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Viewed Papers: Yin K, Baillie GJ and Vetter I A Pharmacological and Transcriptomic approach to exploring Novel Pain Targets Kathleen Yin Bachelor of Pharmacy A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 Institute for Molecular Bioscience i Abstract Ever since the discovery that mutations in the voltage-gated sodium channel 1.7 protein are responsible for human congenital insensitivity to pain, the voltage-gated sodium channel (NaV) family of ion channels has been the subject of intense research with the hope of discovering novel analgesics. We now know that NaV1.7 deletion in select neuronal populations yield different phenotypes, with the deletion of NaV1.7 in all sensory neurons being successful at abolishing mechanical and heat-induced pain. However, it is rapidly becoming apparent that a number of pain syndromes are not modulated by NaV1.7 at all, such as oxaliplatin-mediated neuropathy. It is particularly interesting to note that the loss of NaV1.7 function is also associated with the selective inhibition of pain mediated by specific stimuli, such as that observed in burn-induced pain where NaV1.7 gene knockout abolished thermal allodynia but did not affect mechanical allodynia. Accordingly, significant interests exist in delineating the contribution of other NaV isoforms in modality-specific pain pathways. Two other isoforms, NaV1.6 and NaV1.8, are now specifically implicated in some NaV1.7-independent conditions such as oxaliplatin-induced cold allodynia. Such selective contributions of specific ion channel isoforms to pain highlight the need to discover other putative protein targets involved in mediating nociception. The aim of my work is therefore to discover selective molecular inhibitors of NaV1.6 and NaV1.8, to find useful cell models for peripheral nociceptors, to investigate the roles of NaV1.6 and NaV1.8 in an animal model of burn-induced pain, and to screen for putative new targets for analgesia in burn-related pain. Chapter 2 of this thesis describes activity-guided discovery of novel NaV1.6 and NaV1.8- modulting peptides from crude spider venoms. Crude venom from Poecilotheria metallica successfully reduced NaV1.8-mediated voltage changes in HEK293-expressing cells. A new NaV1.8-inhibitory peptide was sequenced from the venom and named Pme1a. However, Pme1a exhibited TRPV1 agonist activity and induced nocifensive behaviours, such as paw licking, after injection into the hind paws of mice, indicating the peptide was not a selective NaV1.8 modulator and was unsuitable as a tool for NaV1.8 investigation. ii With the unsuccessful exploration of spider venoms for new specific inhibitors, I then investigated in vitro cell models as tools to research specific nociceptor subtypes that express NaV1.6 or NaV1.8. In Chapter 3, I provide the first complete transcriptome of three common in vitro neuronal cell lines (SH-SY5Y, F-11, and ND7/23) to examine whether they were appropriate for investigating the functions of NaV1.6 and NaV1.8, and to identify if the cell lines resemble in vivo dorsal root ganglion (DRG) neuronal subclasses. The three cell lines examined all expressed proteins belonging to similar pathways and of similar proportions to native DRG neurons. However, they did not express any cellular markers in a fashion that represented any known subclasses of DRG neurons. Therefore, it is unlikely that any of the cell lines examined are appropriate models for NaV1.6 or NaV1.8 function, and highlights the need for careful selection of models in in vitro work. As investigations for selective NaV1.6 and NaV1.8 modulators and appropriate in vitro cell models both proved to be unsuccessful, I then turned to animal models to study modality- specific contributions of NaV isoforms. Chapter 4 describes the establishment of a murine model of peripheral burn injury to assess the involvement of NaV1.6 and NaV1.8 in burn- induced pain. The inhibition of NaV1.6 and no other NaV isoforms significantly reduced burn- induced mechanical allodynia, a NaV1.7-independent pain modality, indicating NaV1.6 plays a vital role in this NaV1.7-independent condition. However, immunofluorescence studies revealed NaV1.6 expression was not changed in the affected DRGs. I therefore decided to conduct a transcriptomic investigation to examine whether the expression of other pain- related ion channel gene were changed, and to discover