
==== Front
Invest Ophthalmol Vis Sci
Invest Ophthalmol Vis Sci
IOVS
Investigative Ophthalmology & Visual Science
0146-0404
1552-5783
The Association for Research in Vision and Ophthalmology

39297806
10.1167/iovs.65.11.31
IOVS-24-40336
Retinal Cell Biology
Retinal Cell Biology
Blue Light-Induced Accelerated Formation of Melanolipofuscin-Like Organelles in Japanese Quail RPE Cells: An Electron Microscopic Study
Light Accelerates the Melanolipofuscin' Formation
Serejnikova Natalia B. 1
Trofimova Natalia N. 2
Yakovleva Marina A. 2
Dontsov Alexander E. 2
Zak Pavel P. 2
Ostrovsky Mikhail A. 2
1 I. M. Sechenov First Moscow State Medical University, Moscow, Russia
2 N. N. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia
* Correspondence: Marina A. Yakovleva, N. N. Emanual Institute of Biochemical Physics, Russian Academy of Sciences, Kosygina str., 4, Moscow 119334, Russia; lina.invers@gmail.com.
19 9 2024
9 2024
65 11 3102 9 2024
02 5 2024
Copyright 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Purpose

The retinal pigment epithelium (RPE) is a monolayer of epithelial cells essential for photoreceptor function and viability. Quail Coturnix japonica is a convenient experimental animal model for the study of age and pathological retina processes to an accelerated time regime. The three main types of pigment granules present in the RPE are melanin-containing melanosomes, lipofuscin-containing lipofuscin granules, and mixed melanolipofuscin granules containing both melanin and lipofuscin. The purpose of this work was to study the process of melanolipofuscinogenesis during aging and under light exposure.

Methods

We examined melanolipofuscin granules in “macular” areas, the area of the retina containing oxycarotenoids, as a function of the macula in humans, of the quail retina by transmission electron microscopy in young, middle-aged, and old birds, and in middle-aged birds irradiated with blue LED light (450 nm, 4 J/cm2).

Results

It has been shown that during photo-oxidative stress caused by the action of blue light on the quail eye, active fusion of melanosomes and lipofuscin granules occurs with formation of various types, including giant, mixed melanolipofuscin-like granules. Increased accumulation of melanolipofuscin-like granules was also observed in non-irradiated old birds.

Conclusions

It is assumed that the decrease in the number of melanosomes in the RPE during aging and photo-oxidative stress is associated with their fusion with lipofuscin granules and subsequent degradation of melanin by reactive oxygen species formed in melanolipofuscin-like granules. The disappearance of melanin deprives the RPE cells of light-filtering and antioxidant protection, and significantly increases the risk of their oxidative stress.

retinal pigment epithelium (RPE)
Japanese quail
melanosomes
melanolipofuscin granules
visible light
superoxide
melanin degradation
==== Body
pmcThe retinal pigment epithelium (RPE) is a polarized monolayer of pigment cells, on the one hand, closely adjacent on one side to the light-sensitive photoreceptor cells of the neural retina, and, on the other hand, to a layer of vascular capillaries, from which it is separated by Bruch's membrane.1 This arrangement of RPE cells also determines its main functions. These functions include phagocytosis of shed photoreceptor outer segments, transport and removal of metabolites from photoreceptor cells, regulation of vitamin A metabolism and control of the visual cycle, absorption of scattered light, regulation of ion currents, production of growth factors for photoreceptors, and maintenance of the blood-retinal barrier.1–3

There are three main types of pigment granules in RPE cells: (1) lipofuscin granules (LGs), containing the fluorescent “age pigment” lipofuscin, melanosomes, (2) specialized organelles of RPE cells containing a protein part and a polymer of the eumelanin type, and (3) complex granules containing simultaneously both types of pigments – melanin and lipofuscin, melanolipofuscin-like granules (MLLGs). Melanosomes in RPE cells are formed during the prenatal period of development. Their functions are screening photoreceptor cells from exposure to excess light and antioxidant protection of cells from free radical oxidation.4–7 Long-term exposure to light irradiation and intracellular oxidants are likely the factors that lead to a decrease in the number of melanosomes with age.8,9

