Cellular and Molecular Mechanisms of Cochlear Innervation

项目总结 听力功能依赖于螺旋神经节神经元(SGN)、橄榄耳蜗区的精确连接模式 传出纤维和耳蜗毛细胞。这些连接的缺陷是听力障碍和 人工耳蜗术的疗效。这项研究的长期目标是确定负责 耳蜗神经元和毛细胞之间的连接事件，因此改进的治疗方法可以被开发出来 治疗人类的听力损失。Pou3f4是一种由耳间充质细胞表达的转录因子。 耳蜗和Pou3f4基因突变会导致人类听力损失。Pou3f4在小鼠模型中的丢失导致 耳间充质和这些模型的形态缺陷是已知的听力损伤的结果 耳蜗内电位降低。我们最近发现Pou3f4在SGN轴突引导中起关键作用。但是，我们 对Pou3f4如何控制听觉神经机制的了解非常有限，因为 Pou3f4在耳蜗中的转录靶点还不是很清楚。这项提案旨在确定 Pou3f4在轴突引导、转录调控和听觉系统神经元存活中的作用。 肾上腺素是细胞表面结合的配体，可激活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在内的细胞间通讯。我们的初步数据表明 Pou3f4调节耳间充质中Ephin基因的表达，这些基因在毛细胞中是关键的 接线。这项拟议工作的结果将展示这些埃弗林蛋白是如何控制耳蜗的。 在Pou3f4突变体中观察到神经支配和贡献神经支配缺陷。我们建议的工作成果 还将生成一组全面的Pou3f4转录靶点，其中将包括所有可能的因素 参与了耳蜗神经的支配。我们还发现耳廓间充质细胞表达Pou3f4到 成年后，Pou3f4缺失的成年人表现出明显的SGN丢失。因此，在这些研究中，我们还将 确定Pou3f4正常情况下介导SGN存活的机制(S)。在这项工作中，我们将使用 一系列体内和体外技术、创新的成像方法和转录图谱方法。 这项研究的贡献将是重大的，因为它将确定Pou3f4及其目标 有助于发展和维持复杂的传入神经支配模式 哺乳动物的听觉系统。此外，这项工作预计将确定新的指导机制 适当的听觉连接所需的，因此它将补充其他人正在进行的神经营养因子方面的工作， 基因疗法，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.