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

Abstract

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.

Measurement of Diurnal Variation in Rod Outer Segment Length In Vivo in Mice With the OCT Optoretinogram

“… 52 , 75 , 76 Ongoing efforts combining AO-OCT, AO-SLO system, 77 , 78 and/or speckle averaging OCT 79 – 81 in mouse photoreceptor imaging also show promise in distinguishing cone backscatter signals from those of the rods in the mouse photoreceptor mosaic. More generally, the approach used here, combined with AO-OCT and AO-SLO should afford efficient use of the ORG in mouse to screen for, and characterize molecular mechanisms that govern rod and cone disc shedding and renewal, 29 , 82 , 83 as well as the mechanisms underlying bleaching-induced outer segments swelling and scattering changes in the posterior eye.…”

Protection of retinal function and morphology in MNU-induced retinitis pigmentosa rats by ALDH2: an in-vivo study

“…Inside the retina, photoreceptors contain a large number of mitochondria, which are highly membranous. In addition, the outer segments of the rod photoreceptors are consist of membranes that have an extremely high proportion of polyunsaturated lipids [13]. These membranous morphologies of retina, plus the frequent formation of reactive oxygen species (ROS) insides, make it extremely susceptible to LPO [14].…”

Biochemistry and physiology of zebrafish photoreceptors

“…They devised a stable line with heat shock–inducible fluorescent membrane protein that allowed them to follow the renewal and shedding of the rod outer segments as an updated experimental approach to the classic radioactive labeling method [ 214 ]. This line was then used in a high-content small-molecule screens that among others identified an involvement of cyclooxygenase in outer segment growth, gamma secretase in outer segment shedding, and mTOR in RPE phagocytosis [ 25 ].…”