This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Cellular/Molecular The role of the microglial Cx3cr1 pathway in the post-natal maturation of retinal photoreceptors Andrew I. Joblinga, Michelle Waugha, Kirstan A. Vesseya, Joanna A. Phippsa, Lidia Trogrlica, Una Greferatha, Samuel A. Millsa, Zhi L. Tana, Michelle M. Warda and Erica L. Fletcher aa aDepartment of Anatomy and Neuroscience. The University of Melbourne, Parkville 3010 Victoria, Australia. DOI: 10.1523/JNEUROSCI.2368-17.2018 Received: 21 August 2017 Revised: 22 March 2018 Accepted: 10 April 2018 Published: 18 April 2018 Author contributions: A.I.J., M.W., and E.F. designed research; A.I.J., M.W., K.A.V., J.A.P., L.T., U.G., S.A.M., Z.L.T., and M.M.W. performed research; K.A.V., J.A.P., and E.F. contributed unpublished reagents/analytic tools; A.I.J., M.W., K.A.V., L.T., U.G., S.A.M., Z.L.T., and M.M.W. analyzed data; A.I.J. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was supported by the National Health and Medical Research Council of Australia (#1061418 to E.L.F. and A.I.J.; #1061419 to E.L.F. and K.A.V.) and the Victorian Science Agenda (E.L.F.). The authors would like to thank Gene Venables for processing and imaging the acetylated #-tubulin and Horace (Wing Hei) Chan for his help with the photoreceptor cilium diagram. We would also like to thank Associate Professor Paulo Ferreira from Duke University Medical centre and Dr Tiansen Li from the National Eye Institute for their donation of antibodies to probe the photoreceptor cilium. Corresponding author: Prof. Erica L. Fletcher. Department of Anatomy & Neuroscience. The University of Melbourne, Grattan St, Parkville 3010, Victoria, Australia. Email: [email protected]; Tel: +61-3-8344-3218; Fax: +61-3-9347-5219 Cite as: J. Neurosci ; 10.1523/JNEUROSCI.2368-17.2018 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: The role of the microglial Cx3cr1 pathway in the post-natal maturation of retinal 2 photoreceptors. 3 Abbreviated title: Microglia regulate photoreceptor maturation. 4 Author affiliation: Andrew I. Joblinga, Michelle Waugha, Kirstan A. Vesseya, Joanna 5 A. Phippsa, Lidia Trogrlica, Una Greferatha, Samuel A. Millsa, Zhi L. Tana, Michelle M. 6 Warda and Erica L. Fletchera 7 a Department of Anatomy and Neuroscience. The University of Melbourne, Parkville 8 3010 Victoria, Australia. 9 Corresponding author: Prof. Erica L. Fletcher. Department of Anatomy & 10 Neuroscience. The University of Melbourne, Grattan St, Parkville 3010, Victoria, 11 Australia. Email: [email protected]; Tel: +61-3-8344-3218; Fax: +61-3-9347-5219 12 Number of pages: 50 13 Number of figures: 10 14 Number of tables: 3 15 Number of words for abstract: 247 16 Number of words for introduction, including citations: 675 17 Number of words for discussion, including citations: 1575 18 Conflict of interest: The authors declare no conflict of interest. 19 Acknowledgments: This work was supported by the National Health and Medical 20 Research Council of Australia (#1061418 to E.L.F. and A.I.J.; #1061419 to E.L.F. 21 and K.A.V.) and the Victorian Science Agenda (E.L.F.). The authors would like to 22 thank Gene Venables for processing and imaging the acetylated α-tubulin and 23 Horace (Wing Hei) Chan for his help with the photoreceptor cilium diagram. We 24 would also like to thank Associate Professor Paulo Ferreira from Duke University 1 25 Medical centre and Dr Tiansen Li from the National Eye Institute for their donation of 26 antibodies to probe the photoreceptor cilium. 27 2 28 Abstract 29 Microglia are the resident immune cells of the CNS and their response to infection, 30 injury and disease is well documented. More recently, microglia have been shown to 31 play a role in normal CNS development, with the fractalkine-Cx3cr1 signalling 32 pathway of particular importance. This work describes the interaction between the 33 light sensitive photoreceptors and microglia during eye opening, a time of post-natal 34 photoreceptor maturation. Genetic removal of Cx3cr1 (Cx3cr1GFP/GFP), led to an early 35 retinal dysfunction soon after eye opening (P17) and cone photoreceptor loss (P30 36 onwards) in mice of either sex. This dysfunction occurred at a time when fractalkine 37 expression was predominantly outer retinal, when there was increased microglial 38 presence near the photoreceptor layer and increased microglial-cone photoreceptor 39 contacts. Photoreceptor maturation and outer segment elongation was coincident 40 with increased opsin photopigment expression in wild-type retina, while this was 41 aberrant in the Cx3cr1GFP/GFP retina and outer segment length was reduced. A 42 beadchip array highlighted Cx3cr1 regulation of genes involved in the photoreceptor 43 cilium, a key structure important for outer segment elongation. This was confirmed 44 with quantitative PCR with specific cilium-related genes, Rpgr and Rpgrip1, 45 downregulated at eye opening (P14). While overall