抑制Notch信号通路对耳蜗毛细胞纤毛再生修复作用的研究

The senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Hair cells transduce mechanical stimuli into electrical activity. The site of mechanotransduction is the hair bundle, an array of stereocilia with different height connected by side- and tip-links. The stereocilia bundle and tip links are susceptible to acoustic trauma and ototoxic drugs. It has been shown that the damaged hair cells can undergo spontaneous repair in chick and frog hair cells. In organ cultures of the bullfrog saccule, hair cells that lost their hair bundles as a result of sublethal antibiotics treatment survived as bundleless cells for at least one week. New hair bundles emerge between 4 and 7 days in the absence of mitosis. Utriclar hair cells from neonatal rodents can also survive gentamicin insult and regenerate the lost stereocilia in culture. Cochlear hair cells, however, no longer retain that capability despite of the fact that the bundleless hair cells can survive up to 12 days in culture. The long-term goal of my laboratory is to explore strategies that can regenerate lost stereocilia in cochlear hair cells. The Notch signaling pathway, an evolutionary conserved molecular mechanism involved in the determination of a variety of cell fates, plays multiple roles during vertebrate inner ear morphogenesis. Notch signaling first specifies prosensory progenitors through lateral induction and subsequently directs progenitors to further differentiate into supporting cells by preventing supporting cells from adopting hair cell fate through lateral inhibition. Our recent study showed that pharmacological inhibition of the canonical Notch pathway in the cultured organ of Corti induced generation of stereocilia bundles in supporting cells and supernumerary stereocilia in hair cells. The newly emerged stereocilia bundles were able to respond to mechanical stimulation with mechanotransduction current, suggesting that suppression of Notch signaling is sufficient for formation of functional stereocilia bundles. In the proposed study, we will test the hypothesis that inhibition of Notch signaling can lead to regeneration of lost stereocilia in cochlear hair cells. We have two specific aims. In Aim 1, we will determine whether blocking Notch signaling by application of LY-411575 (a γ-secretase inhibitor) will promote stereocilia regeneration in cochlear hair cells in the tissue culture of the organ of Corti from neonatal mice where stereocilia bundles were removed by the suction pipette technique. We will also examine the molecular mechanisms of stereocilia regeneration by examining changes in gene expression of hair cells at different time points during regeneration using RNA-seq-based transcriptome analysis of hair cells during bundle regeneration. Hair bundle regeneration will be examined using confocal and scanning electron microscopy, whereas the function of the regenerated bundles will be examined by measuring mechanotransduction current using the whole-cell voltage-clamp technique. In Aim 2, we will explore whether application of LY-411575 will lead to regeneration and repair of lost and damaged stereocilia bundles and restore hearing in adult mice after exposed to impulsive noise. Hair bundle morphology will be examined using confocal and electron microscopy while auditory function will be evaluated by measuring auditory brainstem response and cochlear microphonic. The proposed research is highly significant since hair cell and hearing loss usually starts with loss and damage of stereocilia bundles after exposure to impulsive noise or low dosage of ototoxic drugs. Therefore, repair and regeneration of stereocilia bundles will be critical for preventing subsequent hair cell loss and for restoring hearing. This study is also expected to provide mechanistic understanding of the genes and pathways underlying stereocilia bundle generation in response to Notch inhibition.

耳蜗毛细胞损伤可导致永久性听力损失，细胞顶端静纤毛束的损伤、脱落是耳蜗毛细胞受损后最常见表现。纤毛受损后，非哺乳动物的毛细胞可自发再生修复，哺乳动物前庭毛细胞也具有一定自发修复能力，哺乳动物的耳蜗毛细胞却丧失自发再生修复功能，对此目前尚无有效干预办法。已有实验证实，纤毛损伤后耳蜗毛细胞仍可存活十天以上，这个时间窗是挽救性治疗听力的关键。Notch信号在内耳细胞发育分化中起到重要作用，我们前期工作已证实，在培养组织中抑制Notch信号，不仅使支持细胞长出纤毛束，且使耳蜗毛细胞生长出更多的静纤毛，这些纤毛同样具有机电转换功能。因此拟用免疫组化、扫描电镜、听力检测等方法，观察抑制Notch信号，在培养组织中能否促使耳蜗毛细胞受损纤毛出现再生修复，在实验小鼠中能否利用这种修复机制挽救其受损听力。如果实验结果肯定，将为挽救性治疗各种原因所致耳蜗毛细胞早期损伤提供重要的依据和思路，具有重要的临床意义。

耳蜗毛细胞损伤可导致永久性听力损失，细胞顶端静纤毛束的损伤、脱落是耳蜗毛细胞受损后最常见表现。纤毛受损后，非哺乳动物的毛细胞可自发再生修复，哺乳动物前庭毛细胞也具有一定自发修复能力，哺乳动物的耳蜗毛细胞却丧失自发再生修复功能。Notch信号在内耳细胞发育分化中起到重要作用，我们发现，在新生小鼠培养组织中抑制Notch信号，不仅使支持细胞长出纤毛束，且使耳蜗毛细胞生长出更多的静纤毛，这些纤毛同样具有机电转换功能。在本课题中，我们建立了新生小鼠内耳基底膜培养组织的毛细胞纤毛机械损伤模型，并观察使用LY-411575抑制Notch信号后促进损伤纤毛再生修复的效果，明确了在培养组织中，抑制Notch信号后可以使纤毛受损的毛细胞有新生纤毛出现。随后，我们建立了耳毒性药物毛细胞损伤小鼠模型，并观察半规管给药抑制Notch信号对腹腔注射庆大霉素导致的内耳毛细胞损伤的影响，确定抑制notch信号对耳毒性药物（庆大霉素）造成的毛细胞损伤的保护作用。（相关文章已发表：Yi Li, et al. Frontiers in Cellular Neuroscience., 2020; Tianying Wang, et al. Frontiers in Neurology ,2022）同时我们观察分析了衰老小鼠的耳蜗内外毛细胞的细胞学及其纤毛、细胞电生理及基因表达的改变，获得了在衰老过程中明显差异性表达的基因的数据库。(相关文章已发表：Huizhan Liu, et al. Cell Reports.,2022) 并对目标基因Slc7a14建立全敲除小鼠模型以及单个氨基酸突变的Knock-in小鼠模型，在此模型基础上采用形态学、分子生物学、共聚焦显微镜、免疫组化等一系列实验方法验证此基因在细胞内的表达部位、功能及其突变对听功能及其他感觉系统的影响。(相关文章已发表：Kimberlee P. Giffen, et al. Science Advances, 2022) 本实验结果可为挽救性治疗各种原因所致耳蜗毛细胞早期损伤提供重要的依据和思路，具有重要的临床意义。