NEUROTROPHIN GENE REGULATION IN THE INNER EAR

内耳机械感觉毛细胞的损伤或丢失 被认为是听力和平衡障碍的主要原因。 因为毛细胞和感官之间的密切关系 支配他们的神经元，严重的继发性感觉丧失 毛细胞受损后可能会出现神经元。感官的生存 神经元是人工耳蜗术有效使用的必要条件 它们依靠这些细胞来传递来自 中枢神经听觉区域的语音处理器 系统。感觉神经元的存活也可能对 在毛细胞再生后恢复功能，据报道可以 发生在哺乳动物的前庭系统中。在哺乳动物的内耳中， 这些神经元的正常发育和存活依赖于 正确表达称为神经营养因子的生长因子。其中两个 神经营养因子、脑源性神经营养因子和 神经营养因子-3(NT-3)是由感觉细胞产生的 上皮细胞，是正常生长和生存所必需的 耳蜗区和前庭感觉神经元。证明这一点的最好证据 需求来自于有针对性地删除编码BDNF的基因 和NT-3，导致耳蜗和前庭感觉完全丧失 神经元。 这里提出的实验是为了鉴定基因启动子。 控制空间、时间和生理的序列 脑源性神经营养因子和NT-3基因在内耳中的转录调控。 然而，与引进外国DNA相关的困难 (转染)有丝分裂后细胞，如毛细胞，支持 细胞和内耳的感觉神经元到目前为止 对这种组织中基因调控机制的详细分析。 为了克服这些问题，我们已经开始应用新开发的 转基因技术在器官培养中基因表达研究中的应用 由小鼠内耳的感觉组织制成。我们建议： 1)制定可靠和一致的方法，以暂时引入 器官培养中外源DNA有序进入内耳细胞 提供分析特定启动子调控的测试系统 2)瞬时分析脑源性神经营养因子和NT-3基因启动子 为了识别需要维持的特定序列而进行的转染法 发育过程中基因表达的正常模式 成年人。脑源性神经营养因子和NT-3mRNA水平对多种生理反应 以及耳毒性休克等环境刺激。的发展。 分析内耳基因表达的新技术至关重要 为了理解这些转录机制，这是第一步 走向设计操纵潜在的表达式的方法 临床上重要的蛋白质，如神经营养因子。

Injury or loss of the mechano-sensory hair cells of the inner ear is considered to be the major cause of hearing and balance impairment. Because of the intimate relationship between hair cells and the sensory neurons that innervate them, substantial secondary loss of sensory neurons can occur following hair cell damage. Survival of the sensory neurons is a requirement for the effective use of cochlear prosthesis which depend on these cells to communicate electrical information from a speech processor to the auditory regions of the central nervous system. Sensory neuron survival is also likely to be important for restoring function following hair cell regeneration that reportedly can occur in the mammalian vestibular system. In the mammalian inner ear, the normal development and survival of these neurons is dependent on the correct expression of the growth factors known as neurotrophins. Two of the neurotrophins, the Brain Derived Neurotrophic factor (BDNF) and Neurotrophic Factor-3 (NT-3) are produced by cells of the sensory epithelium and required for the normal growth and survival of the cochlear and vestibular sensory neurons. The best evidence for this requirement comes from targeted deletion of the genes that code for BDNF and NT-3 which causes a total loss of cochlear and vestibular sensory neurons. The experiments proposed here are designed to identify the gene promoter sequences that control the spatial, temporal and physiological regulation of BDNF and NT-3 gene transcription in the inner ear. However, difficulties associated with the introduction of foreign DNA (transfection) into post-mitotic cells such ass the hair cells, support cells, and the sensory neurons of the inner ear have so far precluded a detailed analysis of the mechanisms of gene regulation in this tissue. To overcome these problems we have begun applying newly developed transfection techniques to study of gene expression in organ cultures made from the sensory tissue of the inner ear of mice. We propose to: 1) Develop reliable and consistent means to transiently introduce foreign DNA into the cells of the inner ear in organ cultures in order to provide a test system for analyzing specific promoter regulatory elements; 2) Analyze the promoters of BDNF and NT-3 genes by transient transfection in order to identify specific sequences needed to maintain the normal pattern of gene expression during development and in the adult. BDNF and NT-3 MRNA levels respond to a variety of physiological and environmental stimuli such as ototoxic shock. The development of new techniques for analyzing gene expression in the inner ear is crucial for understanding these transcriptional mechanisms and is a first step towards designing means of manipulating the expression of potentially clinically important proteins such as neurotrophins.