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

项目摘要 非哺乳类脊椎动物，如鱼类，恢复感觉毛细胞的主要途径是通过 支持细胞的剩余群体的增殖和分化。相反，支持细胞失去了 哺乳动物出生后增殖的能力，以及阻止细胞周期重新进入的分子机制 仍然知之甚少。 我们的工作已经确定，河马信号作为一个主要的抑制机制，阻止 支持哺乳动物内耳中的细胞增殖和可塑性。在三个目标中，我们将识别分子 Hippo抑制促进成年椭圆果外植体中有丝分裂感觉受体再生的机制 (Aim 1）;评估Hippo信号传导的可逆药理学失活是否刺激真正的前庭神经系统。 毛细胞再生以支持体内功能恢复（目标2）;并评估该途径与 细胞周期抑制剂p27 Kip 1，特异性Corti器官，在成年内耳体内（目的3）。长期 该提案的目标是确定通过控制听力和平衡恢复的治疗策略， 操纵河马通路 由于其相对较新的发现，对内耳中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，以帮助 内耳通路的研究。最值得注意的是，我们发现了第一个Lats激酶的小分子抑制剂， - Hippo信号传导中的核心酶--我们证明它能有效地诱导支持细胞增殖， 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 并利用几种尖端的遗传和表观遗传技术（例如 多基因组测序，CUT&RUN）。 所提出的基础研究是有意义的，因为了解分子机制阻止细胞 内耳中的周期折返可能会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Lats激酶的短暂药理学抑制诱导支持细胞 在成年胞果中增殖，允许后代重新退出细胞周期并自发上调感觉神经元的表达。 药物戒断后的受体基因。总的来说，我们的数据表明，海马信号的暂时失活是 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.