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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Laird, Dale W.

Abstract

Connexin and pannexin large-pore channels allow the regulated passage of small molecules at sites of cell-cell contacts, and from the cytosol to the extracellular milieu, respectively. Since it has been known for many years that Cx26 and Cx30 gap junction proteins are crucial in hearing we propose that Cx43 might also be important in hearing. Here we used two different genetically modified mouse lines that contain systemic Cx43 gene mutations that reduces gap junctional intercellular communication (GJIC) to examine whether Cx43 is also important for proper hearing function. Furthermore, since pannexins have also been postulated to be involved in auditory function we used three different Panx global knock-out mice to evaluate their hearing profiles. We showed that Cx43 mutant mice that had severe loss of Cx43 channel function had hearing loss, while mutant mice with a modest loss of Cx43 function exhibited normal hearing. Surprisingly, Panx1-/-, Panx3-/-, and double knock-out (dKO) mice did not have hearing loss, suggesting that pannexins do not play an important role in hearing. To evaluate whether large-pore channels played a role in noise-induced hearing loss (NIHL), we challenged Cx43 mutant and pannexin knock-out mice with a loud noise-exposure and examined their permanent hearing loss. Interestingly, only Panx3-/- and Panx dKO mice were slightly protected against permanent hearing damage. Finally, organotypic cochlear cultures from Cx43 mutant mice and a CRISPR Cas9 Cx43 ablated cochlear-derived cell line, revealed that GJIC does not exacerbate drug-induced hearing loss but does cause supporting cell reorganization. Collectively, our results highlight the importance of Cx43 GJIC in hearing function, but not noise- or drug-induced ototoxicity. Furthermore, our studies support the notion that Panxs are not involved in baseline hearing, but loss of Panx3 may lead to slight protection against permanent NIHL.

Summary for Lay Audience

The cells of our body are in constant communication with each other to facilitate tissue health. Gap junction channels form the basis of direct cell to cell communication allowing the transfer of ions and small signaling molecules in a process known as gap junctional intercellular communication (GJIC). It has been known for many years now that GJIC is essential for proper hearing as many mutations in genes that encode connexin (Cx) gap junction proteins cause the majority of inherited hearing loss. Another large-pore channel type found in the inner ear that has been proposed to be important in hearing is made from pannexins (Panx) that allow small molecules to exit the cell. We evaluated the importance of Cx43, a poorly studied gap junction protein found in the auditory track, as well as Panx channels in baseline hearing and in noise-induced hearing loss. We found that Cx43 function was important in baseline hearing function, however, moderate levels of Cx43 function was enough to maintain proper hearing. In addition, Panxs were not found to be involved in hearing function. Loss of either Cx43 or Panx1 did not result in more or less permanent noise-induced hearing loss. Interestingly, loss of Panx3 led to a slight protection of hearing loss after a loud noise-exposure, however, the mechanisms behind this remains unknown. Cisplatin, a commonly used therapeutic agent, is effectively used to treat solid cancer tumors but results in permanent hearing loss in 75-100% of patients. Blocking and/or loss of GJIC with cisplatin treatment did not result in any differences in inner ear cell death, however, gap junction proteins were reorganized after cisplatin treatment. Overall, we examined the importance of large-pore channels in the context of three different kinds of hearing impairments including genetics, noise, and drug induced hearing loss. Interestingly, we found that Cx43 was essential for baseline hearing, but not NIHL or cisplatin-induced hearing loss. Further Panxs were not involved in any type of hearing function. These studies will directly contribute to our knowledge of the mechanisms that could ultimately contribute to molecular therapies in treatment of hearing loss.

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