During life, lipofuscin granules accumulate in the RPE, which are a product of incomplete degradation of the outer segments of photoreceptor cells of the retina. LGs have strong fluorescence and photosensitizing properties, which are due to the bisretinoids present in their composition. The most studied bisretinoid is N-retinylidene-N-retinylethanolamine (A2E). When irradiated with visible light, LGs are capable of generating reactive oxygen species (ROS), in particular, the generation of superoxide radicals.10–14 Photodegradation of bisretinoids produces toxic water-soluble products.15–19

With aging, melanosomes can fuse with LGs20,21 or with partially degraded phagosomes22–24 to form mixed melanolipofuscin-like granules. During aging, changes in the density of all pigment granules are observed. Similar processes occur when various retinal pathologies occur, such as Stargardt disease and age-related macular degeneration (AMD).25 In this case, a decrease in the number of melanosomes occurs, which is accompanied by a simultaneous increase in the number of LGs and MLLGs.26,27 There is evidence showing that the total number of granules increases with age.28–31

The total amount of lipofuscin-containing granules can occupy up to a third of the RPE cell volume in people over 70 years of age.26 Melanin in MLLGs can undergo photo-oxidative degradation caused by ROS generated by bisretinoids of the lipofuscin part of MLLGs with the formation of water-soluble fluorescent products.9,32,33 Oxidative degradation of RPE melanosomes leads, on the one hand, to a decrease in their antioxidant activity, and, on the other hand, to the appearance of products with pro-oxidant properties and increased photoreactivity.34–36 Moreover, as we have previously shown, the products of photooxidative degradation of melanin are themselves photoinduced superoxide generators and contain active carbonyl compounds.33 It can be assumed that the accumulation of pro-oxidant factors in the MLLGs will contribute to their accelerated damage upon irradiation with light and the destruction of both the melanin and lipofuscin parts of the granule. In this case, the age-related decrease in the concentration of melanin in the RPE cell will be the result of a decrease in the number of melanosomes due to their fusion with LGs and the subsequent degradation of melanin already inside these complex granules, that is, in the MLLG. Degradation of melanin will lead to a decrease in the specific gravity of MLLGs in the RPE cells and to the accumulation of mixed granules with small impurities of melanin that have not yet had time to be destroyed under the influence of superoxide radicals. In Reference 33, we showed that a decrease in the concentration of melanin in RPE cells of the human eye with age is due to its oxidative degradation by ROS generated under the influence of light by LGs in the composition of MLLGs.

The purpose of this work was to study the process of melanolipofuscinogenesis during aging and under light exposure. Studying such processes in laboratory conditions seems convenient and possible when using the Japanese quail Coturnix japonica as an experimental model. The advantages of this model are due to the short life span of birds, and the structure of the retina and carotenoid metabolism similar to humans. In this model, human-like pathological and age-related changes occurring in the RPE and macular region of the retina can be observed on an accelerated time scale. In addition, the Japanese quail has been quite well studied and is widely used in experimental studies.28,29

Human and quail RPE cells have a common structure and are similarly heterogeneous in size and packing density in the central and peripheral retina, which determines more efficient light absorption in this area.28,37,38 The content, ratio, and distribution of melanin and lipofuscin granules, as well as changes in these parameters depending on the location (macular or peripheral areas of the retina), with aging and light exposure are well comparable in human and quail RPE cells, as evidenced by literature data on human RPE cells26,39,40 and quail RPE cells.28–30

The aging process of Japanese quail is accompanied by continual accumulation of the aging pigment lipofuscin in RPE cells. This accumulation (by the end of a quail's life, the amount of LGs in RPE cells increases 5–8 times) occurs approximately 50 times faster than in human RPE cells. The accumulation of lipofuscin in RPE cells and the sensitivity of photoreceptors to light damage correlate with each other. Thus, in quails, with aging and prolonged exposure to light, the death of photoreceptors increases. In the human retina with age, the highest levels of lipofuscin correspond to the highest density of rods in the perifoveal region.41 In quails, a similar correlation is observed between lipofuscin content and rod density.28 In both humans and quails, the melanin content correlates with the maximum concentration of cones in the central zone of the retina.38

We have previously described age-related changes in the ultrastructure of melanosomes in the pigment cells of the Japanese quail choroid, namely, a violation of the correct shape and homogeneity of the contents of the granules.42,43 It has been shown that the total number of LGs and MLLGs in the RPE of the Japanese quail increases with age.28 In this work, we studied the dynamics of changes in morphology, surface area, and number of pigment granules in Japanese quail RPE cells depending on age and the damaging effects of blue irradiation.

