Inducing cochlear sensory cell differentiation

 描述（由申请人提供）：不可修复的神经变性是许多神经系统疾病的特征，并导致神经系统功能随年龄增长而下降。因此，揭示神经元替代的机制是许多临床表现的治疗的核心挑战。由于神经元、神经胶质及其连接的多样性和复杂性，这一挑战在CNS中是艰巨的。因此，我们正在研究一个更简单的模型：听觉系统的神经元样感觉毛细胞（HC），它由神经胶质样细胞支持，并通过听觉神经元将声音刺激传递到大脑。HC的遗传和环境损伤导致感觉神经性听力损失（SNHL），这是一种常见的疾病，需要大量的医疗费用。无论起始原因如何，在大多数SNHL病例中，HC死亡，而支持细胞（SC）和听觉神经元可以持续存在。然而，由于哺乳动物耳蜗没有再生能力，HC的丧失导致永久性SNHL，其通过目前的疗法得到改善，但不能治愈。Notch信号的抑制已经成为诱导新的HC从残留的SC在remaened动物的手段，但在小鼠中，它是低效的胚胎阶段后，当HC正常出现，表明额外的发育信号的调节可能是至关重要的HC再生和重新布线。HC和SC发生所需的关键信号之一是成纤维细胞生长因子（FGF）家族的成员，它们激活不同FGF受体的能力不同。FGF 20/FGFR 1信号传导促进外毛细胞（OHC）的发育，外毛细胞（OHC）放大由内毛细胞（IHC）转导的声音刺激; FGF 8/FGFR 3信号传导促进柱细胞（PC）的分化，柱细胞是将IHC与OHC分开的SC。奇怪的是，尽管FGFR 3信号传导的遗传丢失和获得对导致SNHL的PC分化具有相反的作用，但这两种变化都诱导由异位Deiters细胞（DC）支持的异位OHC。Fgfr 3-/-小鼠在妊娠晚期显示异位OHC和DC。我们对小鼠FGFR 3功能获得性突变与配体结合特异性改变的初步研究表明，异位OHC和DC在出生后出现。此外，它们的发生取决于FGF 10，这是一种意想不到的配体，它改变RAS/MAPK靶基因并诱导DC到PC的命运转化和可塑性。在这个建议中，我们测试的假设，FGF/RAS/MAPK信号足以诱导OHC分化的DC祖细胞胚胎和出生后，包括在体内模型的OHC损失。我们将1）确定Fgfr 3-/-小鼠中异位功能获得机制是否驱动异位OHC和DC; 2）确定FGFR 3功能丧失和获得突变体中异位OHC的谱系;和3）确定正常和OHC缺失耳蜗DC中RAS/MAPK信号传导的强制激活是否会诱导OHC。我们的研究结果将推动未来的研究，解决利用FGF通路与其他信号一起诱导受损耳蜗中强大的哺乳动物听觉HC再生和听力恢复的长期目标，并将为操纵CNS胶质细胞分化为神经元的努力提供信息。

 DESCRIPTION (provided by applicant): Irreparable neurodegeneration characterizes many neurologic diseases and contributes to the decline in nervous system function with age. Thus, uncovering mechanisms for neuronal replacement is a central challenge of therapeutics for many clinical presentations. This challenge is formidable in the CNS because of the variety and complexity of neurons, glia and their connections. Therefore, we are investigating a simpler model: the neuron-like sensory hair cells (HCs) of the auditory system, which are supported by glia-like cells and relay sound stimuli through auditory neurons to the brain. Genetic and environmental damage to HCs cause sensorineural hearing loss (SNHL), a common condition with significant healthcare costs. Regardless of initiating cause, in most cases of SNHL, the HCs die, whereas supporting cells (SCs) and auditory neurons can persist. However, as mammalian cochleae have no regenerative capacity, loss of HCs leads to permanent SNHL that is ameliorated, but not cured, by current therapies. Inhibition of Notch signaling has emerged as a means of inducing new HCs from residual SCs in deafened animals, but in mice it is inefficient after the embryonic stages when HCs normally arise, suggesting that regulation of additional developmental signals may be critical to HC regeneration and rewiring. Among the critical signals required for genesis of both HCs and SCs are members of the Fibroblast Growth Factor (FGF) family, which differ in their ability to activate different FGF receptors. FGF20/FGFR1 signaling promotes development of outer hair cells (OHCs), which amplify sound stimuli transduced by inner hair cells (IHCs); and FGF8/FGFR3 signaling promotes differentiation of pillar cells (PCs), SCs that separate IHCs from OHCs. Curiously, although genetic loss and gain of FGFR3 signaling have opposing effects on PC differentiation that cause SNHL, both changes induce ectopic OHCs supported by ectopic Deiters' cells (DCs). Fgfr3-/- mice show ectopic OHCs and DCs by late gestation. Our preliminary studies of a mouse FGFR3 gain-of-function mutation with altered ligand-binding specificity show that ectopic OHCs and DCs arise postnatally. Furthermore, their genesis depends on FGF10, the same unexpected ligand that alters RAS/MAPK target genes and induces the DC-to-PC fate transformation and plasticity we described previously. In this proposal we test the hypothesis that FGF/RAS/MAPK signaling is sufficient to induce OHC differentiation from DC progenitors both embryonically and postnatally, including in an in vivo model of OHC loss. We will 1) determine whether an ectopic gain-of-function mechanism drives ectopic OHCs and DCs in Fgfr3-/- mice; 2) determine the lineage of ectopic OHCs in FGFR3 loss- and gain-of-function mutants; and 3) determine whether forced activation of RAS/MAPK signaling in DCs of normal and OHC-depleted cochleae will induce OHCs. Our results will drive future studies addressing the long-term goal of harnessing the FGF pathway together with other signals to induce robust mammalian auditory HC regeneration and hearing restoration in damaged cochleae, and will inform efforts to manipulate CNS glia to differentiate as neurons.