Christopher L. Cunningham, Ph.D. - US grants

Project Summary/Abstract Hearing loss is the most prevalent sensory deficit in humans and causes a significant toll on the quality of life of those afflicted. The mammalian cochlea initiates the perception of sound by converting sound waves into electrical signals with exquisite sensitivity, speed and precision. Mechanotransduction in sensory hair cells leads to electrochemical signals that induce neurotransmitter release from specialized ribbon synapses, initiating the neural signal for auditory processing. The molecular composition of the auditory processing machinery in hair cells is not well understood, and the nature of how specific molecular interactions lead to physiological events for the processing of sound is unclear. Over 100 nonsyndromic forms of genetic hearing loss have been described but many of the genes linked with these mutations are unknown or not well characterized. Mutations in Catechol-O- methyltransferase 2 (COMT2) cause nonsyndromic recessive deafness (DFNB63) in humans. Our lab generated by ENU-mutagenesis a mouse mutant (termed `Add') in which a point mutation in Comt2, the mouse homolog of the human gene, leads to profound deafness. Comt2 mRNA is expressed by hair cells in the inner ear but the manner in which COMT2 contributes to auditory processing in mice and humans is completely unknown. The objective of this proposal is to investigate the role of COMT2 in the peripheral auditory system and the contribution of COMT2 to auditory processing. The hypothesis is that COMT2 contributes to hair cell function and auditory processing by regulating synapse function in the cochlea. Aim 1 will utilize in situ hybridization, immunohistochemistry and immunogold electron microscopy to precisely characterize the cell-specific and temporal expression patterns of Comt2 mRNA in the mouse inner ear and auditory brainstem and the subcellular localization of COMT2 protein in sensory hair cells. Aim 2 will use CRISPR/Cas9 technologies to generate and validate constitutive Comt2 null mice and floxed Comt2 mice to facilitate studies that will examine loss of Comt2 function on peripheral auditory function and auditory processing. Aim 3 will analyze Comt2 mutants to determine the mechanism by which Comt2 contributes to auditory processing in sensory hair cells. By interrogating Comt2 expression patterns and functions within the auditory system, this work will contribute to a better understanding of mechanisms of auditory processing, physiological roles of COMT2, and etiologies of DFNB63 in humans.  

Project Summary/Abstract The overall goal of the project is to define and characterize the organelles and specialized molecular machinery for protein assembly, processing and transport in hair cells, and the defects in this process that cause deafness. We propose here to investigate the mechanisms by which TOMT regulates transport of a subset of functionally important proteins in hair cells, and to begin to uncover TOMT-associated proteins that participate. Based on our preliminary data, we hypothesize that TOMT is an endoplasmic reticulum protein that is critical for assembly and transport of a specific set of transmembrane proteins in hair cells. To test our hypothesis, we will: i) use immunolocalization and immunogold transmission electron microscopy to precisely localize TOMT to hair-cell specific transport organelles; ii) identify and characterize important TOMT functional domains; and iii) look for interaction partners for TOMT that functionally cooperate in hair cell protein transport. Our preliminary data show the feasibility of our approach. We anticipate that our studies will elucidate new insights into the unique molecular machinery of hair cell protein transport that establishes distinctive functional specializations critical for auditory processing. Furthermore, a better understanding of protein transport mechanisms in hair cells has the potential to provide important context for protein transport deficits that are common in hearing loss.