Materials and Methods

Animals

The work was carried out on sexually mature individuals of Japanese quail aged from 2 to 17 months. All studies were conducted in accordance with the principles of biomedical ethics, as set out in the 1964 Declaration of Helsinki and its later amendments. Animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (http://oacu.od.nih.gov/regs/index.htm) and handled according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Breeding birds and monitoring their life support was carried out at the Institute of Biomedical Problems of the Russian Academy of Sciences within the framework of the Agreement on Scientific and Technical Cooperation No. 59-23 dated March 4, 2023. The quails were kept under standard lighting with an incandescent lamp with a daily cycle of 15 hours of light and 9 hours of darkness at the experimental base of the State Research Center of the Russian Federation, Institute of Biomedical Problems, Russian Academy of Sciences. The birds were fed balanced feed for adult quails PK1P-18154-776 (JSC Istra-Khleboprodukt, veterinary certificate No. 10015 dated September 19, 2017, GOST R 51851-2001). The keeping of birds and their removal from the experiment by decapitation was carried out in accordance with the “Rules of Laboratory Practice” approved by Order of the Ministry of Health of the Russian Federation dated April 1, 2016 N 199n.

Experiments were conducted on females quails, which age faster than male quails and exhibit higher sensitivity to photodamage.28,29 Based on data on the egg production of birds and the lipofuscin content in RPE cells depending on age, the following age groups were identified: young (9–25 weeks, n = 3), middle aged (35–40 weeks, n = 6), and old age (52–78 weeks, n = 5).

Blue Light Irradiation

For light irradiation, a blue LED source (λmax = 450 nm; LED450) was used in a photodamaging (sublethal) dose for the retina (4.0 J/cm2 of the corneal surface when irradiated for 40 minutes).44

There are literature data on experiments with blue light irradiation of other animals (mice, rats, rabbits, and primates), in which significant structural and functional changes in RPE cells were identified, leading to disruption of the integrity of the external hematoretinal barrier and cell death.45 The literature contains data on vacuolization of the cytoplasm of RPE cells after irradiation, on the disappearance of their apical processes and basal folds,46 about mitochondrial damage,47 about the appearance of compressed cell nuclei with peripheral chromatin condensation and enlarged nuclear pores, about the uneven distribution of melanin granules,48 about disorganization of the actin cytoskeleton of cells, destruction of tight intercellular contacts, increase in cell size, and the formation of syncytium.49 There was also an increase in the intracellular level of ROS and pro-inflammatory cytokines in the RPE and the recruitment of macrophages to the blood-retinal barrier after irradiation with blue light.50 According to the study by Van Norren and Gorgels,44 the blue part of the spectrum we selected (λmax = 450 nm) has the most active photobiological effect. Threshold doses for photodamage to the retina by blue light are 50 to 100 times lower than for long-wavelength light (λ = 500–650 nm).44 In the work of Fite and others,34 they used a light dose of 3000 lx for 18 hours, which caused significant morphological changes. It should be noted that quite often the cage containing the animal is irradiated, whereas in our experiments the light was focused directly into the eye of the bird. Because a fairly high radiation dose was chosen, the result of the light appears quite quickly.

Eye irradiation was performed on intact animals. The right eye of the birds was exposed to irradiation with “manual” fixation of the bird's head in relation to the light flux and dilated eyelids, the left eye remained without exposure (control). Manual restraint is when the researcher holds the bird with his hands to prevent it from twitching, turning, or closing its eye toward the light. The eyelid was fixed by holding the head with one hand to avoid displacement relative to the beam of light, as well as self-harm of the bird, and with the other hand, they held the beak and woke it up, preventing it from closing the eye, by touching the head with their fingers. Manual fixation is necessary because the bird, when exposed to bright light, constantly falls asleep and closes the eye exposed to the light (a normal protective reaction), so it has to be woken up and forced to open the eye manually. The effects of photodamaging light were assessed 24 hours after irradiation. The birds were removed from the experiment by decapitation, the eyes were enucleated, the posterior hemisphere was isolated, cut into fragments with an area of approximately 1.0 cm2, containing the central part of the retina with RPE, and then placed in fixative solutions.

