Improving chemotherapeutic regimens by mitigating hearing loss as a limiting side-effect and examining transition metal ions as a ligand for zebrafish Tlr4
Creators: Fox, Aaron
Publisher: name: University of Alberta Library
Year: 2023

<p>Cancer is often a debilitating disease associated with the uncontrolled growth of cells within an organism. We have made significant strides over the past few decades in treating many cancers, unfortunately, many of these treatments have unfavourable side effects. Cisplatin is a revolutionary drug in cancer treatment that contributes to an approximately 80% five-year survival rate in pediatric patients, but its use is limited by several adverse side effects, particularly ototoxicity. Cisplatin-induced ototoxicity (CIO) is permanent, bilateral, sensorineural hearing loss found in up to 90% of patients treated with cisplatin. There are currently very few treatments to mitigate CIO, and only one is approved by the FDA. Our collaborators in the Bhavsar Lab have recently determined a novel pathway through which cisplatin contributes to the generation of CIO and have demonstrated its potential as a therapeutic target.<br>
Toll-like receptor 4 is the first discovered innate immune pattern recognition receptor. It is crucial in signalling to the body when to initiate an inflammatory response following a gram-negative bacterial infection. The function of TLR4 in mammals is well characterized, but its function in other species, particularly fish, is still under debate. A major problem with understanding its function arises in the confusion behind its origins. Not all living things bind to the same TLR4 ligand, and in many cases, we are still unsure of what it binds to entirely. TLR4 was recently determined to bind to nickel and other heavy metals to induce a proinflammatory response and mediate an allergic response. Furthermore, it has recently been shown to bind to the platinum-containing drug cisplatin directly, mediating the generation of CIO. Due to the unique binding mechanism of these metals to TLR4, we believe it would be possible to selectively inhibit cisplatin binding while maintaining the regular immune function of TLR4. This novel mechanism would distinguish it from several CIO therapies, demonstrating its promise for treating CIO.<br>
Zebrafish make a great animal model for disease and share greater than 80% of disease-related genes with humans. Their small size, optical transparency, and high throughput nature also provide an advantage over other model systems. Zebrafish are known to possess three TLR4 paralogs, Tlr4ba, Tlr4al, and Tlr4bb, but like many fish, their function and origins remain controversial. However, their utility in research related to CIO is still particularly valuable due to the presence of hair cell clusters on the exterior of their bodies called neuromasts. These hair cells are homologous to the hair cells within the inner ear of mammals. Their position and similarity make them an excellent model for CIO and particularly suited for novel therapeutic drug discovery.<br>
This thesis aims to tease apart some of the uncertainties in the evolutionary history of TLR4 while advancing our understanding of its activities in zebrafish. I also aim to utilize zebrafish as an effective model of CIO to screen novel selective therapeutics. To achieve these objectives, we created larval genetic mutants of Tlr4, allowing us to test its interaction with specific ligands in vivo. By creating a heterologous cell system using zebrafish Tlr4, we extended these findings in vitro. Finally, by creating a novel vibration-based behavioural assay, we demonstrated the end-point effects of a novel therapeutic capable of selectively inhibiting Tlr4 to mitigate CIO. The research presented in this thesis establishes the foundation for future investigations that aim to selectively inhibit TLR4 in order to reduce CIO while identifying potential ligands for a presently orphaned receptor.</p>