Comparison of rod photoreceptor outer segment renewal in wild type and Tmem138-deficient mice using AAV-delivered Dendra2-tagged rhodopsin.

Visualization of photoreceptor outer segment (OS) renewal dynamics is essential for vision research but has proved difficult in mice due to the small size and dense packing of their photoreceptors. Lack of effective protein “trackers” and the time-consuming generation of transgenic reporter lines add to these challenges. In this study, we evaluated AAV-mediated delivery of photoconvertible Rhodopsin/Dendra2 and Peripherin2/Dendra2 fusion proteins as a means to track OS renewal in mouse photoreceptors. OS renewal was assessed by two approaches: (1) comparing the lengths of native (green) Dendra2 domains at two post-infection time points, and (2) using photoconversion of Dendra2 to distinguish newly synthesized discs from preexisting ones within the same cell. We validated this method in both wild type mice and a Tmem138-deficient ciliopathy mutant, in which a reduced OS renewal rate was observed. Taken together, this method offers a rapid genetic tool for “real-time” evaluation of OS renewal dynamics in mice, overcoming limitations posed by the compact and densely organized photoreceptor architecture.

SummaryVertebrate photoreceptors are specialized light sensing neurons. The photoreceptor outer segment is a highly modified cilium where photons of light are transduced into a chemical and electrical signal. The outer segment has the typical cilary axoneme but, in addition, it has a large number of densely packed, stacked, intramembranous discs. The molecular and cellular mechanisms that contribute to vertebrate photoreceptor outer segment morphogenesis are still largely unknown. Unlike typical cilia, the outer segment is continuously regenerated or renewed throughout the life of the animal through the combined process of distal outer segment shedding and proximal outer segment growth. The process of outer segment renewal was discovered over forty years ago, but we still lack an understanding of how photoreceptors renew their outer segments and few, if any, molecular mechanisms that regulate outer segment growth or shedding have been described. Our lack of progress in understanding how photoreceptors renew their outer segments has been hampered by the difficulty in measuring rates of renewal. We have created a new method that uses heat-shock induction of a fluorescent protein that can be used to rapidly measure outer segment growth rates. We describe this method, the stable transgenic line we created, and the growth rates observed in larval and adult rod photoreceptors using this new method. This new method will allow us to begin to define the genetic and molecular mechanisms that regulate rod outer segment renewal, a crucial aspect of photoreceptor function and, possibly, viability.

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.

Morphogenesis and renewal of cone outer segments

PurposeGalectin-3 (gal-3) is a soluble glycoprotein that has been associated with diverse forms of phagocytosis, including some mediated by the engulfment receptor MerTK. Retinal pigment epithelium (RPE) in vivo uses MerTK (or the related Tyro3) for phagocytosis of shed outer segment fragments during diurnal outer segment renewal. Here, we test if gal-3 plays a role in outer segment renewal in mice and if exogenous gal-3 can promote MerTK-dependent engulfment of isolated outer segment fragments by primary RPE cells in culture.MethodsWe explored age- and strain-matched wild-type (wt), lgals3−/− and mertk−/− mice. Immunofluorescence and immunoblotting characterized gal-3 and RPE/retina protein expression, respectively. Outer segment renewal was investigated by live imaging of phosphatidylserine (PS) exposure on photoreceptor outer segment distal tips and by microscopy of rhodopsin-labeled RPE phagosomes in tissue sections. Retinal function was assessed by recording electroretinograms (ERGs). Phagocytosis assays feeding purified outer segment fragments (POS) were conducted with added recombinant proteins testing unpassaged primary mouse RPE.ResultsGal-3 localizes to neural retina and RPE in wt mice. The lgals3−/− photoreceptor outer segments display normal diurnal PS exposure at distal tips. The number of rhodopsin-positive phagosomes in wt and lgals3−/− RPE does not differ at peak or trough of diurnal phagocytosis activity. lgals3−/− mice show light responses like wt, and their eyes contain wt levels of retinal and RPE proteins. Unlike purified protein S, recombinant gal-3 fails to promote POS engulfment by mouse primary RPE in culture.ConclusionsGal-3 has no essential role in MerTK-dependent outer segment renewal in mice.