Like humans, quails have a central area of acute vision,51 containing a high concentration of cones of different types.52 Naturally, the effect of light on this area of the retina is much greater than on the periphery and, therefore, the number of granules here is also greater. In our experiments, we irradiated the central part and therefore studied morphological changes there. It is known28,29 that the highest content of melanin granules also occurs in the central part of the retina and therefore it makes sense to look at changes in their content in this area.

Microscopic Studies

Tissue samples were fixed in a mixture of 2.5% glutaraldehyde (Pan Real, Spain) with 2% formaldehyde (MP Biomedicals, France) in 0.1 M PBS (Amresco, USA; pH 7.2–7.4) for 6 to 8 hours at +4°C. Then the material was postfixed with 1% OsO4 (Electron Microscopy Sciences, USA), contrasted with 70% alcohol with 2% uranyl acetate (Electron Microscopy Sciences, USA), dehydrated in alcohols of increasing concentration and in acetone (ChimMed, Russia), and filled with a mixture of epoxy resins (Epon 812, Fluka, Germany). To orient the material, semi-thin sections (1–2 µm thick) were prepared on Pyramitome LKB microtome with glass knives (LKB Bromma 11800 Pyramitome, Sweden), and stained with a 1% aqueous solution of methylene blue (Isolab, Germany). Ultrathin transverse sections of the retina were additionally contrasted with 2% uranyl acetate (Electron Microscopy Sciences, USA) and lead citrate (Electron Microscopy Sciences, USA), according to Reynolds, and then viewed in a JEM-1011 transmission electron microscope (JEOL, Japan) at magnifications of 8000x to 25,000x.

Morphometric Analysis

The number and area of different types of lipofuscins and melanolipofuscin granules were measured using electron microscopy photographs in RPE cells using ImageJ software (Wayne Rasband, USA). At least 10 RPE cells were analyzed for each animal. To count granules of different types in each eye, three ultrathin sections were taken, passing through different RPE cells of the central zone. Statistical data are presented as averages.

Statistical Analysis

Statistical processing was performed using GraphPad Prism version 8.00 software (GraphPad Software, USA). Normality of distribution was determined using the Shapiro-Wilk test (ɑ ˃ 0.05). Between-group differences were assessed using 1-way ANOVA with Tukey’s multiple comparison test. The P values ≤ 0.05 were considered statistically significant.

Results

In all micrographs in Figure 1, it shows the same basal domains of the cytoplasm of RPE cells with basal folds adjacent to Bruch's membrane, and the main organelles (nuclei, mitochondria, lysosomes, etc.) and granules located in this part of the cytoplasm. In different cells, granules were counted in the same areas of the RPE (basal domains), so the polarity of the RPE cells cannot bias the results. It is known28 that granules are located mainly in the basal part of the RPE cell. In our work, not all photographs showed cell nuclei in the section, but, at the same time, these photographs are most demonstrative regarding the distribution of granules of different types, and therefore we included them in this paper. Electron microscopic analysis of RPE cells showed (see Fig. 1) that with aging and irradiation with blue light, pronounced changes occur in the subcellular level: the structure of the main cellular components – nuclei and mitochondria – was disrupted, LGs and MLLGs were formed and accumulated, and the basal infoldings were deformed.

Figure 1. Ultrastructure of RPE cells (basal domains) in birds of different ages under normal conditions and after light exposure. (A, B) These are the young quails, (C, D) the middle aged quails, (E, F) and the old quails. (A, C, D) These are the control quails that received no irradiation, (B, D, F) these are the quails after irradiation. Scale bars in all photographs correspond to 1 µm. N-nuclear, M-mitochondria, B-basal infoldings. Stars indicate melanin granules, and the arrows point to different types of melanolipofuscin granules: yellow = type I, blue = type II, purple = type III, green = type IV, and red = type V.

