON) is a mitochondrially inherited form of visual dysfunction caused by mutations in several genes encoding subunits of the mitochondrial respiratory NADH-ubiquinone oxidoreductase complex (complex I). Development of gene therapies for LHON has been impeded by genetic heterogeneity and the need to deliver therapies to the mitochondria of retinal ganglion cells (RGCs), the cells primarily affected in LHON. The therapy under development entails intraocular injection of a nuclear yeast gene NADH-quinone oxidoreductase (NDI1) that encodes a single subunit complex I equivalent and as such is mutation independent. NDI1 is imported into mitochondria due to an endogenous mitochondrial localisation signal. Intravitreal injection represents a clinically relevant route of delivery to RGCs not previously used for NDI1. In this study, recombinant adenoassociated virus (AAV) serotype 2 expressing NDI1 (AAV-NDI1) was shown to protect RGCs in a rotenone-induced murine model of LHON. AAV-NDI1 significantly reduced RGC death by 1.5-fold and optic nerve atrophy by 1.4-fold. This led to a significant preservation of retinal function as assessed by manganese enhanced magnetic resonance imaging and optokinetic responses. Intraocular injection of AAV-NDI1 overcomes many barriers previously associated with developing therapies for LHON and holds great therapeutic promise for a mitochondrial disorder for which there are no effective therapies.
Asunto(s)
Dependovirus/genética , Complejo I de Transporte de Electrón/genética , Vectores Genéticos/administración & dosificación , Atrofia Óptica Hereditaria de Leber/patología , Atrofia Óptica Hereditaria de Leber/terapia , Animales , Modelos Animales de Enfermedad , Humanos , Inyecciones Intravítreas , Ratones , Proteínas Asociadas a Microtúbulos/genética , Atrofia Óptica/patología , Atrofia Óptica/terapia , Células Ganglionares de la Retina/patología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Translational read-through-inducing drugs (TRIDs) promote read-through of nonsense mutations, placing them in the spotlight of current gene-based therapeutic research. Here, we compare for the first time the relative efficacies of new-generation aminoglycosides NB30, NB54 and the chemical compound PTC124 on retinal toxicity and read-through efficacy of a nonsense mutation in the USH1C gene, which encodes the scaffold protein harmonin. This mutation causes the human Usher syndrome, the most common form of inherited deaf-blindness. We quantify read-through efficacy of the TRIDs in cell culture and show the restoration of harmonin function. We do not observe significant differences in the read-through efficacy of the TRIDs in retinal cultures; however, we show an excellent biocompatibility in retinal cultures with read-through versus toxicity evidently superior for NB54 and PTC124. In addition, in vivo administration of NB54 and PTC124 induced recovery of the full-length harmonin a1 with the same efficacy. The high biocompatibilities combined with the sustained read-through efficacies of these drugs emphasize the potential of NB54 and PTC124 in treating nonsense mutation-based retinal disorders.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Aminoglicósidos/farmacología , Codón sin Sentido , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Enfermedades de la Retina/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular , Proteínas del Citoesqueleto , Femenino , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Oxadiazoles/farmacología , Retina/efectos de los fármacos , Retina/metabolismo , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/metabolismoRESUMEN
PURPOSE: Human Usher syndrome (USH) is the most frequent cause of inherited deaf-blindness. It is clinically and genetically heterogeneous, assigned to three clinical types of which the most severe type is USH1. No effective treatment for the ophthalmic component of USH exists. Gene augmentation is an attractive strategy for hereditary retinal diseases. However, several USH genes, like USH1C, are expressed in various isoforms, hampering gene augmentation. As an alternative treatment strategy, we applied the zinc-finger nuclease (ZFN) technology for targeted gene repair of an USH1C, causing mutation by homologous recombination. METHODS: We designed ZFNs customized for the p.R31X nonsense mutation in Ush1c. We evaluated ZFNs for DNA cleavage capability and analyzed ZFNs biocompatibilities by XTT assays. We demonstrated ZFNs mediated gene repair on genomic level by digestion assays and DNA sequencing, and on protein level by indirect immunofluorescence and Western blot analyses. RESULTS: The specifically designed ZFNs did not show cytotoxic effects in a p.R31X cell line. We demonstrated that ZFN induced cleavage of their target sequence. We showed that simultaneous application of ZFN and rescue DNA induced gene repair of the disease-causing mutation on the genomic level, resulting in recovery of protein expression. CONCLUSIONS: In our present study, we analyzed for the first time ZFN-activated gene repair of an USH gene. The data highlight the ability of ZFNs to induce targeted homologous recombination and mediate gene repair in USH. We provide further evidence that the ZFN technology holds great potential to recover disease-causing mutations in inherited retinal disorders.