Computational approaches to studying peripheral auditory system development and protection against ototoxicity.

摘要

The nicotinic cholinergic receptor (nAChR) composed of alpha9 and alpha10 subunits and functionally coupled to calcium-activated potassium channels, mediate the inhibitory transmission between medial efferent fibers originating from the brainstem and cochlear hair cells. When activated by sound, this system reduces cochlear sensitivity, and protects the auditory periphery from excessive noise. However, little is known regarding the specific genes downstream of alpha9alpha10 nAChRs, and represents an important gap in our knowledge of molecules involved in cochlear efferent neurotransmission. The first part of this thesis describes molecular mechanisms associated with alpha9alpha10 nAChRs by utilizing microarrays to compare wild type and alpha9 knockout (alpha9-/-) transcriptomes over the critical period of synapse maturation and at 2 months. Gene expression profiling along with subsequent experiments revealed a previously unrecognized link between the cholinergic and the GABAergic system within the cochlea. Altered gene expression patterns pointed to a depressed state of cochlear maturation in alpha9-/- mice suggesting a role for alpha9 subunit and the efferent system in postnatal cochlear development. We also describe a computational approach developed to identify sets of functionally related genes, by combining biclustering with gene set enrichment methods. This approach highlighted that the peripheral alpha9alpha10 nAChRs may be used as a model system to study neuronal nAChRs.;The second part of this work details the link between CRF receptor 2 (CRFR2) with aminoglycoside-induced ototoxicity in vitro. CRFR2, operating through G-protein coupled receptors, modulates hearing sensitivity and provides protection against noise-induced hearing loss; potentially imparting long-lasting cochlear protection. Using biochemical assays and proteomics screening, we demonstrated that activity via CRFR2 protects against oxidative stress and activation of cell death pathways. Bioinformatic analyses identified protein candidates potentially involved in protection against cytotoxicity.;Given the clinical importance of both systems in auditory function preservation, an understanding of the molecular mechanisms that contribute to their activity is crucial for developing effective therapies designed for maintenance of normal hearing. Our findings illustrate new avenues to explore the functional consequences following loss of alpha9 subunits in terms of cochlear maturation, and potential therapeutic targets for protection against metabolic stress-induced cochlear damage with the CRFR2 class of receptors.