Endosomal sorting by Semaphorin 4A in retinal pigment epithelium supports photoreceptor survival.

Photoreceptor cell death is the hallmark of a group of human inherited retinal degeneration. Although the causative genetic mutations are often known, the mechanisms leading to photoreceptor degeneration remain poorly defined. Here, we show that Semaphorin 4A (Sema4A), a member of axonal guidance molecule semaphorin, plays a role in Rab11/FIP2-mediated endosomal sorting in retinal pigment epithelial cells to support photoreceptor function. In response to oxidative stress, Sema4A switches the endosomal sorting of the lysosomal precursor protein prosaposin from the lysosome to the exosomal release, which prevents light-induced photoreceptor apoptosis. In the absence of oxidative stress, Sema4A sorts retinoid-binding proteins with retinoids between the cell surface and endoplasmic reticulum, by which 11-cis-retinal, a chromophore for phototransduction, is regenerated and transported back to photoreceptors. Owing to defects in these processes, Sema4A-deficient mice exhibit marked photoreceptor degeneration. Our findings therefore indicate that Sema4A regulates two distinct endosomal-sorting pathways that are critical for photoreceptor survival and phototransduction during the transition between daylight and darkness.

Molecular characterization of visual pigments in Branchiopoda and the evolution of opsins in Arthropoda.

Studies on color vision in invertebrates have focused primarily on insect visual pigments, with little attention given to crustacean visual pigments. None of the blue-green-, blue-, or ultraviolet (UV)-sensitive-opsins have been identified in crustaceans. In addition, the discussion of visual pigments has been limited to long-wavelength-sensitive opsins in Pancrustacea. Here, we focused on Branchiopoda (Crustacea), which is a sister group of Hexapoda including insects. In the tadpole shrimp Triops granarius, the visual pigment chromophore was retinal. Multiple opsins were isolated from each of three branchiopod species, T. granarius, Triops longicaudatus, and the fairy shrimp Branchinella kugenumaensis (five, five, and four opsins from these species, respectively). Phylogenetic analyses and the presence of a lysine residue corresponding to position 90 in bovine rhodopsin suggested that three of the branchiopod opsins comprise UV-sensitive pigments. In addition, the phylogenetic relationships between insect and branchiopod UV-sensitive opsins revealed that the divergence of blue- and UV-sensitive pigments predates the Branchiopoda and Insecta divergence. The other branchiopod opsins show distant relationships to other known insect opsins and form novel clusters. The present results strongly suggest that the ancestral arthropod of the Chelicerata-Pancrustacea lineages possessed at least four types of opsins. The ancestors of Pancrustacea and the Insecta-Branchiopoda lineages possessed at least five and six types of opsins, respectively. Our results suggest that in the evolutionary process associated with each lineage, several opsins appeared and diversified with repeated gene duplication, of which some have been lost in some taxa.

Photoreactivation in Paramecium tetraurelia under conditions of various degrees of ozone layer depletion.

Photoreactivation (PR) is an efficient survival mechanism that helps protect cells against the harmful effects of solar-ultraviolet (UV) radiation. The PR mechanism involves photolyase, just one enzyme, and can repair DNA damage, such as cyclobutane-pyrimidine dimers (CPD) induced by near-UV/blue light, a component of sunlight. Although the balance of near-UV/blue light and far-UV light reaching the Earth's surface could be altered by the atmospheric ozone layer's depletion, experiments simulating this environmental change and its possible effects on life have not yet been performed. To quantify the strength of UVB in sunlight reaching the Earth's surface, we measured the number of CPD generated in plasmid DNA after UVB irradiation or exposure to sunlight. To simulate the increase of solar-UV radiation resulting from the ozone layer depletion, Paramecium tetraurelia was exposed to UVB and/or sunlight in clear summer weather. PR recovery after exposure to sunlight was complete at a low dose rate of 0.2 J/m2 x s, but was less efficient when the dose rate was increased by a factor of 2.5 to 0.5 J/m2 x s. It is suggested that solar-UV radiation would not influence the cell growth of P. tetraurelia for the reason of high PR activity even when the ozone concentration was decreased 30% from the present levels.

