Cellular and Molecular Mechanisms of Cochlear Innervation

项目总结 螺旋神经节神经元(SGN)和耳蜗毛细胞之间的突触联系是至关重要的 听力障碍，并在多种形式的听力障碍中丧失。这些突触在听力发作之前形成，并且 由支持细胞诱导的、内在产生的活动激活，这在发育中是突出的 耳蜗骨。这种周期性和自发的突触活动启动SGN突发放电，从而促进SGN存活， SGN的成熟和中枢听觉回路频率调谐的发展。我们之前的研究揭示了 耳间充质细胞(OMC)在建立适当的SGN-毛细胞连接中的意外作用 通过激活与X连锁耳聋相关的转录因子POU3F4。表达了POU3F4 只有耳蜗中的OMCs。我们发现，在发育中的SGN附近的OMC中，POU3F4上调Eph 受体-A4(EphA4)促进SGN颤动。随后，我们发现POU3F4也是 是SGN生存所必需的。钙离子成像和单细胞RNA测序(ScRNAseq)的最新数据 实验支持OMCs通过调节自发活动促进SGN发育的模型 通过POU3F4、胰岛素样生长因子(IGF)和信号素(SEMA)信号转导。在这里，我们将测试 假设OMCs表达这些因子既能促进听前自发活动，又能促进 建立毛细胞-SGN突触连接，以使听力。这一假设将在三年内得到检验 目标。在目标1中，我们将确定POU3F4和OMCs在产生听前自发活动中的作用。 在目标2中，我们将确定SEMA5A抑制SGN自发活动的机制。在《目标3》中，我们将 确定POU3F4如何促进IHC神经支配。这些研究将包括一系列的钙离子成像， 生理学、分子图谱和组织培养技术。 我们和其他人已经记录了SGN指导和生存的机制，但仍然有限 了解SGN如何分化并与毛细胞形成突触。声学过度暴露后，SGN 细胞体可以存活很长一段时间，但它们的外围突起远离毛细胞。 而不会轻易地重新连接。目前，如何重新建立这些联系还没有得到很好的理解。成功 这些目标的完成将定义OMC表达的基因如何控制第一批突触的形成 在听觉通路中。通过了解耳蜗神经在发育过程中的机制，我们将 开始建造一个“工具箱”，可以用来开发重新连接受损成人的分子疗法。 耳蜗骨。这项研究还将揭示耳蜗发育和调节所需的关键机制 自发活动。由于神经活动被认为是电路形成的关键方面，因此有可能 活动可能是耳蜗线重排中的一个重要考虑因素。

PROJECT SUMMARY Synaptic connections between spiral ganglion neurons (SGNs) and hair cells in the cochlea are critical for hearing and lost in many forms of hearing impairment. These synapses form prior to hearing onset and are activated by supporting cell-induced, intrinsically generated activity that is prominent within the developing cochlea. This periodic and spontaneous synaptic activity initiates SGN burst firing, which promotes SGN survival, SGN maturation, and development of frequency tuning in central auditory circuits. Our prior studies revealed an unexpected role for otic mesenchyme cells (OMCs) in establishing appropriate SGN-hair cell connectivity through activation of POU3F4, a transcription factor associated with X-linked deafness. POU3F4 is expressed only by OMCs in the cochlea. We found that, in OMCs adjacent to developing SGNs, POU3F4 upregulates Eph receptor-A4 (EphA4) to promote SGN fasciculation. Subsequently, we discovered that POU3F4 is also necessary for SGN survival. Recent data from Ca2+ imaging and single cell RNA sequencing (scRNAseq) experiments support a model whereby OMCs promote SGN development by regulating spontaneous activity through POU3F4, insulin-like growth factor (IGF) and Semaphorin (SEMA) signaling. Here, we will test the hypothesis that expression of these factors by OMCs promotes both prehearing spontaneous activity and the establishment of hair cell–SGN synaptic connections to enable hearing. This hypothesis will be tested in three aims. In Aim 1, we will determine the role of POU3F4 and OMCs in generating prehearing spontaneous activity. In Aim 2, we will define the mechanisms by which SEMA5A inhibits SGN spontaneous activity. In Aim 3, we will determine how POU3F4 promotes IHC innervation. These studies will incorporate a range of Ca2+ imaging, physiology, molecular profiling, and tissue culture techniques. We and others have documented mechanisms of SGN guidance and survival, but there is still limited understanding of how SGNs differentiate and form synapses with hair cells. After acoustic overexposure, SGN cell bodies can survive for long periods of time, but their peripheral processes retract away from the hair cells without easily reconnecting. At present, how to re-establish these connections is not well understood. Successful completion of these aims will define how genes expressed by OMCs control the formation of the first synapses in the auditory pathway. By understanding the mechanisms of cochlear innervation during development, we will begin to build a “toolbox” that could be used to develop molecular therapies for rewiring the damaged adult cochlea. This research will also reveal key mechanisms required for the development and regulation of cochlear spontaneous activity. Since neural activity is recognized as a crucial aspect of circuit formation, it is possible that activity could be an important consideration in cochlear rewiring.