Regeneration of stereocilia of cochlear hair cells

描述(申请人提供)：听觉和平衡的感觉依赖于毛细胞，内耳的感觉感受器。毛细胞将机械刺激转化为电活动。非先天性听力和平衡障碍的主要原因是毛细胞老化或接触噪声和耳毒性药物导致毛细胞丢失或传导装置受损，毛细胞表面的立体纤毛束。过去二十年的研究表明，在鸟类和低等脊椎动物中，丢失的毛细胞可以通过产生新的毛细胞来替代。哺乳动物的毛细胞再生是有限的，但有证据表明，受损的毛细胞可能会存活并进行细胞内修复。在这项提案中，我们将确定哺乳动物耳蜗毛细胞在失去立体纤毛后是否还能存活。如果毛细胞可以存活一段时间，这一机会之窗对于修复发束的遗传或化学治疗干预至关重要。因此，我们将探索利用基因治疗方法再生立体纤毛的可行性。我们对这项探索性/发展性研究有两个具体目标。在目标1中，我们将研究哺乳动物毛细胞在其立体纤毛被故意移除后的命运，以确定毛细胞是否能够存活和发育。在目标2中，我们将确定参与立体纤毛形态发生的两个基因-肌球蛋白-15a和Whirlin的过度表达是否可以促进毛束修复/再生，以及新再生的立体纤毛是否具有功能。实验将在新生沙土鼠的Corti培养器官中进行。位于科尔蒂器官指定区域的外毛细胞发束将通过一个小吸管被移除。毛细胞的存活/发育和立体纤毛的形态/功能将使用免疫组织化学(共聚焦显微镜)、时间推移成像、扫描电子显微镜和电生理学进行检测。该项目具有非常重要的意义，因为探索维持、拯救和修复受损毛细胞的策略对于毛细胞相关性听力损失和耳聋的生物修复和预防至关重要。公共卫生相关性：由于大多数美国听力损失患者(总计约3000万人)存在某种毛细胞损伤，了解损伤修复的细胞过程，探索抢救和修复受损毛细胞的可能性，对于毛细胞相关的听力损失和耳聋的生物修复和预防至关重要。

DESCRIPTION (provided by applicant): 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. Loss of hair cells or damage to the transduction apparatus, the stereocilium bundle on the surface of the hair cell, as a result of aging or exposure to noise and ototoxic drugs is the major cause of noncongenital hearing and balance deficits. Studies in the last two decades have demonstrated that lost hair cells can be replaced by production of new hair cells in birds and lower vertebrates. Hair cell regeneration is limited in mammals, but there is evidence to suggest that damaged hair cells may survive and undergo intracellular repair. In this proposal, we will determine whether mammalian cochlear hair cells could survive after loss of stereocilia. If the hair cells can survive for some time, this window of opportunity can be crucial for genetic or chemical therapeutic interventions in order to repair hair bundles. Thus, we will explore the feasibility of regenerating stereocilia using a gene therapy approach. We have two specific aims for this Exploratory/Developmental research. In Aim 1, we will examine the fate of mammalian hair cells after their stereocilia have been intentionally removed to determine whether hair cells can survive and develop. In Aim 2, we will determine whether over-expression of myosin-15a and whirlin, two genes that are involved in stereocilium morphogenesis, can promote hair-bundle repair/regeneration and whether the newly regenerated stereocilia are functional. The experiments will be carried out in the cultured organ of Corti from neonatal gerbils. The hair bundles of outer hair cells in a designated area of the organ of Corti will be removed by a small suction pipette. Hair-cell survival/development and stereocilium morphology/function will be examined using immunohistochemistry (with confocal microscopy), time-lapse imaging, scanning electron microscopy, and electrophysiology. This project is highly significant, since exploring the strategies of maintaining, rescuing, and repairing injured hair cells are essential to the biological remediation and prevention of hair cell-related hearing loss and deafness. PUBLIC HEALTH RELEVANCE: Because the majority of Americans with hearing loss, some 30 million in all, have some kind of hair cell damage, understanding the cellular process of damage repair and exploring the possibilities of rescuing and repairing injured hair cells are essential to the biological remediation and prevention of hair cell-related hearing loss and deafness.