Investigating the mechanisms of stereocilia length regulation and innovative strategies for restoring hearing

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Abstract Sensorineural hearing loss is a debilitating condition with no cure that directly impacts >30 million people. A common cause of inherited hearing loss is the disruption of cochlear hair cell stereocilia, which are essential for transducing sound vibrations to the brain. For proper function, stereocilia must be tall enough to reach the tectorial membrane (TM) and form stereocilia-TM junctions (STJs). Stereocilia elongation greatly depends on actin regulation; as such, disruption of actin regulatory proteins and other cytoskeletal elements cause profound hearing loss in humans and model organisms. Thus, it is critical to elucidate the dynamic regulation of stereocilia lengths to understand normal hearing and determine how to rescue stereocilia-related hearing loss. Previous work demonstrated that stereocilia actin filament elongation depends on epidermal growth factor pathway substrate 8 (Eps8), an actin-regulatory protein that is critical for hearing in both mice and humans. Eps8 paradoxically contains domains with both actin filament capping and bundling activities, which are typically associated with shortening or elongation of actin filaments, respectively. Moreover, it was found that two other deafness-associated proteins essential for stereocilia elongation, Myosin-XVa (MyoXVa) and whirlin, bind to Eps8 at stereocilia tips in a tripartite complex. Preliminary data show that stereocilin, another deafness-associated protein which links stereocilia to the TM at STJs, is not properly targeted to stereocilia tips in Eps8, MyoXVa, or whirlin knockout (KO) mice, and that all these mice lack normal STJs. Moreover, early (≤postnatal day 1) adeno-associated virus (AAV)-mediated delivery of Eps8 can rescue stereocilia elongation, stereocilin localization, and STJs in Eps8 KO mouse apical hair cells, suggesting that it may be possible to rescue hearing function. This proposal tests the hypothesis that Eps8 regulates stereocilia elongation and the proper formation of STJs by directly regulating actin bundle growth through its C-terminal actin capping and bundling regions and indirectly regulating stereocilia growth through interactions with MyoXVa and whirlin. Further, it is proposed that there is a critical window of hair cell maturation during which stereocilia plasticity is sufficient for full rescue. To test these hypotheses, an AAV-mediated delivery of Eps8 mutants lacking either or both capping and bundling domains (Aim 1) or Eps8 mutants lacking MyoXVa-, whirlin-, or actin-binding activity (Aim 2) in Eps8 KO models will be used. In addition, novel light- and chemically-inducible Eps8 mice will be employed to explore cochlear plasticity and define the critical window for restoring hair cell function in vivo (Aim 3A). The potential for partial reprogramming to expand or restore the critical window for rescuing hair cell function (Aim 3B) will be investigated. Thus, by combining advanced genetic tools, high-resolution imaging, and hearing assays, the basic cell biological mechanisms of stereocilia length regulation will be elucidated and innovative strategies for restoring hearing will be developed.