A high content, small molecule screen identifies candidate molecular pathways that regulate rod photoreceptor outer segment renewal

www.nature.com/scientificreports OPEN Received: 26 February 2018 Accepted: 5 September 2018 Published: xx xx xxxx A high content, small molecule screen identifies candidate molecular pathways that regulate rod photoreceptor outer segment renewal Leah J. Campbell 1,3 , Megan C. West1 & Abbie M. Jensen1,2 The outer segment of the vertebrate rod photoreceptor is a highly modified cilium composed of many discrete membranous discs that are filled with the protein machinery necessary for phototransduction. The unique outer segment structure is renewed daily with growth at the base of the outer segment where new discs are formed and shedding at the distal end where old discs are phagocytized by the retinal pigment epithelium. In order to understand how outer segment renewal is regulated to maintain outer segment length and function, we used a small molecule screening approach with the transgenic (hsp70:HA-mCherryTM) zebrafish, which expresses a genetically-encoded marker of outer segment renewal. We identified compounds with known bioactivity that affect five content areas: outer segment growth, outer segment shedding, clearance of shed outer segment tips, Rhodopsin mislocalization, and differentiation at the ciliary marginal zone. Signaling pathways that are targeted by the identified compounds include cyclooxygenase in outer segment growth, γ-Secretase in outer segment shedding, and mTor in RPE phagocytosis. The data generated by this screen provides a foundation for further investigation of the signaling pathways that regulate photoreceptor outer segment renewal. Vertebrate photoreceptors are specialized light-sensing neurons with unique morphology that is essential for function. The compartmentalized structure includes a highly modified cilium called the outer segment, which contains densely stacked membranous discs. These discs are packed with the phototransduction machinery that absorbs and converts light into the membrane potential change that alters neurotransmitter release. Blinding diseases such as retinitis pigmentosa and macular degeneration are characterized by degeneration and loss of photoreceptors1,2. Therefore, a better understanding of the cellular maintenance of the photoreceptor outer segment may provide guidance for the design and optimization of treatments to prevent vision loss and restore or prolong vision. The rod photoreceptor outer segment (ROS) contains on the order of 1,000 discrete discs that are stacked perpendicularly to the ciliary axoneme3. In order to supply the ROS with fresh membrane and protein, the ROS undergoes a unique process of continuous renewal. Using autoradiography to detect pulse-labeled H3-proteins, it was observed that new protein-packed discs are regularly added to the base of the ROS4,5. Recent studies give strong evidence to the evagination model of disc formation where new discs develop as evaginations of the ciliary plasma membrane and successive evaginations fuse to form the discrete discs6–8. The tips of outer segments, which contain the oldest discs and associated proteins, are recognized, phagocytosed, and digested diurnally by the neighboring retinal pigment epithelium (RPE)9,10. ROS renewal is regulated, in part, by light as demonstrated by reports that exposure to light inhibits delivery of Rhodopsin to the ROS11 and that the shedding event is initiated by illumination12,13. In addition, phosphodiesterase inhibitors can mimic the dark state to prevent ROS shedding14. Beyond this, our understanding of the mechanisms that regulate renewal are limited. Stress from insults to the system, such as mutations in the protein 1 Biology Department, University of Massachusetts, Amherst, MA, 01003, USA. 2Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, MA, 01003, USA. 3Present address: Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, 46556, USA. Correspondence and requests for materials should be addressed to A.M.J. (email: ) SCientifiC REPOrTs | (2018) 8:14017 | DOI:10.1038/s41598-018-32336-y 1 www.nature.com/scientificreports/ trafficking or ciliary trafficking machinery that disrupt the delivery of molecules to the OS, results in photoreceptor degeneration15. Disruption of RPE phagocytic function caused by mutations in the Mertk receptor in the RPE16–18 and mutations in the Mertk ligands, Gas6 and Protein S19, also result in degeneration. Understanding the regulation of growth and shedding at a molecular level will provide better insight of how renewal is balanced for healthy maintenance of the outer segment. A major obstacle in the progress towards identifying the mechanisms regulating ROS renewal has been the inability to easily and quantitatively measure growth and shedding kinetics. To accelerate progress, we created a transgenic zebrafish, Tg(hsp70:HA-mCherryTM), that allows us to quickly and quantitatively measure ROS growth and shedding20. Given the high amenability of zebrafish to chemical screens21, we screened a library of compounds with known bioactivity using Tg(hsp70:HA-mCherryTM) fish to identify modulators of ROS renewal. In addition to the primary objective of identifying molecular pathways involved in ROS renewal, we also examined Rhodopsin localization, clearance of shed ROS material by the RPE, and the addition of new rod photoreceptors from the ciliary marginal zone (CMZ) of the retina. Results High content screen of 1351 bioactive compounds on ROS renewal. ROS renewal occurs only in the intact retina, and efforts to recapitulate the process by culturing rods have been unsuccessful22. The zebrafish offers several unique advantages as an in vivo model for ROS renewal studies, including the amenability for compound screening. The zebrafish retina develops rapidly with large numbers of ROS present as early as 5 days post fertilization (dpf)23. It is easy to generate the large numbers of individual animals needed for a bioactive compound screen, and compounds can be delivered by bathing the zebrafish in small volumes of water that require small amounts of compound. Finally, the Tg(hsp70:HA-mCherryTM) line provides a quicker method for measuring ROS renewal kinetics than the time-consuming and cumbersome autoradiography, which relies on detection of radioactively-labeled proteins and has been rarely used since the 1970s4,5,20. To identify potential pathways that regulate ROS renewal, we tested 1351 compounds with known bioactivity. Figure 1 describes the screening approach. At 6 dpf, Tg(hsp70:HA-mCherryTM); Tg(Xla.rho:EGFP); alb−/− fish were heat shocked to induce expression of the HA-mCherryTM marker of ROS renewal (mCherryTM stripe) and then immediately transferred to tissue culture wells containing 20 μM of compound or 0.2% DMSO (vehicle control). At 10 dpf (4 days post heat shock), fish were fixed and processed for immunofluorescence (Fig. 1a). Antibodies against GFP and the HA-epitope in the mCherryTM stripe were used to exten (...truncated)