Identification of pain genes contributing to ciguatoxin-induced pain using haplotype-based computational genetic mapping in mice by Julian Soh Bachelor of Medicine/Bachelor of Surgery Submitted for the degree of Masters of Philosophy at The University of Queensland in 2018 School of Medicine Abstract Pain is a complex process involving numerous underlying genetic contributions still not completely appreciated, which may explain the high degree of interindividual differences seen with nociception. Chronic pain is responsible for a significant socioeconomic burden on the individual as well as society and is affected by several patient specific aspects including physiological, psychological and environmental factors. Interindividual response to current analgesics is exceedingly inconsistent and the functional basis for the unpredictable efficacies of medications has not been elucidated. It is highly probable that variation seen in nociception is due to undiscovered genetic mechanisms, and specific polymorphisms within genes can be potentially singled out by observing deviations in spontaneous pain responses between animals of differing genotypes using the ciguatoxin marine poison. Ciguatoxin is isolated from the Gambierdiscus toxicus dinoflagellate, a specific type of plankton, which is shown to produce spontaneous pain as well as cold allodynia in a dose dependent manner. The objectives of this thesis are centred on the demonstration of the genotypic influence on pain perception, identifying possible genetic sources implicated in nociception, and validating these potential targets with in vivo methods. In Chapter 2, pain behaviours of sixteen mouse strains with recognised genotypes were quantified after administration of the ciguatoxin, which is known to induce spontaneous flinching and licking pain reactions after intraplantar injection. The mechanism of this interaction is reliant on the actions of mainly voltage-gated sodium channels, particularly NaV1.6 and NaV1.7, but also voltage-gated potassium channels. Utilisation of inbred mouse strains with differing genotypes provides the foundation for the study, as the established SNP characteristics can be contrasted against the pain phenotype observed. Inoculation of numerous mouse strains employed with ciguatoxin produced measurable pain responses and a significant difference in the number of flinches as well as the time spent licking between each mouse strain was seen. The contrasting pain responses between the different strains implicates genotype discrepancies as the likely source of variation, therefore, variation between flinching and licking responses were analysed to ascertain further details. In Chapter 3, the enumerated pain phenotypes were interrogated using the i haplotype-based computational genetic mapping (HBCGM) technique aimed at matching phenotypic differences to the unique SNP fingerprint established when utilising a combination of mouse strains of diversified genotypes. Genes recognised to have correlation to the pain phenotype based on their SNP profile were screened according to their likelihood to impact nociception using various techniques. Gene expression sites and gene ontology were considered in order to direct the search towards specific genes or gene clusters with potential ramifications in pain transmission. Via these methods, it was seen that the HBCGM gene lists exhibited features very similar to that of the native mouse genome, signifying that specific genetic loci have not been clearly attributed to the phenotype differences using this approach, and search for potential pain genes will need to be continued further. Despite this finding, the Nedd4-2 gene was selected as a putative target for further validation with knockout mice in Chapter 4, given that the NEDD4-2 E3 ubiquitin ligase product has been implicated in the trafficking of multiple NaV subtypes at the dorsal root ganglia. The native function of E3 ubiquitin ligases involves maintaining membrane populations of specific proteins, which includes NaV1.6-1.8, but the roles of Nedd4-2 with respect to ciguatoxin-induced pain have not yet been investigated. During observation, Nedd4-2 knockout mice demonstrated a significant increase in licking times after administration of ciguatoxin compared to their wild-type counterparts, the possible