The visual system is essential for humans to perceive the environment. In the retina, rod and cone photoreceptor neurons are the initial sites where vision forms. The apical region of both cone and rod photoreceptors contains a light-sensing organelle known as the outer segment (OS), which houses tens of thousands of light-sensitive opsins. The OSs of photoreceptors are not static; they require rhythmic renewal to maintain normal physiological functions. Disruptions in OS renewal can lead to various genetic disorders, such as retinitis pigmentosa (RP). Understanding the patterns and molecular mechanisms of photoreceptor OS renewal remains one of the most intriguing topics in visual biology. This review aims to elucidate the structure of photoreceptor OSs, the molecular mechanisms underlying photoreceptor OS renewal, and the retinal diseases resulting from defects in this renewal process. Additionally, we will explore retinal diseases related to photoreceptor OS renewal and potential therapeutic strategies, concluding with a discussion on future research directions for OS renewal.

Visual performance depends in the initial stages on the properties of retinal photoreceptors. In this context we consider the effects of rod outer segment (OS) renewal and OS response gradient on performance. The OS renews itself by forming new membrane near the cilium, at the base of the OS. This membrane then advances towards the tip where it is shed and phagocytosed in the pigment epithelium. Thus the OS is older at the tip than the base, and it has been proposed that it is due to this aging that the observed light responses are smaller and slower at the tip than at the base. We have tested this hypothesis by exposing Xenopus laevis in a controlled manner to altered temperature and lighting conditions which are known either to slow down or to accelerate OS renewal. We have recorded the light responses of the OS base and tip and found that they are, indeed, correlated with the age differences. In addition to OS membrane age we have also investigated the effects of animal age on the response gradient along the OS. Here we found that during development the response differences decrease and then stabilise at maturity. Concurrently with the decrease of the gradient the response kinetics are slowed down. There is thus a changing trade-off between response gradient and kinetics up to maturity in this animal. These results have important implications for visual performance. Visual reaction times depend on photoreceptor kinetics; and since photons can be absorbed anywhere along the OS, the response gradient, especially at low light levels, directly affects the reliability of detecting stimulus intensity. We conclude that visual performance in Xenopus laevis is related to the rates of OS renewal and the resulting response gradient in an age-dependent manner.

Vision begins in the outer segment compartment of photoreceptor cells, which is constantly renewed through the addition of membrane material at its base and ingestion of mature membranes at its tip by the retinal pigment epithelium (RPE). The close apposition of outer segments to the RPE is believed to be critical for maintaining this renewal process. Yet, in several retinal diseases, expansion of the subretinal space separating photoreceptors from the RPE does not immediately impact photoreceptor functionality. Here, we analyzed outer segment function and renewal in the Adam9 knockout mouse characterized by a major expansion of the subretinal space. Surprisingly, photoreceptor-RPE separation affected neither the sensitivity of photoreceptor light-responses nor the normal rate of outer segment renewal in this mouse prior to the onset of photoreceptor degeneration. The latter is achieved through the formation of elongated RPE “pseudopods” extending across the enlarged subretinal space to ingest outer segment tips. This work suggests that pseudopod formation may underlie the persistence of photoreceptor function in human diseases accompanied by photoreceptor-RPE separation, such as vitelliform macular dystrophy or age-related macular degeneration associated with subretinal drusenoid deposits.

