Inducing cochlear sensory cell differentiation

 描述(申请人提供)：不可修复的神经退行性变是许多神经系统疾病的特征，并导致神经系统功能随年龄下降。因此，揭示神经元替代的机制是许多临床表现的治疗学的中心挑战。由于神经元、神经胶质细胞及其连接的多样性和复杂性，这一挑战在中枢神经系统是令人敬畏的。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(HCS)，它由胶质样细胞支持，通过听神经元将声音刺激传递到大脑。HCS的遗传和环境损害会导致感音神经性听力损失(SNHL)，这是一种常见的疾病，具有巨大的医疗成本。无论起病原因如何，在大多数SNHL病例中，HCs死亡，而支持细胞(SCs)和听神经元可以存活。然而，由于哺乳动物耳蜗没有再生能力，失去HCS会导致永久性的SNHL，目前的治疗方法可以改善这种情况，但并不能治愈。抑制Notch信号已成为从耳聋动物残留的干细胞中诱导新的HC的一种手段，但在小鼠胚胎阶段之后，当HC正常出现时，抑制Notch信号的作用是无效的，这表明对额外发育信号的调节可能对HC的再生和重新连接至关重要。成纤维细胞生长因子(成纤维细胞生长因子)家族的成员是HCS和SCs发生所需的关键信号之一，它们激活不同的成纤维细胞生长因子受体的能力不同。FGF20/FGFR1信号促进外毛细胞的发育，外毛细胞放大内毛细胞传递的声音刺激；FGF8/FGFR3信号促进柱状细胞的分化，柱状细胞是将内毛细胞与内毛细胞分开的干细胞。奇怪的是，尽管FGFR3信号的遗传缺失和获得在导致SNHL的PC分化中具有相反的作用，但这两种变化都可以诱导由异位Deiters细胞(DC)支持的异位OHC。FGFR3-/-小鼠在妊娠晚期出现异位的内分泌细胞和树突状细胞。我们对具有配体结合特异性改变的小鼠FGFR3功能获得突变的初步研究表明，异位的毛细胞和树突状细胞是在出生后产生的。此外，它们的起源依赖于FGF10，同样是一个意想不到的配体，改变了RAS/MAPK的靶基因，并诱导了我们前面描述的DC到PC的命运转换和可塑性。在这项研究中，我们验证了一种假设，即成纤维细胞生长因子/RAS/MAPK信号足以诱导DC祖细胞在胚胎和出生后分化为OHC，包括在体内OHC缺失的模型中。我们将1)确定异位功能获得机制是否驱动FGFR3-/-小鼠的异位OHC和DC；2)确定FGFR3功能缺失和功能获得突变体中异位OHC的谱系；3)确定在正常和OHC耗竭的耳蜗树突状细胞中强制激活RAS/MAPK信号是否会诱导OHC。我们的结果将推动未来的研究，以实现利用成纤维细胞生长因子途径与其他信号一起在受损的耳蜗中诱导强大的哺乳动物听觉HC再生和听力恢复的长期目标，并将为操纵中枢神经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.