cilium structure was unaffected, 46 expression of Rpgr, Rpgrip1 and centrin were restricted to more proximal regions of 47 the transitional zone. This study highlighted a novel role for microglia in post-natal 48 neuronal development within the retina, with loss of fractalkine-Cx3cr1 signalling 49 leading to an altered distribution of cilium proteins, failure of outer segment 50 elongation and ultimately cone photoreceptor loss. 3 51 Significance Statement 52 Microglia are involved in CNS development and disease. This work highlights the 53 role of microglia in post-natal development of the light detecting photoreceptor 54 neurons within the mouse retina. Loss of the microglial Cx3cr1 signalling pathway 55 resulted in specific alterations in the cilium, a key structure in photoreceptor outer 56 segment elongation. The distribution of key components of the cilium transitional 57 zone, Rpgr, Rpgrip1 and centrin were altered in retina lacking Cx3cr1, with reduced 58 outer segment length, and cone photoreceptor death observed at later post-natal 59 ages. This work identifies a novel role for microglia in the post-natal maturation of 60 retinal photoreceptors. 61 4 62 Introduction 63 Microglia are the resident immune cells of the central nervous system (CNS), 64 forming part of the innate immune system (Waisman et al., 2015). Microglia arise 65 from myeloid progenitor cells in the yolk sac and colonise the brain and retina early 66 during embryonic development (Santos et al., 2008; Ginhoux et al., 2010). Once 67 established, they survey the local environment, extending and contracting their 68 processes, contacting neurons, blood vessels and other glia. As modulators of the 69 immune environment, their response to injury and infection has been well studied 70 and is characterised by the presence of an amoeboid phenotype, production of 71 inflammatory cytokines and / or macrophage-like phagocytic activity (Colton, 2009; 72 Kettenmann et al., 2011). Given the importance of inflammation in CNS pathology, 73 the role of microglia in neurodegeneration within the brain, spinal cord and retina has 74 been extensively explored (Combadiere et al., 2007; Kigerl et al., 2009; Fuhrmann et 75 al., 2010). 76 77 More recently, the role of microglia in establishing tissue architecture and 78 maintaining homeostasis has received some attention. During CNS development, 79 neuronal number is tightly regulated, with a subset of cells targeted for programmed 80 cell death. Microglia actively regulate key aspects of this process, in addition to 81 removing dead or dying cells (Frost and Schafer, 2016). Studies performed in the 82 brain, retina and spinal cord have shown that depletion of microglia, reduce the 83 number of dead or dying cells thereby increasing neuronal cell number (Frade and 84 Barde, 1998; Sedel et al., 2004; Hose et al., 2005; Cunningham et al., 2013). While 85 the exact mechanism remains to be fully elucidated, microglial-derived factors such 86 as nerve growth factor, insulin-like growth factor and interleukin-1b have been 5 87 identified as key trophic factors (Frade and Barde, 1998; Ueno et al., 2013; 88 Shigemoto-Mogami et al., 2014). In addition to modulating neuronal fate, microglia 89 are known to make transient, activity-dependent contact with synapses thereby 90 regulating neuronal circuitry (Wake et al., 2009; Tremblay et al., 2010). Through this 91 interaction, microglia eliminate immature, less active synapses via the complement 92 system (Schafer et al., 2012; Bialas and Stevens, 2013). A recent study also 93 suggests that adult retinal synapses are dependent upon microglial involvement 94 (Wang et al., 2016). Because of this modulation, it is perhaps not surprising that 95 several reports suggest microglia affect overall brain function and behaviour (Bilbo et 96 al., 2005; Parkhurst et al., 2013). 97 98 Microglial-neuronal interactions are regulated by several signalling pathways, 99 including fractalkine-Cx3cr1. Fractalkine (Cx3cl1) can exist in transmembrane and 100 soluble forms and is mostly expressed by neurons, while its sole receptor Cx3cr1, is 101 predominantly found on microglia within the CNS. Work undertaken in the retina has 102 indicated that the dynamics of microglial processes are modulated by 103 fractalkine/Cx3cr1 signalling (Liang et al., 2009), while microglial migration is 104 impaired in the somatosensory barrel cortex of Cx3cr1-deficient mice (Hoshiko et al., 105 2012; Arnoux and Audinat, 2015). These data suggest that fractalkine/Cx3cr1 106 signalling has a key role in regulating microglia-neuronal interaction. This is 107 supported by work showing that loss of Cx3cr1 signalling leads to altered synapse 108 formation in the brain and retina, impaired trophic support and impaired or delayed 109 synaptic pruning within the hippocampus and barrel cortex (Paolicelli et al., 2011; 110 Hoshiko et al., 2012; Ueno et al., 2013; Wang et al., 2016).