This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Neurobiology of Disease Gdnf, Neurturin and Artemin Activate and Sensitize Bone Afferent Neurons and Contribute To Inflammatory Bone Pain Sara Nencini, Mitchell Ringuet, Dong-Hyun Kim, Claire Greenhill and Jason J Ivanusic Department of Anatomy and Neuroscience, University of Melbourne. DOI: 10.1523/JNEUROSCI.0421-18.2018 Received: 15 February 2018 Revised: 20 March 2018 Accepted: 22 April 2018 Published: 30 April 2018 Author contributions: S.N. and J.J.I. edited the paper; J.J.I. wrote the first draft of the paper; J.J.I. designed research; S.N., M.R., D.-H.K., C.G., and J.J.I. performed research; J.J.I. contributed unpublished reagents/ analytic tools; S.N., M.R., D.-H.K., C.G., and J.J.I. analyzed data; J.J.I. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was supported by funding from the Australian National Health and Medical Research Council, a Clive and Vera Ramaciotti Foundation Establishment Grant, and a Medical Research and Technology Grant from Australian and New Zealand Charitable Trustees. The authors have no conflicts of interest. The authors acknowledge Associate Professor James Brock for his comments on the manuscript. Corresponding Author: Associate Professor Jason Ivanusic, Department of Anatomy and Neuroscience, University of Melbourne, Melbourne, VIC 3010, Australia, Tel: +61 3 8344 7254, Fax +61 3 9347 5219, Email: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0421-18.2018 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 TITLE: 2 Gdnf, Neurturin and Artemin Activate and Sensitize Bone Afferent Neurons and Contribute 3 To Inflammatory Bone Pain 4 5 ABBREVIATED TITLE: 6 Gdnf, Neurturin and Artemin Signaling In Bone Pain 7 8 AUTHOR NAMES: Sara Nencini, Mitchell Ringuet, Dong-Hyun Kim, Claire Greenhill and 9 Jason J Ivanusic 10 11 AFFILIATIONS: 12 Department of Anatomy and Neuroscience, University of Melbourne. 13 14 CORRESPONDING AUTHOR: 15 Associate Professor Jason Ivanusic, 16 Department of Anatomy and Neuroscience, 17 University of Melbourne, 18 Melbourne, VIC 3010, Australia 19 Tel: +61 3 8344 7254 20 Fax +61 3 9347 5219 21 Email: [email protected] 22 23 NUMBER OF PAGES: 38 24 NUMBER OF FIGURES: 6 25 NUMBER OF TABLES: 3 26 NUMBER OF WORDS: Abstract = 155, Introduction = 649, Discussion = 1510 1 27 28 CONFLICT OF INTEREST: 29 The authors declare no competing financial interests. 30 31 ACKNOWLEDGEMENTS: 32 This work was supported by funding from the Australian National Health and Medical 33 Research Council, a Clive and Vera Ramaciotti Foundation Establishment Grant, and a 34 Medical Research and Technology Grant from Australian and New Zealand Charitable 35 Trustees. The authors have no conflicts of interest. The authors acknowledge Associate 36 Professor James Brock for his comments on the manuscript. 37 38 KEY WORDS 39 Bone; Bone pain; Skeletal; Skeletal Pain; Artemin, Neurturin, GDNF; GFL; Inflammatory 40 pain 41 42 ABSTRACT 43 Pain associated with skeletal pathology or disease is a significant clinical problem, but the 44 mechanisms that generate and/or maintain it remain poorly understood. In this study, we 45 explored roles for GDNF, neurturin and artemin signaling in bone pain using male Sprague- 46 Dawley rats. We have shown that inflammatory bone pain involves activation and 47 sensitization of peptidergic, NGF-sensitive neurons via artemin/GFRD3 signaling pathways, 48 and that sequestering artemin might be useful to prevent inflammatory bone pain derived 49 from activation of NGF-sensitive bone afferent neurons. In addition, we have shown that 50 inflammatory bone pain also involves activation and sensitization of non-peptidergic neurons 51 via GDNF/GFRD1 and neurturin/GFRD2 signaling pathways, and that sequestration of 2 52 neurturin, but not GDNF, might be useful to treat inflammatory bone pain derived from 53 activation of non-peptidergic bone afferent neurons. Our findings suggest that GDNF family 54 ligand signaling pathways are involved in the pathogenesis of bone pain and could be targets 55 for pharmacological manipulations to treat it. 56 57 SIGNIFICANCE STATEMENT 58 Pain associated with skeletal pathology, including bone cancer, bone marrow edema 59 syndromes, osteomyelitis, osteoarthritis, and fractures causes a major burden (both in terms 60 of quality of life and cost) on individuals and health care systems worldwide. We have shown 61 the first evidence of a role for GDNF, neurturin and artemin in the activation and 62 sensitization of bone afferent neurons, and that sequestering these ligands reduces pain 63 behavior in a model of inflammatory bone pain. Thus, GDNF family ligand signaling 64 pathways are involved in the pathogenesis of bone pain and could be targets for 65 