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Codón sin Sentido , ADN/genética , Endonucleasas/genética , Reparación del Gen Blanco/métodos , Síndromes de Usher/terapia , Dedos de Zinc/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Proteínas del Citoesqueleto , Roturas del ADN de Doble Cadena , División del ADN , Recombinación Homóloga , Humanos , Retina/metabolismo , Retina/patología , Síndromes de Usher/genética , Síndromes de Usher/metabolismoRESUMEN
The human Usher syndrome (USH) is the most frequent cause of combined hereditary deaf-blindness. USH is genetically heterogeneous with at least 11 chromosomal loci assigned to 3 clinical types, USH1-3. We have previously demonstrated that all USH1 and 2 proteins in the eye and the inner ear are organized into protein networks by scaffold proteins. This has contributed essentially to our current understanding of the function of USH proteins and explains why defects in proteins of different families cause very similar phenotypes. We have previously shown that the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) contributes to the periciliary protein network in retinal photoreceptor cells. This study aimed to further elucidate the role of SANS by identifying novel interaction partners. In yeast two-hybrid screens of retinal cDNA libraries we identified 30 novel putative interacting proteins binding to the central domain of SANS (CENT). We confirmed the direct binding of the phosphodiesterase 4D interacting protein (PDE4DIP), a Golgi associated protein synonymously named myomegalin, to the CENT domain of SANS by independent assays. Correlative immunohistochemical and electron microscopic analyses showed a co-localization of SANS and myomegalin in mammalian photoreceptor cells in close association with microtubules. Based on the present results we propose a role of the SANS-myomegalin complex in microtubule-dependent inner segment cargo transport towards the ciliary base of photoreceptor cells.
Asunto(s)
Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Retina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Células COS , Bovinos , Células Cultivadas , Chlorocebus aethiops , Proteínas del Citoesqueleto , Humanos , Macaca mulatta , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Unión Proteica/fisiología , Levaduras/genética , Levaduras/metabolismoRESUMEN
PURPOSE: The human Usher syndrome (USH) is the most frequent cause of inherited combined deaf-blindness. USH is clinically and genetically heterogeneous, assigned to three clinical types. The most severe type is USH1, characterized by profound inner ear defects and retinitis pigmentosa. Thus far, no effective treatment for the ophthalmic component of USH exists. The p.R31X nonsense mutation in USH1C leads to a disease causing premature termination of gene translation. Here, we investigated the capability of the novel synthetic aminoglycoside NB30 for the translational read-through of the USH1C-p.R31X nonsense mutation as a retinal therapy option. METHODS: Read-through of p.R31X by three commercial, clinically applied aminoglycosides and the synthetic derivative NB30 was validated in vitro, in cell culture, and in retinal explants. Restoration of harmonin functions was monitored in GST pull-downs (scaffold function) and by F-actin bundling analysis in HEK293T cells. Biocompatibility of aminoglycosides was determined in retinal explants by TUNEL assays. RESULTS: In vitro translation and analyses of transfected HEK293T cells revealed a dose-dependent read-through by all aminoglycosides. In addition, gentamicin, paromomycin, and NB30 induced read-through of p.R31X in mouse retinal explants. The read-through of p.R31X restored harmonin protein function. In contrast to all commercial aminoglycosides NB30 showed good biocompatibility. CONCLUSIONS: Commercial aminoglycosides and NB30 induced significant read-through of the USH1C-p.R31X nonsense mutation. However, the observed read-through efficiency, along with its significantly reduced toxicity and good biocompatibility, indicate that the novel derivate NB30 represents a better choice than commercial aminoglycosides in a read-through therapy of USH1C and other ocular diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Aminoglicósidos/farmacología , Codón sin Sentido/efectos de los fármacos , Retina/efectos de los fármacos , Animales , Western Blotting , Técnicas de Cultivo de Célula , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Relación Dosis-Respuesta a Droga , Electroporación , Expresión Génica/fisiología , Gentamicinas/farmacología , Células HEK293/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Paromomicina/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Retina/metabolismo , TransfecciónRESUMEN
