Cellular and Molecular Mechanisms of Cochlear Innervation

项目摘要 听觉功能依赖于螺旋神经节神经元（SGN）的精确连接模式，橄榄耳蜗 传出神经和耳蜗中的毛细胞。这些连接的缺陷是听力障碍的基础， 人工耳蜗的功效这项研究的长期目标是确定负责 神经元和耳蜗毛细胞之间的连接事件，以便开发改进的疗法， 治疗人类听力损失。Pou3f4是一种转录因子，由耳中的间充质细胞表达。 耳蜗和Pou3f4突变导致人类听力损失。小鼠模型中Pou3f4的缺失导致 耳间充质中的形态缺陷，并且已知这些模型由于 耳蜗内电位降低。我们最近发现Pou3f4在SGN轴突引导中至关重要。但我们 我对Pou3f4如何控制听觉神经支配机制的理解非常有限， Pou3f4在耳蜗中的转录靶点还不清楚。本建议旨在确定 Pou3f4在听觉系统中的轴突引导、转录调节和神经元存活中的功能。 肝配蛋白是细胞表面结合的配体，其激活Ephs（受体酪氨酸激酶）以促进多种形式 包括轴突引导和突触发生。我们的初步数据显示， Pou3f4调节耳间充质中Ephrin基因的表达，这些基因在毛细胞中至关重要 接线.从拟议的工作结果将证明这些肝配蛋白如何控制耳蜗 在Pou3f4突变体中观察到神经支配和促成神经支配缺陷。我们拟议工作的结果 还将产生一套全面的Pou3f4转录靶点，其中包括所有可能的因子 参与耳蜗神经支配。我们还发现，耳间充质细胞表达Pou3f4， 成年期和Pou3f4无效的成年人显示出显著的SGN损失。因此，在这些研究中，我们也将 确定Pou3f4正常介导SGN存活的机制。在这项工作中，我们将使用一个 一系列体内和体外技术、创新的成像方法和转录谱分析方法。 这项研究的贡献将是重大的，因为它将确定如何Pou3f4和它的目标， 有助于发展和维持复杂的传入神经支配模式内的 哺乳动物的听觉系统此外，这项工作还有望确定新的指导机制 因此，它将补充其他人正在进行的关于神经营养素的工作， 基因治疗或细胞替代策略。

PROJECT SUMMARY Hearing function depends on precise connectivity patterns of spiral ganglion neurons (SGNs), olivocochlear efferents, and hair cells in the cochlea. Deficits in these connections underlie hearing impairment and the efficacy of cochlear implants. The long-term goal of this research is to define the mechanisms responsible for wiring events between neurons and hair cells in the cochlea, so that improved therapies can be developed to treat hearing loss in humans. Pou3f4 is a transcription factor expressed by otic mesenchyme cells in the cochlea and mutations in Pou3f4 cause human hearing loss. Loss of Pou3f4 in mouse models leads to morphological defects in otic mesenchyme and these models are known to be hearing impaired as a result of reduced endocochlear potential. We showed recently that Pou3f4 is critical in SGN axon guidance. But, we have a very limited understanding of how Pou3f4 controls auditory innervation mechanisms because the transcriptional targets of Pou3f4 in the cochlea are not well understood. This proposal seeks to determine the function of Pou3f4 in axon guidance, transcriptional regulation, and neuronal survival in the auditory system. Ephrins are cell-surface bound ligands that activate Ephs (receptor tyrosine kinases) to facilitate diverse forms of intercellular communication including axon guidance and synaptogenesis. Our preliminary data suggest that Pou3f4 regulates the expression of Ephrin genes in otic mesenchyme and that these are critical in hair cell wiring. Results from the proposed work will demonstrate how these Ephrin proteins control cochlear innervation and contribute innervation defects observed in Pou3f4 mutants. Results from our proposed work will also generate a comprehensive set of Pou3f4 transcriptional targets, which will include all possible factors involved in cochlear innervation. We have also found that otic mesenchyme cells express Pou3f4 into adulthood and that Pou3f4 null adults show a significant loss of SGNs. Thus, in these studies, we will also determine the mechanism(s) by which Pou3f4 normally mediates SGN survival. In this work, we will use a range of in vivo and in vitro techniques, innovative imaging approaches, and transcriptional profiling methods. The contribution of this research will be significant because it will determine how Pou3f4 and its targets contribute to the development and maintenance of the complex afferent innervation patterns within the mammalian auditory system. In addition, this work is expected to determine new guidance mechanisms required for appropriate auditory connectivity, thus it will complement ongoing work by others on neurotrophins, gene therapy, or cell replacement strategies.