SANS (USH1G) expression in developing and mature mammalian retina

AbstractThe 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

Direct interaction of the Usher syndrome 1G protein SANS and myomegalin in the retina

AbstractThe 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

Analysen zur molekularen Charakterisierung von Proteinen des humanen Usher-Syndroms und Evaluation genbasierter Therapiestrategien

Analysen zur molekularen Charakterisierung von Proteinen des humanen Usher-Syndroms und Evaluation genbasierter Therapiestrategien rnDas humane Usher Syndrom (USH) ist die häufigste Form vererbter Taub-Blindheit. In der vorliegenden Dissertation wurde diese komplexe Erkrankung auf verschiedenen Ebenen analysiert: in Arbeiten zur Expression und Lokalisation von USH-Proteinen, der Analyse der USH-Proteinnetzwerke und deren Funktionen sowie darauf aufbauend die Entwicklung von Therapiestrategien für USH.rnIm Rahmen der Arbeit wurde die Expression und (sub)-zelluläre Lokalisation des USH1D-Genproduktes CDH23 in der Retina und Cochlea analysiert. CDH23-Isoformen werden in der Maus zeitlich und räumlich differentiell exprimiert. In den Retinae von Mäusen, nicht humanen Primaten und Menschen zeigten Analysen eine unterschiedliche Expression und Lokalisation des Zell-Zelladhäsionsmoleküls CDH23, was auf Funktions-unterschiede der einzelnen Isoformen in den analysierten Spezies hindeutet.rnAnalysen zur Aufklärung der USH-Proteinnetzwerke ergaben eine potentielle Interaktion des USH1G-Gerüstproteins SANS mit dem Golgi- und Centrosom-assoziierten Protein Myomegalin. Die direkte Interaktion der Proteine konnte durch unabhängige Experimente verifiziert werden. Beide Interaktionspartner sind in den Retinae verschiedener Spezies partiell ko-lokalisiert und partizipieren im periciliären USH-Proteinnetzwerk. Die Assoziation von SANS und Myomegalin mit dem Mikrotubuli-Cytoskelett weist auf eine Funktion des Proteinkomplexes in gerichteten Transportprozessen innerhalb der Photorezeptoren hin und bekräftigt die Hypothese einer Rolle von SANS und assoziierten Netzwerken mit Transportprozessen.rnDas hier gewonnene erweiterte Verständnis der molekularen Grundlagen sowie die Aufklärung der zellulären Funktion der Proteinnetzwerke ermöglichen die Entwicklung therapeutischer Strategien für USH. Ein Fokus der vorliegenden Arbeit lag auf der Entwicklung genbasierter Therapiestrategien und deren Evaluation, wobei der Schwerpunkt auf der Therapiestrategie der Genreparatur lag. Die mit Hilfe von Zinkfinger-Nukleasen (ZFN) induzierte Homologe Rekombination für die Genkorrektur wurde exemplarisch an der 91C>T/p.R31X-Mutation im USH1C-Gen gezeigt. Effiziente ZFN wurden identifiziert, generiert und erfolgreich im Zellkulturmodellsystem eingesetzt. Die Analysen demonstrierten eine Reparatur der Mutation durch Homologe Rekombination auf genomischer Ebene und die Expression des wiederhergestellten Proteins. Durch die Genkorrektur im endogenen Lokus sind Größe des Gens, Isoformen oder die Art der Mutation keine limitierenden Faktoren für die Therapie. Die in der vorliegenden Arbeit durchgeführten Experimente unterstreichen das enorme Potential ZFN-basierter Therapiestrategien hin zu personalisierten Therapieformen nicht nur für USH sondern auch für andere erbliche Erkrankungen, deren genetische Grundlagen bekannt sind.rnSummary PhD thesisrnThe human Usher syndrome is the most common form of combined hereditary deaf-blindness. This complex disease was analyzed on different levels: by the analysis of expression and localization of USH proteins, the analysis of USH protein networks and their function and based on this the elaboration of therapeutic strategies for the treatment of USH.rnThe expression and (sub) cellular localization of the USH1D gene product cadherin23 was analyzed in the retina and cochlea. In the mouse CDH23 isoform expression is temporally and spatially regulated. In the retina of mice, non-human primates and humans CDH23 is expressed and localized differentially, indicating different functions of the cell-cell adhesion molecule in the analyzed species. rnAnalyses for the decipherment of the USH protein network revealed a potential interaction of the USH1G protein SANS with the Golgi and centrosome associated protein myomegalin. The direct interaction of the proteins was validated by independent methods. Both proteins are partially co-localized in the retina of different species and participate in the periciliary USH protein network. The association of SANS and myomegalin with the microtubule cytoskeleton indicates a function of the complex in photoreceptor cell transport and strengthens the hypothesis for a role of SANS and associated networks in transport processes. rnThe gathered knowledge of the molecular basis and the decipherment of the cellular function of USH proteins enable the establishment of therapeutic strategies for USH. One focus of the present work was the development of gene-based therapies and their evaluation, with the emphasis on a strategy for gene repair by homologous recombination. This represents a powerful method to correct genetic defects. The strategy of zinc finger nuclease (ZFN) induced homologous recombination was used to exemplarily correct the 91C>T/p.R31X nonsense mutation in the USH1C gene. Screening the mutated USH1C sequence for plausible zinc finger binding sites revealed six promising pairs of ZFNs. Potential ZFNs were identified, generated by modular assembly, and the nuclear localization was verified upon transfection in HEK293T cells. Efficient ZFN were successfully tested in a 91C>T/p.R31X cell culture model system. PCR demonstrated cleavage of the target sequence in vitro by ZFN. Successful gene repair mediated by ZFN induced homologous recombination and an exogenously introduced rescue plasmid encoding for the healthy USH1C gene was shown on genomic level by TseI digestion and sequencing. Expression of the repaired USH1C gene product harmonin demonstrated ZFN activated gene repair on protein level, analyzed by indirect immunofluorescence and Western blot. rnDue to the repair of the gene in its endogenous locus the size of the gene, isoforms and the type of mutation are no limiting factors for therapy. The performed experiments underline the enormous potential of ZFN based strategies in the direction of personalized therapies not only for USH but also other inherited diseases

Gene repair of an Usher syndrome causing mutation by zinc-finger nuclease mediated homologous recombination

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, 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 ZFN induced cleavage of their target sequence. We showed that simultaneous application of ZFN and rescue DNA induce 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

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A novel Usher protein network at the periciliar

A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells.

Contains fulltext : 69178.pdf (publisher's version ) (Closed access)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