Cochlear Nucleus Proteomics in a Mouse Model of Age-Related Hearing Loss

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DESCRIPTION (provided by applicant): People that have had AHL will have changes in their central auditory pathways. Our studies (and some other recent studies) have shown that the critical "gateway" cells in the cochlear nucleus that are involved in processing and transmitting timing information used for sound localization and some aspects of speech perception have altered synaptic function and electrical excitability in deaf animals. These adaptations result from changes in the expression of specific proteins (receptors and ion channels), as well as other proteins that have not yet been identified. Identifying the underlying protein changes will help to understand how the brain responds to deafness, and should help to design stimulus protocols, as well as possibly inform future gene therapies, that optimize the stimulation and rehabilitation of the central auditory pathway. The focus of this proposal is to characterize the proteomic changes in the anterior ventral cochlear nucleus (AVCN) in a murine model of age-related hearing loss (AHL). While many studies have focused on defects in the cochleus, the primary auditory organ, less is known about the proteomic changes in the auditory brainstem. This proposal consists of a two targeted-proteomic approaches. Our first aim is to characterize a membrane protein that changed during AHL progression based on our electrophysiological data -- the ionotropic glutamate receptor 2. Since isoform variation and posttranslational modification of this protein is known to affect its activity, we will investigate this protein with top-down and bottom-up mass spectrometric analysis after gel-based and affinity-based protein purification. An in-depth characterization of this protein should indicate pathways which are disrupted in AHL. Our second aim is identification of any membrane proteins that change during AHL progression. This will be accomplished by using traditional subcellular fractionation combined with comparative 1 D-gel electrophoresis and and novel FTICR-based mass spectrometry-based approaches for differential protein expression analysis. Using high-resolution multi-dimensional liquid chromatography coupled to FTICR-MS, peptides from fractionated membrane proteins from normal and AHL mice are acetylated with either acetic anhydride or 03-acetic anhydride, and combined. The ratios of these peptide doublets indicate relative protein expression levels, while the peptide masses and MS/MS fragmentation patterns identify the protein.