To study the dynamics of melanolipofuscinogenesis in RPE cells of different age groups of quails depending on age and light exposure, we identified and characterized five types of granules containing lipofuscin and melanin (Fig. 2). By the term “dynamics of melanolipofuscinogenesis” we mean those changes in the content of pigment granules of different types that are observed at different time points (in young, mature, and old birds).

Figure 2. Subtypes of LG and MLLG granules in Japanese quail RPE cells. Scale bars in all photographs correspond to 0.5 µm. Type I granules are typical lipofuscin granules, consisting of homogeneous light-colored contents, surrounded by a membrane and containing no melanin. Granules of types II to V contain both lipofuscin and melanin (melanolipofuscin-like granules [MLLG]). Type II granule is a melanolipofuscin granule, which is surrounded by a dense rim of melanin nature and has an internal content similar to lipofuscin. Melanolipofuscin granule type III is a lipofuscin granule containing melanin inside, which may be partially degraded. Type IV granule is a type III cohesive melanolipofuscin granule and is characterized by optically highly heterogeneous content with a large number of inclusions of different shapes and sizes. Type V granule is a megamelanolipofuscin granule (˃0.5 µm2), apparently formed as a result of the fusion of smaller type IV granules.

Whereas a lipofuscin granule in humans is about 1 µm, these granules in quails seem much smaller. Apparently, this is due to the peculiarities in the structure of the quail RPE, in contrast to the human RPE. Thus, in the quail, there is a greater variety of cones in the RPE and longer apical processes than in humans. Hence, faster and more intense phagocytosis and smaller LGs.

Visual electron microscopic analysis revealed, after light exposure, increased formation of MLLGs due to the fusion of melanosomes and LGs in RPE cells of quails of different ages (see Fig. 1). It can be assumed that there are two mechanisms for this process: in the first case, the melanosome absorbs LGs, enveloping them (see type II in Fig. 2), and, in the second case, on the contrary, the lipofuscin granule absorbs the melanosome (see type III in Fig. 2). Similar results were obtained in Reference 53, in which the authors discovered MLLGs containing lipofuscin both in the center and at the periphery of the granule. These two mechanisms appear to occur with equal probability throughout the life of the organism. When irradiated with intense blue light, LGs begin to actively generate ROS, which, in turn, are deactivated by melanosomes. As a result, during the interaction of melanin with ROS, melanin pigment is degraded, and the damaged melanosome can be absorbed by the LGs.

The most pronounced effect of the formation and accumulation of MLLGs as a result of light exposure is observed in middle-aged quails (see Figs. 1C, 1D). This is probably due to the fact that the RPEs of young birds still contains too little LGs necessary for the formation of MLLGs (see Figs. 1A, 1B), whereas in old birds, the number of melanosomes becomes significantly smaller and, as a consequence, their protective function, such as the formation of MLLGs, is weakened (see Figs. 1E, 1F). Therefore, middle-aged quails, which, on the one hand, still have many intact melanosomes, and, on the other hand, have accumulated quite a lot of LGs, were chosen for a detailed study of the effect of light irradiation on morphological changes in RPE cells.

Morphometric analysis showed (Fig. 3) that in middle-aged birds the content of MLLGs of types II, III, and IV per cell decreased after light irradiation, and, for type V, the granules were reliably increased almost twice (see Fig. 3). This is probably due to the fact that these granules further fuse with each other to form giant MLLGs.

Figure 3. The content of MLLG of different types in RPE cells in normal and after light exposure. (A) Green color = young, control quails (n = 3); blue color = middle age of quails, control (n = 6); and red color = old, control quails (n = 5). (B) Blue color = middle age of quails, control (n = 6), yellow color = middle age of quails, that were irradiated (n = 6). * - P ˂ 0.05. The number of granules was normalized for all groups per 1500 µm22 RPE cell area.

The histogram of the distribution of areas of individual granules showed an enlargement of MLLGs of different types with age and, mainly, after light exposure, as a result of which the area of MLLGs increased by two or more times (Fig. 4).