PURPOSE: We compared cadherin 23 (Cdh23) mRNA and protein variants in the inner ear and retina of wild-type and mutant mice and primates to better understand the pleiotropic effects of Cdh23 mutations, and specifically to understand the absence of retinal degeneration in Cdh23 mutant mice. METHODS: Semiquantitative real-time PCR was used to compare the level of expression of Cdh23 alternative transcripts in the inner ear and retina of wild-type and homozygous Cdh23(v-6J) (waltzer) mice. Antibodies generated against CDH23 isoforms were used in immunohistochemistry, immunohistology, electron microscopy, and western blot analyses of mouse and primate inner ear and retina to study the distribution of these isoforms in various cellular compartments. RESULTS: Cdh23 mRNA alternative splice variants were temporally and spatially regulated in the inner ear and retina. In the mature mouse retina, CDH23 isoforms were broadly expressed in various cellular compartments of the photoreceptor layer. The wild-type CDH23_V3 protein isoform, which has PDZ binding motifs but neither extracellular domains nor a transmembrane domain, localized exclusively to the outer plexiform layer of the retina containing photoreceptor cell synapses and to the synaptic region of auditory and vestibular hair cells. The longest CDH23 protein isoform, CDH23_V1, appeared by western blotting to be the only one affected by the Cdh23(v-6J) mutation; it was expressed in the wild-type mouse inner ear, but not in the mouse retina. However, CDH23_V1 was detected in western blot analyses of monkey and human retinas. CONCLUSIONS: The time- and tissue-dependent expression patterns that we have shown for Cdh23 alternative transcripts suggest developmental roles and tissue-specific functions for the various transcripts. Many of these isoforms continue to be expressed in waltzer mice. The longest CDH23 isoform (CDH23_V1), however, is not expressed in mutant mice and is necessary for normal inner ear function. The longest isoform is expressed in the retinas of primates, but not detected in the mouse retina. This species difference suggests that the mouse may not be a suitable model for studying the retinitis pigmentosa phenotype of human Usher syndrome type 1D.
Asunto(s)
Cadherinas/metabolismo , Síndromes de Usher/metabolismo , Animales , Anticuerpos , Western Blotting , Cadherinas/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Vestibulares/citología , Células Ciliadas Vestibulares/metabolismo , Haplorrinos , Células HeLa , Humanos , Ratones , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestructura , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Fracciones Subcelulares/metabolismo , Sinapsis/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
The human Usher syndrome (USH) is the most common form of combined deaf-blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction and prepubertal-onset of retinitis pigmentosa. Five corresponding genes of the six USH1 genes have been cloned so far. The USH1G gene encodes the SANS (scaffold protein containing ankyrin repeats and SAM domain) protein which consists of protein motifs known to mediate protein-protein interactions. Recent studies indicated SANS function as a scaffold protein in the protein interactome related to USH. Here, we generated specific antibodies for SANS protein expression analyses. Our study revealed SANS protein expression in NIH3T3 fibroblasts, murine tissues containing ciliated cells and in mature and developing mammalian retinas. In mature retinas, SANS was localized in inner and outer plexiform retinal layers, and in the photoreceptor cell layer. Subcellular fractionations, tangential cryosections and immunocytochemistry revealed SANS in synaptic terminals, cell-cell adhesions of the outer limiting membrane and ciliary apparati of photoreceptor cells. Analyses of postnatal developmental stages of murine retinas demonstrated SANS localization in differentiating ciliary apparati and in fully developed cilia, synapses, and cell-cell adhesions of photoreceptor cells. Present data provide evidence that SANS functions as a scaffold protein in USH protein networks during ciliogenesis, at the mature ciliary apparatus, the ribbon synapse and the cell-cell adhesion of mammalian photoreceptor cells. Defects of SANS may cause dysfunction of the entire network leading to retinal degeneration, the ocular symptom characteristic for USH patients.
