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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
5R01DC004844-10
Application #
8277962
Study Section
Special Emphasis Panel (ZRG1-IFCN-B (03))
Program Officer
Watson, Bracie
Project Start
2001-04-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
10
Fiscal Year
2012
Total Cost
$297,319
Indirect Cost
$93,676
Name
Father Flanagan's Boys' Home
Department
Type
DUNS #
073136806
City
Boys Town
State
NE
Country
United States
Zip Code
68010
Tian, Mei; Wang, Weimin; Delimont, Duane et al. (2014) Photoreceptors in whirler mice show defective transducin translocation and are susceptible to short-term light/dark changes-induced degeneration. Exp Eye Res 118:145-53
Zallocchi, Marisa; Binley, Katie; Lad, Yatish et al. (2014) EIAV-based retinal gene therapy in the shaker1 mouse model for usher syndrome type 1B: development of UshStat. PLoS One 9:e94272
Cosgrove, Dominic; Zallocchi, Marisa (2014) Usher protein functions in hair cells and photoreceptors. Int J Biochem Cell Biol 46:80-9
Tian, Mei; Zallocchi, Marisa; Wang, Weimin et al. (2013) Light-induced translocation of RGS9-1 and G?5L in mouse rod photoreceptors. PLoS One 8:e58832
Zallocchi, Marisa; Meehan, Daniel T; Delimont, Duane et al. (2012) Role for a novel Usher protein complex in hair cell synaptic maturation. PLoS One 7:e30573
Peng, You-Wei; Zallocchi, Marisa; Wang, Wei-Min et al. (2011) Moderate light-induced degeneration of rod photoreceptors with delayed transducin translocation in shaker1 mice. Invest Ophthalmol Vis Sci 52:6421-7
Zallocchi, Marisa; Sisson, Joseph H; Cosgrove, Dominic (2010) Biochemical characterization of native Usher protein complexes from a vesicular subfraction of tracheal epithelial cells. Biochemistry 49:1236-47
Zallocchi, Marisa; Meehan, Daniel T; Delimont, Duane et al. (2009) Localization and expression of clarin-1, the Clrn1 gene product, in auditory hair cells and photoreceptors. Hear Res 255:109-20
Cosgrove, Dominic; Meehan, Daniel T; Delimont, Duane et al. (2008) Integrin alpha1beta1 regulates matrix metalloproteinases via P38 mitogen-activated protein kinase in mesangial cells: implications for Alport syndrome. Am J Pathol 172:761-73
Peng, You-Wei; Zallocchi, Marisa; Meehan, Daniel T et al. (2008) Progressive morphological and functional defects in retinas from alpha1 integrin-null mice. Invest Ophthalmol Vis Sci 49:4647-54

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