Figure 4. Distribution of areas (µm22) of individual melanolipofuscin granules in RPE cells of birds of different ages under normal conditions and after light exposure. (A) Shows the young quails, control (n = 3), (B) are the old quails, control (n = 5), (C) are the middle-aged quails, control (n = 6), (D) are the middle-aged quails, that were irradiated (n = 6).

The number of different types of MLLGs formed may reflect the priority mechanism of their formation. Based on our data, we can assume that the process of formation of melanolipofuscin granules during the aging process in the initial stage occurs with the formation, mainly, of type II MLLGs, which then transforms into granules of types III, IV, and V. In old birds, the MLLG type III predominates in RPE cells. The damaging effect of light in the RPE of middle-aged birds (see Fig. 3B) leads to a decrease in the number of granules of types II, III, and IV, whereas the number of granules of type V increases. The development of photo-oxidative stress, accompanied by the generation of ROS by lipofuscin granules, probably causes accelerated formation of MLLGs, destruction of melanin in the composition of these complex granules, and their further fusion with the formation of mega granules (Fig. 5).

Figure 5. Interaction of melanosomes with MLLG in RPE cells of middle-aged quails after light exposure. Ellipse indicate interaction zones. The scale bar corresponds to 1 µm.

In our study, Figure 5 shows the maximum approach of melanosome membranes and type II melanolipofuscin granules, potentially preceding their fusion. Although our work did not conduct an immunohistochemical study, we make this assumption based on literature data. Our electron microscopic morphological picture of this interaction of different types of granules visually corresponds to the electron microscopic data of other authors, who, in RPE cells, using immunofluorescence and labeling with gold particles, clearly demonstrated the active movement and convergence of melanosomes and phagosomes (future lipofuscin granules) along microtubules54,55 and their fusion.56

Discussion

The main result of the work comes down to the following: (a) when blue light acts on the quail's eye, active fusion of melanosomes and lipofuscin granules occurs with the formation of various types of mixed MLLGs; (b) irradiation leads to a twofold decrease in the amount of LGs (dark = 19.6 ± 0.8 and light = 9.8 ± 0.5) and a synchronous increase in the amount of MLLGs in the quail RPE cells; and (c) increased accumulation of MLLGs was also observed in non-irradiated quails with age, as in old birds.

The results of our morphometric analysis of granules in the quail RPE cells are somewhat different from the results obtained previously in works.35,36 Thus, in Reference 35, when assessing morphological changes in the RPE, not only canonical LGs but also all types of melanolipofuscin-like granules were taken into account as LGs. For this reason, the diameters of the granules were averaged in such a way that, as a result of irradiation, the total area of the LGs per unit cell area decreased. We took into account all subtypes of LGs and MLLGs, including those whose an area that increased after exposure to light. It turned out that it was with these granules that the greatest changes, both qualitative and quantitative, occurred (see Figs. 3, 4). At the same time, the ultrastructure of RPE cells, both in our work and in the works of References 28 and 29 were visually identical.

In Reference 53, the authors assessed the number of LGs and MLLGs in the RPE of the human eye using the structured illumination microscopy (SIM) method based on the intensity of the fluorescence signal. In this case, it is possible that some of the melanosomes containing partially oxidized, fluorescent products could be classified as MLLGs, because there was no confirmation of the type of granules by electron microscopy. It is possible that the visualization parameters were not set quite correctly during the calculation, as a result of which a significant number of granules could be taken into account repeatedly and, as a result, distort the final value of the number of granules per cell. According to the same authors,53 more MLLGs are present in the foveal region, whereas more LGs are present in the periphery of the retina, which is consistent with the results presented in Reference 57 about more intense fluorescence of retinal areas located closer to the center of the fundus. This fluorescence is most likely due to the fluorescence of oxidized bisretinoids,58 whereas more unoxidized bisretinoids are located in the periphery.

