Usher syndrome is the leading genetic cause of deaf/blindness in the world. Progress in genetic research has revealed a high degree of heterogeneity for the disorder, with nine specific genes identified as causal for the three clinical sub-types. The genes encode an array of products and preliminary functional analysis points to a number of specific interactions that suggest the existence of a functional usher protein """"""""interactome"""""""". The existence of the """"""""interactome"""""""" in vivo has never been proven, and the critical subcellular compartments of usher protein function are still controversial. Mouse models for the various usher genes all display a developmental defect in stereocilia associated with disorganized structure in the cuticular plate, abnormal numbers of actin paracrystals, and variable lengths and shapes, all associated with deafness. None of the usher mouse models develop retinal degeneration, which has hampered progress in understanding RP associated with Usher syndrome. In the preliminary results of this proposal we demonstrate that the usher proteins are present in the vesicular sub-cellular fraction of tracheal epithelial cells and do indeed form a complex in vivo that sediments on sucrose gradients at around 50 Svedbergs. For three different usher mouse models (all that have been tested thus far) we show a defect in arrestin and transducin protein translocation in photoreceptors following exposure to light in dark adapted animals. We also demonstrate light-induced photo- receptor cell degeneration in these same animals, suggesting that the usher mice do indeed possess a robust retinal phenotype that is not revealed in the environment of animal housing facilities. These findings form the foundation for the central hypothesis of this proposal that usher proteins function to mediate translocation of proteins in ciliated neuroepithelial cells of the retina and the cochlea. Defects in this process lead to stereo- ciliary defects and photoreceptor cell degeneration. We will test this hypothesis in three specific aims. In the first aim we perform biochemical analysis of the vesicular complex in ciliated tracheal epithelium and retinal extracts, and perform subcellular localization experiments in photoreceptors and hair cells. In the second aim we will examine mouse models for all of the known subtypes of Usher syndrome for defects in protein trans- location and light induced photoreceptor cell degeneration and test the hypothesis directly using vesicle transport inhibitors in organotypic cultures of neural retina. In the third aim we will examine the role of vesicular transport in stereociliary development and maintenance by blocking vesicle transport in cultures of mouse organ of Corti and by immunohistological analysis of usher proteins in usher mouse models. Successful completion of these aims will define a cellular process that explains the defective development and function of ciliated neuroepithelium in the eye and ear that define Usher syndrome pathology. This work will help align the field and promote an acceleration of work aimed at understanding the specific disease mechanism, which is an essential first step towards the development of effective therapeutic strategies.
Usher syndrome is the leading cause of deaf/blindness, and results in the destruction of sensory cells in the eye and the retina. Nine different genes have been identified, and the encoded proteins interact with each other, but the functional connection with hearing and vision is still unknown. This proposal tests the hypothesis that the functional connection is a process called protein translocation, which may pinpoint the defective process that causes deafness and blindness in Usher patients.
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