Asunto(s)
Proteínas del Ojo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Retina/metabolismo , Animales , Especificidad de Anticuerpos , Western Blotting , Centrosoma/metabolismo , Cilios/metabolismo , Cuerpo Ciliar/metabolismo , Proteínas del Ojo/inmunología , Fibroblastos/metabolismo , Sueros Inmunes/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/inmunología , Células Fotorreceptoras de Vertebrados/metabolismo , Ratas , Ratas Endogámicas WKY , Retina/crecimiento & desarrollo , Sinapsis/metabolismo , Sinaptosomas/metabolismo , Síndromes de Usher/metabolismo , Xenopus laevisRESUMEN
The human Usher syndrome (USH) is the most frequent cause of combined deaf-blindness. USH is genetically heterogeneous with at least 12 chromosomal loci assigned to three clinical types, USH1-3. Although these USH types exhibit similar phenotypes in human, the corresponding gene products belong to very different protein classes and families. The scaffold protein harmonin (USH1C) was shown to integrate all identified USH1 and USH2 molecules into protein networks. Here, we analyzed a protein network organized in the absence of harmonin by the scaffold proteins SANS (USH1G) and whirlin (USH2D). Immunoelectron microscopic analyses disclosed the colocalization of all network components in the apical inner segment collar and the ciliary apparatus of mammalian photoreceptor cells. In this complex, whirlin and SANS directly interact. Furthermore, SANS provides a linkage to the microtubule transport machinery, whereas whirlin may anchor USH2A isoform b and VLGR1b (very large G-protein coupled receptor 1b) via binding to their cytodomains at specific membrane domains. The long ectodomains of both transmembrane proteins extend into the gap between the adjacent membranes of the connecting cilium and the apical inner segment. Analyses of Vlgr1/del7TM mice revealed the ectodomain of VLGR1b as a component of fibrous links present in this gap. Comparative analyses of mouse and Xenopus photoreceptors demonstrated that this USH protein network is also part of the periciliary ridge complex in Xenopus. Since this structural specialization in amphibian photoreceptor cells defines a specialized membrane domain for docking and fusion of transport vesicles, we suggest a prominent role of the USH proteins in cargo shipment.
Asunto(s)
Células Fotorreceptoras de Vertebrados/metabolismo , Síndromes de Usher/genética , Síndromes de Usher/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Proteínas de Ciclo Celular , Chlorocebus aethiops , Proteínas del Citoesqueleto , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Técnicas In Vitro , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Inmunoelectrónica , Modelos Biológicos , Células 3T3 NIH , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestructura , Mapeo de Interacción de Proteínas , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Fracciones Subcelulares/metabolismo , Transfección , Síndromes de Usher/clasificación , Xenopus/genética , Xenopus/metabolismoRESUMEN
Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. USH is clinically and genetically heterogeneous with at least 11 chromosomal loci assigned to the three USH types (USH1A-G, USH2A-C, USH3A). Although the different USH types exhibit almost the same phenotype in human, the identified USH genes encode for proteins which belong to very different protein classes and families. We and others recently reported that the scaffold protein harmonin (USH1C-gene product) integrates all identified USH1 molecules in a USH1-protein network. Here, we investigated the relationship between the USH2 molecules and this USH1-protein network. We show a molecular interaction between the scaffold protein harmonin (USH1C) and the USH2A protein, VLGR1 (USH2C) and the candidate for USH2B, NBC3. We pinpoint these interactions to interactions between the PDZ1 domain of harmonin and the PDZ-binding motifs at the C-termini of the USH2 proteins and NBC3. We demonstrate that USH2A, VLGR1 and NBC3 are co-expressed with the USH1-protein harmonin in the synaptic terminals of both retinal photoreceptors and inner ear hair cells. In hair cells, these USH proteins are also localized in the signal uptaking stereocilia. Our data indicate that the USH2 proteins and NBC3 are further partners in the supramolecular USH-protein network in the retina and inner ear which shed new light on the function of USH2 proteins and the entire USH-protein network. These findings provide first evidence for a molecular linkage between the pathophysiology in USH1 and USH2. The organization of USH molecules in a mutual 'interactome' related to the disease can explain the common phenotype in USH.