Vision begins in the outer segment compartment of photoreceptor cells, which is constantly renewed through the addition of membrane material at its base and ingestion of mature membranes at its tip by the retinal pigment epithelium (RPE). The close apposition of outer segments to the RPE is believed to be critical for maintaining this renewal process. Yet, in several retinal diseases, expansion of the subretinal space separating photoreceptors from the RPE does not immediately impact photoreceptor functionality. Here, we analyzed outer segment function and renewal in the Adam9-knockout mouse characterized by a major expansion of the subretinal space. Surprisingly, photoreceptor-RPE separation affected neither the sensitivity of photoreceptor light responses nor the normal rate of outer segment renewal in this mouse prior to the onset of photoreceptor degeneration. The latter is achieved through the formation of elongated RPE pseudopods extending across the enlarged subretinal space to ingest outer segment tips. This work suggests that pseudopod formation may underlie the persistence of photoreceptor function in human diseases accompanied by photoreceptor-RPE separation, such as vitelliform macular dystrophy or age-related macular degeneration associated with subretinal drusenoid deposits.

Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal pigment epithelial (RPE). It is a strictly rhythmic, synchronized process that underlies in part circadian regulation. We highlight findings from recently developed methods that quantify distinct phases of outer segment renewal in retinal tissue. At light onset, outer segments expose the conserved "eat-me" signal phosphatidylserine exclusively at their distal, most aged tip. A coordinated two-receptor efferocytosis process follows, in which ligands bridge outer segment phosphatidylserine with the RPE receptors αvβ5 integrin, inducing cytosolic signaling toward Rac1 and focal adhesion kinase/MERTK, and with MERTK directly, additionally inhibiting RhoA/ROCK and thus enabling F-actin dynamics favoring outer segment fragment engulfment. Photoreceptors and RPE persist for life with each RPE cell in the eye servicing dozens of overlying photoreceptors. Thus, RPE cells phagocytose more often and process more material than any other cell type. Mutant mice with impaired outer segment renewal largely retain functional photoreceptors and retinal integrity. However, when anti-inflammatory signaling in the RPE via MERTK or the related TYRO3 is lacking, catastrophic inflammation leads to immune cell infiltration that swiftly destroys the retina causing blindness.

Skeletal muscle atrophy occurs under a variety of conditions and can result from alterations in both protein synthesis and protein degradation. The muscle-specific E3 ubiquitin ligases, MuRF1 and MAFbx, are excellent markers of muscle atrophy and increase under divergent atrophy-inducing conditions such as denervation and glucocorticoid treatment. While deletion of MuRF1 or MAFbx has been reported to spare muscle mass following 14 days of denervation, their role in other atrophy-inducing conditions is unclear. The goal of this study was to determine whether deletion of MuRF1 or MAFbx attenuates muscle atrophy after 2 weeks of treatment with the synthetic glucocorticoid dexamethasone (DEX). The response of the triceps surae (TS) and tibialis anterior (TA) muscles to 14 days of DEX treatment (3 mg kg(-1) day(-1)) was examined in 4 month-old male and female wild type (WT) and MuRF1 or MAFbx knock out (KO) mice. Following 14 days of DEX treatment, muscle wet weight was significantly decreased in the TS and TA of WT mice. Comparison of WT and KO mice following DEX treatment revealed significant sparing of mass in both sexes of the MuRF1 KO mice, but no muscle sparing in MAFbx KO mice. Further analysis of the MuRF1 KO mice showed significant sparing of fibre cross-sectional area and tension output in the gastrocnemius (GA) after DEX treatment. Muscle sparing in the MuRF1 KO mice was related to maintenance of protein synthesis, with no observed increases in protein degradation in either WT or MuRF1 KO mice. These results demonstrate that MuRF1 and MAFbx do not function similarly under all atrophy models, and that the primary role of MuRF1 may extend beyond controlling protein degradation via the ubiquitin proteasome system.

