Hippo-mediated control of growth and regeneration in the inner ear sensory organs

项目概要 非哺乳动物脊椎动物（例如鱼类）恢复感觉毛细胞的主要方式是通过 剩余支持细胞群的增殖和分化。相反，支持细胞失去 哺乳动物出生后增殖的能力，以及防止细胞周期重新进入的分子机制 仍然知之甚少。 我们的工作已经证实，Hippo 信号传导是一种主要的抑制机制，可以阻止 支持哺乳动物内耳的细胞增殖和可塑性。在三个目标中，我们将确定分子 Hippo抑制促进成年椭圆囊外植体有丝分裂感觉受体再生的机制 （目标 1）；评估 Hippo 信号的可逆药理失活是否会刺激真正的前庭 毛细胞再生以支持体内功能恢复（目标 2）；并评估该途径的相互作用 细胞周期抑制剂 p27Kip1，对成人内耳体内的柯蒂氏器具有特异性（目标 3）。长期来看 该提案的目标是通过控制来确定听力和平衡恢复的治疗策略 Hippo 通路的操纵。 由于其相对较新的发现，对内耳 Hippo 通路的研究本身就是创新的。 此外，我们的团队开创了这一领域并开发了几种专门的研究工具来帮助 研究内耳通路。最值得注意的是，我们发现了第一个 Lats 激酶小分子抑制剂 – Hippo 信号传导中的核心酶 – 我们证明它可以有效诱导支持性细胞增殖和 体外和体内毛细胞再生的初始阶段。我们还优化了后半规管入路 将 LKI 传递到内耳并利用多种尖端遗传和表观遗传技术（例如 多组测序、CUT&RUN）。 所提出的基础研究意义重大，因为了解阻断细胞的分子机制 内耳的循环再进入可能确定诱导毛细胞再生的新治疗靶点。 值得注意的是，我们证明了对 Lats 激酶的短暂药理学抑制会诱导支持细胞 成体椭圆囊增殖，使后代重新退出细胞周期并自发上调感觉 停药后的受体基因。总的来说，我们的数据表明 Hippo 信号传导的暂时失活是 足以通过支持细胞分裂促进毛细胞再生的初始阶段——一个过程思想 在成年哺乳动物内耳中被永久抑制。

Project Summary The main way in which non-mammalian vertebrates, such as fish, restore sensory hair cells is through proliferation and differentiation of the residual population of supporting cells. In contrast, supporting cells lose the capacity to proliferate postnatally in mammals, and the molecular machinery preventing cell cycle reentry remains poorly understood. Our work has established that Hippo signaling serves as a major repressive mechanism that blocks supporting cell proliferation and plasticity in the mammalian inner ear. In three Aims, we will identify the molecular mechanism by which Hippo inhibition promotes mitotic sensory receptor regeneration in the adult utricle explants (Aim 1); assess whether reversible pharmacologic inactivation of Hippo signaling stimulates bona fide vestibular hair cell regeneration to support functional recovery in vivo (Aim 2); and assess the pathway’s interaction with the cell cycle inhibitor p27Kip1, specific to the organ of Corti, in the adult inner ear in vivo (Aim 3). The long-term goal of this proposal is to identify therapeutic strategies for hearing and balance restoration through controlled manipulation of the Hippo pathway. Due to its relatively recent discovery, study of the Hippo pathway in the inner ear is innovative in itself. Furthermore, our group has pioneered this field and developed several specialized research tools to aid the study of the pathway in the inner ear. Most notably, we identified the first small-molecule inhibitor of Lats kinases – the core enzymes in Hippo signaling – that we show to potently induce supporting cell proliferation and the initial stages of hair cell regeneration in vitro and in vivo. We also optimized posterior semicircular canal approach for LKI delivery into the inner ear and utilize several cutting-edge genetic and epigenetic techniques (e.g multiome sequencing, CUT&RUN). The proposed basic research is significant because understanding the molecular machinery blocking cell cycle reentry in the inner ear may determine new therapeutic targets for induction of hair cell regeneration. Remarkably, we demonstrate that brief pharmacologic inhibition of Lats kinases induces supporting cell proliferation in the adult utricle, allowing progeny to re-exit the cell cycle and spontaneously upregulate sensory receptor genes upon drug withdrawal. Collectively our data show that temporal inactivation of Hippo signaling is sufficient to promote the initial stages of hair cell regeneration through supporting cell division – a process thought to be permanently suppressed in the adult mammalian inner ear.