Using a novel database of proteins actively synthesized in mouse inner ear sensory hair cells shortly after birth, we have identified a novel hearing loss protein, Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1), previously not shown to participate in hair cell function, or localized to hair cell stereocilia bundles. By specifically removing PKHD1L1 from mouse inner ear sensory hair cells, we show that absence of PKHD1L1 results in hearing loss. In order to understand the mechanism by which PKHD1L1-deficient mice develop hearing loss, three closely coupled aims are proposed for this study. Since PKHD1L1 has not been previously shown to participate in hair cell function, or been localized to hair cell stereocilia bundles, in Aim 1 we will localize PKHD1L1 on the surface of mouse hair cell stereocilia during bundle maturation (i.e. within the first three weeks after birth), using highly precise electron microscopy protein localization techniques. Our preliminary immunogold antibody labeling results localize PKHD1L1 to the tips of stereocilia at postnatal day 4.
In Aim 2 we test the function of PKHD1L1 by evaluating the hearing deficit of the hair cell-specific PKHD1L1 knock-out mouse. We show that ?stereocilia coat? is absent from the tips of PKHD1L1-deficient stereocilia. We will now specifically focus on the implications of PKHD1L1-deficiency on development of tectorial membrane attachment crowns, bundle cohesion, bundle motion upon stimulation, and the properties of the hair cell transduction current. By studying a PKHD1L1- deficient mouse line without the ?coat? at the tips of stereocilia, we propose a clean experimental paradigm to test PKHD1L1?s (hence, the coat?s) contribution to bundle cohesion, and sliding adhesion. Lastly, in our Aim 3, based on the predicted domain structure of PKHD1L1 suggesting possible homomeric (i.e. with itself) and heteromeric (with other proteins) interactions, we will perform protein interaction experiments to reveal possible interactions with other proteins involved in forming the tectorial membrane or its attachment to stereocilia, like stereocilin, CEACAM16 and tectorin.

Public Health Relevance

? Public Health Relevance The goal of this study is to understand the function of Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1), a novel hearing loss protein with no previous implications in hearing function. We show that absence of PKHD1L1 from the mouse inner ear sensory cells leads to a hearing loss, and understanding the mechanisms by which PKHD1L1-deficient mice develop a hearing loss is the central aim of this project. Identification and characterization of new hearing loss genes enhances the diagnostic potential for undiagnosed hereditary deafness cases in humans, and serves a foundation for developing candidate-based hearing restoration therapies.

National Institute of Health (NIH)
National Institute on Deafness and Other Communication Disorders (NIDCD)
Research Project (R01)
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Auditory System Study Section (AUD)
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Freeman, Nancy
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Massachusetts Eye and Ear Infirmary
United States
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