pharmacological manipulations to treat it. 3 66 INTRODUCTION 67 Pain associated with skeletal pathology, including bone cancer, bone marrow edema 68 syndromes, osteomyelitis, osteoarthritis, and fractures causes a major burden (both in terms 69 of quality of life and cost) on individuals and health care systems worldwide. This burden is 70 expected to increase with advances in modern medicine that prolong life expectancy, because 71 many of the conditions that cause bone pain develop late in life. A dominant feature common 72 to almost all conditions that produce bone pain is the release of inflammatory mediators by 73 cells associated with the disease (Bennett, 1988; Goldring, 2000; Haegerstam, 2001; Mantyh, 74 2004; Urch, 2004; Starr et al., 2008; Berenbaum). Opioids and non-steroidal anti- 75 inflammatory drugs (NSAIDs) are generally used to treat mild to severe inflammatory pain, 76 but therapeutic use for bone pain is limited by side effects associated with prolonged use, and 77 because they may interfere with bone remodeling/healing (Bove et al., 2009; Pountos et al., 78 2012; Chrastil et al., 2013). There is a clear need to find alternative strategies to treat bone 79 pain that do not involve the use of NSAIDs or opioids, and are targeted more specifically at 80 mechanisms that generate and/or maintain inflammatory bone pain. 81 82 Nociceptors can be classified into two groups based on their response to nerve growth factor 83 (NGF) or glial cell line-derived neurotrophic factor (GDNF) (Molliver et al., 1995; Snider 84 and McMahon, 1998). Much is known of the role of NGF in inflammatory pain, and this has 85 led to the development of NGF sequestration to treat inflammatory pain, including that 86 associated with bone pathologies. Sequestering NGF by systemic administration of anti-NGF 87 antibodies reduces pain-like behaviors in animal models of bone cancer and fracture-induced 88 pain (Sevcik et al., 2005; Jimenez-Andrade et al., 2007; Koewler et al., 2007), and also 89 inflammatory pain of other tissue systems (Woolf et al., 1994; Ma and Woolf, 1997; Woolf et 90 al., 1997). Furthermore, clinical trials reveal that they resolve pain in at least some human 91 inflammatory pain conditions (Kumar and Mahal, 2012). However, many osteoarthritic 4 92 patients receiving anti-NGF antibodies in a clinical trial developed rapidly progressive 93 osteoarthritis, particularly when used in conjunction with NSAIDs (Seidel et al., 2013; 94 Hochberg, 2015). We propose that manipulation of signaling through the GDNF family of 95 ligands might constitute an alternative target for therapeutic treatment of inflammatory bone 96 pain. 97 98 The GDNF family of ligands (GFLs: GDNF, neurturin, artemin and persephin) act through 99 the receptor tyrosine kinase RET and one of four accessory subunits (GFRD1-4) which confer 100 ligand specificity for the receptor complexes (GDNF/GFRD1, neurturin/GFRD2, 101 artemin/GFRD3, persephin/GFRD4) (Lindsay and Yancopoulos, 1996; Sah et al., 2005). 102 Further specificity of ligand actions is conferred by restricted expression patterns: GFRD1 103 and GFRD2 are confined mostly to non-peptidergic small diameter GDNF-sensitive sensory 104 neurons (Bennett et al., 1998) whereas GFRD3 is found principally in a sub-population of 105 peptidergic small diameter NGF-sensitive sensory neurons (Orozco et al., 2001) that are 106 likely to be artemin-sensitive. GFRD4 is not found in peripheral sensory neurons. There is 107 documented heterogeneity of GFL receptor expression in sensory neurons projecting to 108 different target tissues (Dolatshad and Saffrey, 2007; Malin et al., 2011), but whether this is 109 true of bone afferent neurons is yet to be determined. There is accumulating evidence for a 110 role of GFL signaling in pain (Lindsay and Yancopoulos, 1996; Sah et al., 2005; Bespalov 111 and Saarma, 2007), and there is evidence that the GFLs and GDNF-sensitive neurons 112 contribute to inflammatory pain (Amaya et al., 2004; Lindfors et al., 2006; Jeon et al., 2008; 113 Malin and Davis, 2008). Of key relevance is the finding that GFL levels are increased by 114 inflammation of peripheral tissues in both humans (Okragly et al., 1999; von Boyen et al., 115 2006) and animals (Amaya et al., 2004; Hashimoto et al., 2005; Malin et al., 2006; Ikeda- 116 Miyagawa et al., 2015). In this study, we show evidence of interactions for each of the GFLs 5 117 with bone afferent neurons, and that blocking some of these interactions prevents 118 inflammatory bone pain. 119 120 METHODS 121 Male Sprague Dawley rats weighing between 200-350 g were used in this study. Animals 122 were housed in pairs or groups of four, in a 12/12 hour light/dark cycle, and were provided 123 with food and water ad libitum.