Identification of 3,4-didehydroretinal isomers in the Xenopus tadpole tail fin containing photosensitive melanophores.

It is well characterized that melanophores in the tail fin of Xenopus laevis tadpoles are directly photosensitive. In order to better understand the mechanism underlying this direct photosensitivity, we performed a retinal analysis of the tail fins and eyes of Xenopus tadpoles at stages 51-56 using high performance liquid chromatography (HPLC). Following the extraction of retinoids by the formaldehyde method, a fraction containing retinal and/or 3,4-didehydroretinal isomers from the first HPLC analysis were collected. These isomers were then reduced by sodium borohydride to convert retinal and/or 3,4-didehydroretinal isomers into the corresponding retinol isomers to prepare for a second HPLC analysis. Peaks of 11-cis and all-trans 3,4-didehydroretinol were detected in the eyes and tail fins containing melanophores, but they were not detected in the tail fins without melanophores. The amounts of 11-cis and all-trans 3,4-didehydroretinol were 27.5 and 5.7 fmol/fin, respectively, and the total quantity of 3,4-didehydroretinal was calculated at approximately 5 x 10(6) molecules/melanophore. These results strongly suggest the presence of 11-cis and all-trans 3,4-didehydroretinal in melanophores of the tadpole tail fin, which probably function as the chromophore of photoreceptive molecules.

Retinoid composition and retinal localization in the eggs of teleost fishes.

Retinoids in the eggs of four teleosts, chum salmon (Oncorhynchus keta), black porgy (Acanthopagrus schlegeli), marbled flounder (Pleuronectes yokohamae), and stingfish (Inimicus japonicus), were analyzed by high performance liquid chromatography. Retinal (RAL1) or both RAL1 and 3,4-didehydroretinal (RAL2) were major or exclusive retinoids in the eggs of every species examined. In O. keta eggs, both RAL1 and RAL2 were present at the ratio of approximately 3:4, whereas RAL1 was the only retinal in the eggs of the other three marine species. RAL1 was the exclusive retinoid in the eggs of P. yokohamae and I. japonicus, whose eggs lack lipid bodies. In the eggs of O. keta and A. schlegeli, which have lipid bodies, retinylesters were also detected, and retinals composed 69% and 93%, respectively, of total retinoids. In O. keta eggs, retinals were present mostly in the aqueous part and were bound to a protein homologous to lipovitellin 1, an amphibian yolk protein, and retinylesters were located in lipids. These results indicate that retinals are the essential mode of retinoid storage in eggs of teleosts and they are the precursors of functional retinoids, such as retinoic acid and visual pigment chromophores. Retinylesters are additional retinoids that accompany lipid accumulation.

Storage of retinal in the eggs of the ascidian, Halocynthia roretzi.

Retinoids in the eggs of the solitary ascidian, Halocynthia roretzi, were analyzed by high performance liquid chromatography. Retinal was the almost exclusive retinoid (>>99%), and the concentration of retinal was 25.9-40.1 (30.6 on average) ng/mg of protein. The egg retinal consisted of four isomers: all-trans (50.9%), 9-cis (6.8%), 11-cis (20.4%) and 13-cis (21.9%). The presence of retinal in the eggs of this ascidian is a characteristic shared with the wide range of oviparous vertebrates, although the isomer composition differs between ascidian eggs and vertebrate eggs; in vertebrate eggs, almost all the retinal is in the all-trans form. The egg retinal was bound to a protein complex via a Schiff base linkage. The electrophoretic characteristics of the protein complex were similar to that of egg yolk proteins of oviparous vertebrates. The results presented in this study strongly suggest that, as is found with oviparous vertebrates, retinal in the ascidian eggs is the essential mode of retinoid storage, and is the precursor of photoreceptive pigment chromophores and retinoic acid during development.

Retinal is the essential form of retinoid for storage and transport in the adult of the ascidian Halocynthia roretzi.

