NEUROTROPHIN GENE REGULATION IN THE INNER EAR

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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.