Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Laird, Dale W.

Abstract

Connexin 26 (Cx26) and Cx30 mediate the intercellular exchange of metabolites and ions within the cochlea in a process known as gap junctional intercellular communication (GJIC). Cochlear cell death and subsequent hearing loss can arise after treatment with ototoxic therapeutics and Cx26 mutant expression. We investigated the role of connexins and GJIC in the development of ototoxicity in HEI-OC1 cochlear-derived cells. The susceptibility of HEI-OC1 cells to aminoglycoside antibiotics and cisplatin-induced cell death was not influenced by the ablation of connexins and GJIC. However, the expression of mitochondrial apoptosis or ER stress markers was altered by the degree of GJIC. Hearing loss linked Cx26 syndromic mutants (N54K and S183F) displayed diverse gain-of-function properties in HEI-OC1 cells. However, non-syndromic mutants (R32H and R184P) displayed similar loss-of-function properties. Collectively, these disease-linked Cx26 mutants exhibited three distinct cellular phenotypes, which may contribute to unique mechanisms for hearing loss in vivo.

Summary for Lay Audience

Cells can communicate by expressing connexin proteins that form gap junction channels between adjacent cells in the majority of tissues throughout the body. These channels allow the transfer of ions and signalling molecules between cells in a process known as gap junctional intercellular communication (GJIC). GJIC is important for cellular health and facilitating in countless physiological processes. Twenty-one different connexin family members exist, including connexin 26 (Cx26), which is highly expressed in the cochlea. The cochlea is located in the inner ear and contains hair cells that allow for sound detection. Cx26 channels and GJIC help maintain hair cell survival in the cochlea. Hair cell death and resulting hearing loss is a result of treatment with particular medications called ototoxic drugs. GJIC can potentially propagate death signals to healthy cells and increase cell death as seen in cancer cells. We found that connexin expression and GJIC did not enhance apoptosis after treatment with ototoxic therapeutics in cochlear-derived cells. However, the degree of GJIC altered the pathways leading to cell death, potentially by altering which molecules could travel between cells. Hearing loss can also arise from mutations in the gene encoding for Cx26. Cx26 mutations result in ~50% of all inherited hearing loss worldwide. Around 135 different Cx26 mutations that induce hearing loss have been identified. Proper connexin trafficking to the cell surface and channel formation is important for connexin function. Cx26 mutants can exhibit loss-of-function properties including improper connexin channel trafficking to the cell surface and defective channel function. Gain-of-function properties include the ability of the Cx26 mutant to interact with other connexin family members impairing or enhancing their function. We analyzed the cellular localization of four hearing loss linked Cx26 mutants in cochlear-derived cells to improve our understanding of the various hearing loss mechanisms. Cx26 mutations that result in hearing loss and a skin disease primarily displayed gain-of-function properties, whereas mutations that only cause hearing loss had loss-of-function properties. Overall, we identified three distinct mechanisms for hearing loss between the mutants studied, suggesting that future therapeutic strategies will need to account for the specific nature of connexin defect.

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