Cellular and Molecular Mechanisms of Cochlear Innervation

项目摘要 耳蜗内螺旋神经节神经元（SGN）与毛细胞之间的突触连接至关重要 听力障碍和各种形式的听力损失。这些突触在听力开始之前形成， 通过支持细胞诱导的内在产生的活动激活，该活动在发育中的 耳蜗这种周期性和自发的突触活动启动SGN爆发放电，促进SGN存活， SGN成熟，以及中枢听觉回路频率调谐的发展。我们之前的研究显示， 耳间充质细胞（OMC）在建立适当SGN-毛细胞连接中的意外作用 通过POU 3F 4的激活，POU 3F 4是一种与X连锁耳聋相关的转录因子。POU 3F 4表达 只有耳蜗中的OMC才能做到。我们发现，在邻近SGN发育的OMC中，POU 3F 4上调Eph 受体-A4（EphA 4）促进SGN成束。随后，我们发现POU 3F 4也是 SGN生存所必需的。Ca 2+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发展的模型 通过POU 3F 4、胰岛素样生长因子（IGF）和脑信号蛋白（SEMA）信号传导。在这里，我们将测试 假设OMC表达这些因子既促进听前自发活动， 毛细胞-SGN突触连接的建立，使听力。这一假设将在三个 目标。在目标1中，我们将确定POU 3F 4和OMCs在产生听前自发活动中的作用。 在目的2中，我们将定义SEMA 5A抑制SGN自发活动的机制。在目标3中，我们 确定POU 3F 4如何促进IHC神经支配。这些研究将纳入一系列钙离子成像， 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。声过度暴露后，SGN 细胞体可以存活很长一段时间，但它们的外周突起会从毛细胞处缩回 而不容易重新连接。目前，如何重新建立这些联系还没有很好的理解。成功 这些目标的完成将确定OMC表达的基因如何控制第一个突触的形成 在听觉通路中。通过了解发育过程中耳蜗神经支配的机制， 开始建立一个“工具箱”，可以用来开发分子疗法，重新连接受损的成人 耳蜗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回路形成的一个关键方面， 活动可能是耳蜗重新布线中的重要考虑因素。

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.