result of increased NaV membrane populations secondary to a reduction in E3 ubiquitin ligase mediated trafficking. This discovery suggests that Nedd4-2 may be responsible for modulation of ciguatoxin-induced spontaneous pain and is therefore a potential pain gene which will require further characterisation. ii DECLARATION BY AUTHOR This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. iii PUBLICATIONS DURING CANDIDATURE No publications. PUBLICATIONS INCLUDED IN THIS THESIS No publications included. iv CONTRIBUTIONS BY OTHERS TO THE THESIS Chapter 2: Prof. Katharina Zimmermann (Friedrich-Alexander-Universität Erlangen-Nürnberg, Bavaria, Germany) for providing mouse strains and for technical assistance. Chapter 3: Dr. Ming Zheng (Peltz’s Lab, Stanford University, California, USA) for inputting phenotype data into the haplotype-based computational genetic mapping software. Chapter 4: Prof. Sharad Kumar (Centre for Cancer Biology, University of South Australia) for providing the Yang Nedd4-2 KO mice. Dr. Jennifer Deuis (Institute for Molecular Bioscience, The University of Queensland) for technical assistance with OD1 experimental steps. STATEMENT OF PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE AWARD OF ANOTHER DEGREE None RESEARCH INVOLVING HUMAN OR ANIMAL SUBJECTS The appropriate ethics approval was obtained from The University of Queensland Animal Ethics Committee, approval number: PHARM/249/14/ARC. FINANCIAL SUPPORT Douglas H.K Lee Family Scholarship (2016) v KEYWORDS Ciguatoxin, pain, nociception, voltage-gated sodium channels, haplotype-based computational genetic mapping, SNP, Nedd4l, Nedd4-2, mice AUSTRALIAN AND NEW ZEALAND STANDARD RESEARCH CLASSIFICATIONS (ANZSRC) ANZSRC code: 060412, Quantitative Genetics (incl. Disease and Trait Mapping Genetics), 40% ANZSRC code: 060408, Genomics, 30% ANZSRC code: 060102, Bioinformatics, 30% FIELDS OF RESEARCH (FoR) CLASSIFICATION FoR code: 0604, Genetics, 70% FoR code: 0601, Biochemistry and Cell Biology, 30% vi Contents Abstract i Declaration by author iii Publications during candidature iv Contribution by others to the thesis v Keywords vi Australian and New Zealand Standard Research Classifications (ANZSRC) vi Fields of Research (FoR) Classification vi Contents vii List of figures x List of tables xii List of abbreviations used in the thesis xiii 1. CHAPTER ONE: Nociception and Ciguatoxin 1.1 Introduction 1 1.2 Literature Review 2 1.2.1 Prevalence of Pain 2 1.2.2 Risk Factors 2 1.2.3 Socioeconomic Impact 3 1.2.4 Anatomy of Pain Pathways 4 1.2.5 Nociception 6 1.2.6 Pain Conduction 8 1.2.7 Peripheral Sensitisation 9 1.2.8 Genetics of Pain 10 1.2.9 Ciguatera 13 1.3 Research hypotheses, aims and significance 15 vii 1.3.1 Hypotheses 16 1.3.2 Aims 16 1.3.3 Significance 16 2. CHAPTER TWO: Strain-dependent Phenotypic Differences in Ciguatoxin-Induced Spontaneous Pain 2.1 Foreword 18 2.2 Introduction 18 2.3 Method 19 2.4 Results 23 2.5 Discussion and Conclusions 32 3. CHAPTER THREE: Haplotype-Based Computational Genetic Mapping 3.1 Foreword 38 3.2 Introduction 38 3.3 Method 40 3.4 Results 44 3.5 Discussion and Conclusions 55 4. CHAPTER FOUR: Nedd4-2 and ubiquitination of voltage-gated sodium channels 4.1 Foreword 64 4.2 Introduction 64 4.3 Method 69 4.4 Results 72 4.5 Discussion and Conclusions 80 5. REFERENCES 88 6. APPENDICES 6.1 Appendix 1: List of flinching genes 98 viii 6.2 Appendix 2: List of licking genes 102 6.3 Appendix 3: List of overlapping genes 108 6.4 Appendix 4: Potential gene candidates for ciguatoxin-induced pain 112 6.5 Appendix 5: Gene ontology for mus musculus genome 116 6.6 Appendix 6: Amino acid structure and properties 118 6.7 Appendix 7: NEDD4-2 amino acid sequence 119 ix LIST OF FIGURES Figure 1.1: Spinothalamic tract ascending pathway. 6 Figure 1.2: Spinothalamic tract descending pathway. 6 Figure 2.1: Setup for mouse observation step. 21 Figure 2.2: Flow diagram of overall experiment. 23 Figure 2.3: Male and female pain responses are highly correlated for both 24 flinching and licking pain behaviours. Figure 2.4: Comparison of flinching and licking for both genders combined. 26 Figure 2.5: Flinches and licking of each individual mouse strain have distinct 26 degrees of responses. Figure 2.6: Mouse strains behave to different extents in terms of flinching and 30 licking pain responses.