Transcriptomic analysis of zebrafish prion protein mutants supports conserved cross-species function of the cellular prion protein; and zebrafish as a model for cisplatin induced ototoxicity and transition metals as potential ligands for Tlr4ba and Tlr4bb

• Author / Creator

  Pollock, Niall MH

• The work presented in this thesis is split between two projects. The first utilises prp1 and prp2 knockout zebrafish to investigate physiological functions of the cellular prion protein (PrPC). The second builds upon the use of zebrafish as a model for hearing loss to confirm a role of toll-like receptor 4 (TLR4) in cisplatin induced ototoxicity (CIO) and to investigate group 10 transition metals as potential ligands for zebrafish Tlr4ba and Tlr4bb. Chapter 1 contains a literature review of systemic and central nervous system (CNS) related amyloid disease and how they relate to each other and prion diseases. Functional amyloids are discussed in the context of how control mechanisms for the formation of functional amyloids may help in developing therapeutics for protein misfolding diseases. In Chapter 2, RNA-sequencing with wild-type and prp1ua5003/ua5003; prp2ua5001/ua5001¬ mutant zebrafish at 3 days post fertilisation was used to identify potential roles of prion protein during zebrafish development. Biological process gene ontology analysis showed the process with the largest number of genes showing a significant decrease in transcript abundance was cell adhesion. Of theses, 31 of the 38 genes belonged to the protocadherin family. Protocadherins are involved in the development and maintenance of the CNS. In addition, ncam1a and st8sia2 both showed a significant reduction in transcript abundance after RNA-sequencing and this was confirmed through RT-qPCR. These results closely match those seen in in vitro experiments in cells lacking PrPC. Abnormal deposition of neuromasts along the posterior lateral line (PLL) was observed in prp1, prp2 and prp1/prp2 knockout zebrafish. In prp1 mutant fish there was a significant decrease in neuromast count along the PLL, in prp2 mutants there was a significant increase. Combined prp1/prp2 mutant zebrafish recovered the loss of neuromasts seen in prp1 mutants but was still higher than wild-type. Together, these results would suggest a cross species conserved role of the cellular prion protein in the early development of organisms.The second part to this thesis investigates the role of TLR4 in CIO in collaboration with the Amit Bhavsar and Fred West labs at the University of Alberta. Transition metals as potential ligands for zebrafish Tlr4ba and Tlr4bb are also explored. Cisplatin is an effective treatment against cancer but has severe side effects. One of these is permanent, bilateral hearing loss and there is currently no co-treatment to prevent this. This has led to a reduction in usage of cisplatin. Zebrafish PLL neuromasts have become an established model for ototoxicity. Recent work has identified TLR4 as a potential mediator for CIO. TAK-242 is a small compound inhibitor of TLR4. In Chapter 3, morpholino knockdown of tlr4ba and tlr4bb and inhibition of zebrafish Tlr4ba and Tlr4bb through TAK-242 or synthetic TAK-242 derivatives, termed syntagonists, was used to confirm the role of TLR4 in mediating CIO. Two syntagonists, 134 and 136 significantly reduced the CIO in neuromast cells in 6-7dpf zebrafish. Morpholino knockdown of tlr4bb through two separate morpholinos and tlr4ba resulted in a significant reduction in CIO. Combined knockout of both tlr4ba and tlr4bb at the same time reduced CIO though not significantly moreso than either individually. These results confirm TLR4 as a mediator for CIO and established zebrafish as a suitable, high-throughput model for investigating inhibition of CIO going forward.Finally, utilising the model established in Chapter 3, Chapter 4 contains results investigating whether transition metals are a ligand for zebrafish Tlr4ba and Tlr4bb. In mammalian TLR4 the canonical ligand is lipopolysacharride, though other ligands such as nickel, cobalt and certain viral proteins can also cause TLR4 signalling. Zebrafish PLL neuromasts were exposed to either NiCl2, PtCl2 or PtCl4 and co-treated with syntagonists. Several syntagonists, 138, 150, 166, 168 and 170 all significantly reduced nickel induced ototoxicity. Of these only one had been also tested against CIO, syntagonist 138, in which it had no effect. After co-treatment of syntagonist 134 with PtCl2 or PtCl4 there was no reduction in platinum-induced ototoxicity. This may be due to the concentrations of platinum salts used, or as with nickel induced ototoxicity, syntagonist 134 is not effective against platinum induced ototoxicity. These results promote optimism that transition metals may activate Tlr4ba or Tlr4bb signalling, though more work is needed to confirm the validity of these results.

• Subjects / Keywords

  • Zebrafish
  • Prion Protein
  • TLR4
  • Transcriptomics
  • Cell Adhesion
  • Cisplatin
  • Ototoxicity

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-1mp9-zz91

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.