Retinoids in the organs (gonad [GND], body wall muscle [BWM], hepatopancreas [HP], gill, hemolymph cells and hemolymph plasma) of the adult ascidian Halocynthia roretzi were analyzed by high performance liquid chromatography. Retinal (RAL) occurred in every organ examined, and most of RAL (>/=99%) was localized in the GND and BWM. None of the organs contained significant amounts of retinol (ROL) or retinyl ester (RE). Lipid droplets, which are characteristic of stellate cells (RE-storing cells of vertebrates), could not be found in the GND, BWM and HP by microscopic observations. These results indicate that this ascidian lacks the RE-storing mechanism, which is ubiquitous in adult vertebrates. The amount and localization of RAL showed the annual change in relation to the reproductive cycle. During summer, the growing season, RAL was present in both GND and BWM at a ratio of about 3:2. From summer to winter, RAL in the GND gradually increased, concomitant with the decrease of RAL in the BWM. In winter, the spawning season, most of RAL was present in the GND (ca. 98%). RAL appears to be accumulated first in the BWM and transported to oocytes accompanying yolk accumulation. ROL and RE were not implicated in the storage and transport of retinoids. The results in the present research strongly suggest that retinoic acid (RA) is produced by the two-step enzymatic reaction: carotenoid cleavage to RAL followed by RAL oxidation to RA and that the prevertebrate chordate lacks ROL-metabolizing systems.

Retinoid storage in the egg of reptiles and birds.

Storage of retinal has been confirmed in eggs from a range of anamniotic vertebrates (teleosts and amphibians) and an ascidian, but the retinoid-storage state in eggs of oviparous amniotic vertebrates (reptiles and birds) has yet to be clarified in detail. We studied four reptilian and five avian species and found that retinal was commonly stored in their egg yolk. Furthermore, retinal was the major retinoid in reptilian eggs, with only low levels of retinol, whereas significant amounts of retinol as well as retinal were stored in avian eggs. In both reptilian and avian eggs, retinal was commonly bound to proteins, which were assumed to be homologous to the proteins that bind retinal in the eggs of anamniotic vertebrates. Despite the common storage state of retinal, retinol would be bound to different proteins. In the reptilian eggs, retinol was found in the yolk-granule fraction, which also contained retinal. However, retinol in avian eggs was found largely in the yolk-plasma fraction, separate from retinal. These results suggest that retinol storage in avian eggs acquired after the divergence of birds from the reptiles, while retinal storage was acquired before the appearance of the vertebrates, and has subsequently been conserved during evolution of oviparous vertebrates.

Distribution of retinylester-storing stellate cells in the arrowtooth halibut, Atheresthes evermanni.

Hepatic stellate cells play a major role in retinylester storage in mammals, but the retinoid-storing state in nonmammalian vertebrates remains to be elucidated. In this study, we examined retinoids and retinoid-storing cells in the arrowtooth halibut, Atheresthes evermanni. High-performance liquid chromatography analyses revealed the highest concentrations of stored retinoids (retinol and retinylester, 6199 nmol/g) in the pyloric cecum, a teleost-specific organ protruding from the intestine adjacent to the pylorus. Considerable amounts of retinoids were also stored in the intestine (3355 nmol/g) and liver (1891 nmol/g), and small amounts in the kidney (102 nmol/g). Very small amounts or no retinoids were detected in the heart, gill, skeletal muscle, and gonads (less than 2 nmol/g). Use of gold chloride staining and fluorescence microscopy to detect retinoid autofluorescence showed that, in the pyloric cecum and intestine, retinoid-storing cells were localized in the lamina propria mucosae. Under electron microscopy, cells containing well-developed lipid droplets, which are common morphological characteristics of the hepatic stellate cells of mammals, were observed in the lamina propria mucosae of the pyloric cecum. Thus, the distribution of stellate cells with retinoid-storing capacity differs between this halibut and mammals, suggesting that the retinoid-storing site has shifted during vertebrate evolution.