Jocelyn Krey, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Dendrite growth is a key step in the development of neuronal circuitry and is perturbed in epilepsy, autism and other neurological diseases. Electrical activity regulates the development and plasticity of dendritic arbors by activating L-type voltage gated Ca2+ channels (LTCs). The goal of this proposal is to understand how LTCs regulate dendritic growth and arborization. To achieve this goal, we have developed a strategy that allows us to introduce mutant LTCs into neurons and to study how these exogenous channels regulate the dendritic arbor. Preliminary studies indicate that LTCs bearing a G406R mutation that causes the autistic disorder Timothy Syndrome (TS), are dramatically impaired in their ability to promote dendrite growth. I plan to characterize the defects of TS mutant LTCs and to investigate how other biophysical and biochemical features of LTCs affect the channel's ability to regulate the dendritic cytoskeleton. These studies will help elucidate the mechanisms by which electrical activity regulates dendritic growth and will also provide critical insight into the molecular underpinnings of autism and other neurological diseases associated with voltage- gated calcium channel dysfunction. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The perception of sound and balance depends on the mechanosensory function of the hair bundle, a group of actin-filled stereocilia that project from the apical surface of auditory and vestibular hair cells. The study of deafness-linked genes has led to the identification of a number of proteins that are localized to the hair bundle and contribute to its development and function [1]. Despite these advances, the complete network of proteins that constitute the hair bundle and regulate its maturation, maintenance and function remain poorly defined. Although the mechanisms by which proteins are specifically transported to the bundle are also poorly understood, accumulated evidence suggests that myosin motor proteins may play important roles [2]. The goal of this proposal is to identify the bundle proteins that are transported to and maintained within the hair bundle during its formation and to test the hypothesis that myosin-VIIa (MYO7A) plays a role in the transport of these proteins. Using proteomics approaches as well as other biochemical techniques, the aims in this proposal will 1) identify in an unbiased manner the protein networks that are enriched within the hair bundle during and after bundle assembly, 2) determine whether MYO7A plays a role in the transport of specific proteins to the bundle and 3) identify which of these bundle-specific proteins directly or indirectly interact with MYO7A. The results of these studies will significantly contribute to our molecular understanding of bundle assembly and function and could help guide the development of therapeutic strategies for hair cell repair and regeneration. PUBLIC HEALTH RELEVANCE: The studies in this proposal will lead to a more comprehensive understanding of the molecular mechanisms underlying the development and function of auditory and vestibular hair cells. The proposed research will also help determine how deafness-causing mutations in specific genes perturb the assembly and function of the sensory apparatus within the hair cell. The results from these studies may guide the future identification of new deafness genes that will aid in improving the diagnosis and treatments of various hearing and balance disorders.

? DESCRIPTION (provided by applicant): Perception of sound and balance depends on mechanotransduction, whereby a molecular complex at the tips of hair cell stereocilia converts an external mechanical stimulus into an electrical signal, which can propagate to the central nervous system. A key unknown in auditory neuroscience is the identity of the molecules that constitute the ion-conducting transduction channel within this complex. By studying deafness- linked genes in humans and mice, investigators have suggested that TMC1 and TMC2 are strong mechanotransduction channel candidates. Unfortunately, biochemical methods with sufficient sensitivity and selectivity to robustly detect and quantify the TMCs or other membrane proteins within the mechanotransduction complex have yet to be developed. Antibody-based methods such as Western blotting and immunocytochemistry often suffer from poor specificity, low multiplexing capabilities, and imprecise quantitation, thus preventing them from providing accurate quantitative information about the composition of the mechanotransduction complex. In addition, the high cost and limited availability of quality antibodies, especially for membrane proteins, limits screening for new transduction-channel candidates identified by other means. The goal of this proposal is to characterize the composition of the mechanotransduction complex in hair cells. To do so, we will develop targeted proteomic assays to identify and accurately quantify transduction proteins and measure how their stereocilia location and mechanotransduction complex association are altered in mouse models of deafness and vestibular dysfunction. We will design two sets of assays, which will allow us to simultaneously monitor the concentrations of known mechanotransduction complex proteins, as well as detect new candidate transduction channel proteins identified in hair cells at the RNA level. These assays, combined with other biochemical approaches, will allow us to 1) determine whether TMC1, TMC2, or other channel-like membrane proteins are localized to the hair bundle and are able to interact with other transduction complex proteins, and 2) determine how the localization of mechanotransduction proteins change in mice lacking TMC1 and TMC2. Together, the results of these studies will significantly contribute to our understanding of the molecular basis of hearing and deafness. In the long term, these assays will continue to be valuable tools for assessing how the composition of the mechanotransduction complex changes across development in various mouse models, as well as in the context of hair cell repair and regeneration.