if genes differentially expressed in the affected DRG neurons could be potential pharmacological targets for analgesia. The results of the transcriptomic investigation of DRGs affected by the burn model are discussed in Chapter 5. A total of 30 genes were found to be differentially expressed, including genes known to be involved in nociception (such as neuropeptide Y) as well as genes with no known association to nociception (such as lipase, family member N). It should be noted that none of the NaV isoforms were differentially expressed, indicating that NaV channels may alter pain sensitivity through modulated function rather than expression. Selective inhibitors of the protein products of the genes found to be up-regulated were then tested in the burn-induced pain mouse model. Proglumide, an inhibitor of the cholecystokinin B receptor, exhibited a significant anti-allodynic effect in burn-induced mechanical allodynia and was synergistic with oxycodone. Some genes found to be differentially expressed in burn iii injury were also identified in the cell lines covered in Chapter 3, and the cell lines can potentially be used as in vitro models to rapidly profile the effects of inhibitors of these proteins in peripheral neurons. iv 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, 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 research higher degree 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. v Publications during candidature Peer-reviewed papers: Yin K, Baillie GJ and Vetter I. Neuronal cell lines as model dorsal root ganglion neurons: A transcriptomic comparison. Mol Pain. 2016; 12: 1-17. Yin K, Zimmermann K, Vetter I, Lewis RJ. Therapeutic opportunities for targeting cold pain pathways. Biochem Pharmacol. 2015;93(2):125-40. Conference abstracts: Yin K, Deuis JR, Vetter I. A Behavioural and Transcriptomic Assessment of a Mouse Burns Model. Oral Presentation. IMB Chemistry and Structural Biology Division Symposium, November 2015, Brisbane, Australia. Yin K, Deuis JR, Vetter I. Development and optimisation of a murine model of burn injury- induced pain. Oral presentation. Australian Pain Society 35th Annual Scientific Meeting, March 2015, Brisbane, Australia. Yin K, Baillie G, Vetter I. Evaluating the molecular expression profiles of common cell lines used as DRG neuron models. Poster presentation. Venoms to Drugs conference, November 2014, Kingscliff, New South Wales, Australia Yin K, Vetter I, Herzig V, Cardoso F, Cabot PJ, Lewis RJ. Discovery and characterisation of a peptide with inhibitory activity at NaV1.8 from the spider P. metallica. Poster presentation. IMB division retreat, March 2014, Brisbane, Queensland, Australia. Publications included in this thesis No publications included. vi Contributions by others to the thesis The thesis is a collection of work primarily conducted by me, with contribution from many other co-workers and collaborators as detailed below. My supervisors, Dr. Irina Vetter, Associate Professor Peter Cabot, and Dr. Fernanda Caldas Cardoso have contributed significantly to the conception and design of many experiments. All crude venoms examined in Chapter 2 were generously provided by Dr. Volker Herzog from the Institute for Molecular Bioscience, and I would like to extend my thanks to Dr. Aihua Jin and Dr. Mu Yu for their provided invaluable support in conducting MS/MS experiments and finalising the sequence of the peptides. Dr. Gregory J. Baillie from the Institute for Molecular Bioscience contributed significantly to bioinformatics data analysis for Chapter 3 and Chapter 5, particularly with gene alignment and mapping. NaV inhibitory peptides used in Chapter 4 were synthesised by Mr. Zoltan Dekan from the Institute for Molecular Bioscience. Dr. Jennifer R. Deuis from the Institute for Molecular Bioscience contributed significantly to mechanical and thermal allodynia measurements in mice for Chapter 4 and Chapter 5, and towards the motor assessment of mice (Figures 4.3 and 5.5). Ms Mathilde Israel and Mr. Bryan Tay also contributed significantly to the fluorescence imaging data present in Figures 4.5 and 4.6, assisting with microscope operation and optimisation. Statement of parts of the thesis submitted to qualify for the award of another degree None vii Acknowledgements First of all, I would like to thank my supervisors for their incessant support during my candidature.
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