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

摘要 感音神经性耳聋是一种无法治愈的衰弱疾病，直接影响3000万人。一个 遗传性听力损失的常见原因是耳蜗毛细胞立体纤毛的破坏，这是必不可少的 用于将声音振动传递到大脑。为了发挥正常的功能，立体纤毛必须足够高，才能达到 覆盖膜(TM)并形成立体纤毛-TM连接(STJ)。立体纤毛的伸长在很大程度上取决于 肌动蛋白调节；因此，肌动蛋白调节蛋白和其他细胞骨架元件的破坏导致 人类和模型生物的严重听力损失。因此，阐明这一动态调控机制至关重要 以了解正常听力并确定如何挽救与立体纤毛相关的听力 损失。以往的工作表明，立体纤毛肌动蛋白细丝的伸长依赖于表皮生长因子 途径底物8(Eps8)，一种肌动蛋白调节蛋白，对小鼠和人类的听力都至关重要。 Eps8矛盾地包含同时具有肌动蛋白细丝封顶和捆绑活性的结构域，这些结构域是 通常分别与肌动蛋白细丝的缩短或延长有关。此外，还发现 另外两种与耳聋相关的蛋白质对立体纤毛的伸长至关重要，它们是肌球蛋白-XVA(MyoXVa)和Whirlin， 在立体纤毛顶端以三元络合物的形式与Eps8结合。初步数据显示，另一种与耳聋相关的蛋白质--立体纤毛与STJ的TM连接，在EPS8中没有正确地针对立体纤毛尖端， MyoXVa，或Whirlin基因敲除(KO)小鼠，所有这些小鼠都缺乏正常的STJ。此外，出生后早期(≤ 第1天)腺相关病毒(AAV)介导的Eps8基因可以挽救立体纤毛的延长，立体定向 在Eps8 KO小鼠根尖毛细胞中的定位和STJ，表明它可能挽救听力 功能。这一建议验证了EPS8调节立体纤毛伸长和正确形成的假说 通过肌动蛋白C端封端和束状区直接调节肌动蛋白束生长 通过与MyoXVa和Whirlin的相互作用间接调节立体纤毛的生长。更进一步说，它是 提出毛细胞成熟存在一个关键的时间段，在这个时间段内，体纤毛的可塑性足以满足 全力救援。为了验证这些假设，AAV介导的Eps8突变体缺乏一个或两个都缺乏 封端和捆绑结构域(目标1)或缺乏MyoXVa、Whirlin或肌动蛋白结合活性的Eps8突变体 (AIM 2)在EPS8中将使用KO模型。此外，新型的光和化学诱导的Eps8小鼠将被 用于探讨耳蜗可塑性和确定体内毛细胞功能恢复的关键窗口(目的 3A)。部分重新编程以扩大或恢复拯救毛细胞的关键窗口的可能性 将对功能(目标3B)进行调查。因此，通过结合先进的基因工具，高分辨率成像， 和听力测试，将阐明立体纤毛长度调节的基本细胞生物学机制和 将制定恢复听力的创新战略。

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.