To determine whether photoreceptor degeneration in the Ozark cave salamander is associated with cessation or changes in the kinetics of outer segment (OS) renewal, an autoradiographic study of 3H-leucine incorporation in photoreceptors was carried out. Six days after isotope injection rods and cones showed labeling in both inner and outer segments. Cone OS were diffusely labeled whereas rods contained a band of radioactivity at the base of the OS. At 13 and 21 days the radioactive band in rods was located progressively nearer the distal tip of the OS. The rate of rod OS renewal ranged from 0.30 to 0.38 mu of OS length per day at 18 degrees C. L-thyroxin induced metamorphosis and light increased the renewal rate compared to larvae in darkness, and adults with photoreceptors in an early stage of degeneration had a slightly higher renewal rate than larvae. Light and electron microscope autoradiographs of degenerate photoreceptors revealed that even in the final stages of degeneration when OS are reduced to small, irregular whorls of membrane, 3H-leucine labeling was present in inner segments and OS membranes. These observations demonstrate that OS renewal occurs in both larvae and adults, and suggest that photoreceptor degeneration may be due to disruption of some aspect of the OS disposal process.

In all mammalian species tested to date, rod photoreceptor outer segment renewal is a circadian process synchronized by light with a burst of outer segment fragment (POS) shedding and POS phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning at light onset. Recent reports show that RPE phagocytosis also increases shortly after dark onset in C57BL/6 (C57) mice. Genetic differences between C57 mice and 129T2/SvEmsJ (129) mice may affect regulation of outer segment renewal. Here, we used quantitative methods to directly compare outer segment renewal in C57 and 129 mouse retina. Quantification of rhodopsin-positive phagosomes in the RPE showed that in 129 mice, rod POS phagocytosis after light onset was significantly increased compared to C57 mice, but that 129 mice did not show a second peak after dark onset. Cone POS phagosome content of RPE cells did not differ by mouse strain with higher phagosome numbers after light than after dark. We further quantified externalization of the “eat me” signal phosphatidylserine by outer segment tips, which precedes POS phagocytosis. Live imaging of retina ex vivo showed that rod outer segments extended PS exposure in both strains but that frequency of outer segments with exposed PS after light onset was lower in C57 than in 129 retina. Taken together, 129 mice lacked a burst of rod outer segment renewal after dark onset. The increases in rod outer segment renewal after light and after dark onset in C57 mice were attenuated compared to the peak after light onset in 129 mice, suggesting an impairment in rhythmicity in C57 mice.

Role of Blood- and Tissue-Associated Inducible Nitric-Oxide Synthase in Colonic Inflammation

PurposeCircadian clocks in the eye are critical for normal visual function and they relate to periodical renewal of photoreceptor outer segments as well as oxidative stress. Impaired rhythm can thus contribute to the pathogenesis of age‐related macular degeneration (AMD). Chronic oxidative stress leads eventually to protein aggregation in combination with impaired autophagy. We hypothesise that pinosylvin, a polyphenolic compound, slows down the progression of AMD through autophagy induction.MethodsWe investigated the relative change in electroretinogram (ERG) metrics of mice raised in 12:12 h light‐dark cycle and after a 24 h two‐week dark period in wild type (WT; n = 10), PGC1‐α knock‐out (KO; n = 7) and Nrf2 KO (n = 7) mice aged 9.7 ± 3.4 months. From these, 9 WT, 5 PGC1‐α and 7 Nrf2 KO mice continued to be raised to an age of 17.3 ± 3.8 months and assigned to a treatment and control group and fed for 2 months with pinosylvin‐feed or regular‐feed prior to recording ERG.ResultsThe two‐week dark period resulted in significantly greater b‐wave amplitudes and shorter b‐wave latencies in WT mice in photopic ERG. Nrf2 KO and PGC1‐α/Nrf2 dKO mice exhibited b‐wave latency changes in the opposite. The dKO also had diminished b‐wave amplitudes. Scotopic ERG yielded no significant changes. Pinosylvin treatment in Nrf2 KO and PGC1‐α KO mice caused significantly increased b‐wave amplitudes in photopic ERG. In scotopic ERG, the pinosylvin treated mice had larger a‐ and b‐wave amplitudes in WT and Nrf2 KO mice, but not in PGC1‐α KO mice.ConclusionsPGC1‐α KO and Nrf2 KO mice showed different circadian rhythmicity. Pinosylvin treatment showed improved ERG signalling in aged NRf2 KO and WT mice.