Thus, based on our data, we assume that during photo-oxidative stress, active fusion of LGs and melanosomes occurs with the formation of MLLGs, because there is an inverse correlation between the content of LGs and MLLGs – the amount of LGs decreases, whereas the amount of MLLGs increases. It is logical to assume that MLLGs contain more oxidized and degraded products than the original LGs, therefore the maximum of their fluorescence should be shifted to a shorter wavelength region. This assumption is confirmed by the data presented in Reference 59, where it was shown that larger granules are located closer to the center of the RPE cell. These were probably melanolipofuscin granules,53 whose fluorescence maximum is in a shorter wavelength region than that of smaller granules (most likely the original LGs) located on the periphery of the fundus.53,59

It is known that in the pathogenesis of AMD, there is an increased accumulation of MLLGs, and not LGs.26,60,61 This is in good agreement with our assumption that under oxidative stress, melanosomes more actively fuse with LGs, which helps prevent the release of ROS generated by lipofuscin bisretinoids into the cell cytoplasm.

To summarize, we can propose a hypothetical scheme for the development of melanolipofuscinogenesis in RPE cells (Fig. 6).

Figure 6. Hypothetical scheme of melanolipofuscinogenesis in Japanese quail RPE cells.

According to the above scheme, the action of light causes the generation of ROS toxic to the RPE cell by LGs, triggering cascades of intracellular signaling mechanisms that promote the movement of melanosomes toward the lipofuscin granules and their fusion with the formation of type II granules and preventing the release of ROS into the cell cytoplasm. If oxidative stress does not stop, then melanin gradually begins to degrade under the influence of superoxide radicals and its water-soluble products contribute to its further destruction, which leads to the formation of type III granules. With further mutual degradation of LGs and melanin in the composition of MLLGs, additional fusion of granules of types I, II, and III occurs with the formation of complex mixed granules of type IV. With the damaging effects of light and with age, all this leads to the formation of giant melanolipofuscin granules of type V. In addition, ultimately, to the gradual disappearance of melanin in the composition of RPE cells.

The mechanism of accelerated formation of complex melanolipofuscin granules under photo-oxidative stress may be due to the activation of melanosome transport toward the source of generation of superoxide radicals, that is, toward LGs. It is known that intracellular organelles do not move by free diffusion.62 For this purpose, the cell uses motor proteins (kinesins, dyneins, and myosins), which move either along microtubules or along actin filaments.63,64 The interaction of organelles with motor proteins is mediated by small GTPases, which are involved in the selection of translocated organelles, as well as in their intracellular transport, docking, and fusion.64–66 In RPE cells, such functions are performed by proteins of the Rab family.64,67,68 It is known that superoxide anion radicals can enhance the dissociation of GDP from the Rab protein molecule with the subsequent addition of a GTP molecule to it, which leads to the activation of Rab GTPase.69,70 It can be assumed that activation of the Rab-melanosome complex in RPE cells causes the movement of the melanosome and its fusion with the lipofuscin granule, which leads to increased accumulation of type II melanolipofuscin granules. At the same time, the release of ROS into the cell cytoplasm is blocked, because the resulting radicals are utilized on the melanin matrix. Thus, this process is a protective reaction that reduces the toxic effect of lipofuscin granules. However, with prolonged exposure to photo-oxidative stress, gradual degradation of melanin occurs in MLLGs under the action of superoxide radicals generated by lipofuscin, with the formation of water-soluble destruction products that can also induce light-dependent degradation of melanin.33 This leads to the formation of various types of melanolipofuscin granules, including giant MLLGs. As part of MLLGs, melanin degrades under natural conditions of the retina both as a result of interaction with ROS generated by lipofuscin bisretinoids, and as a result of reaction with ROS generated by melanin destruction products.

Our study has some limitations that may be addressed in future studies. First, our study was focused exclusively on electron microscopic analysis of the morphological aspects of melanolipofuscinogenesis, and therefore the future use of additional analytical methods, for example, immunocytochemistry, may provide a more complete understanding of the mechanisms of the interaction between different types of granules. Second, despite the similarity of structure and functional processes, the quail RPE cell is not fully comparable with the human RPE cell due to some of its unique features (presence of myeloid bodies, long apical processes, and more intense phagocytosis). Therefore, the size, distribution of different types of granules, and the mechanism of their formation may differ somewhat from those in humans and it would be interesting to conduct such a comparative study in the future. Third, the peculiarities of sample preparation and the labor intensity of electron microscopy analysis have so far made it possible to obtain results on the content and distribution of different types of granules only in the basal part, and not in the whole RPE cell, which will be advisable to focus on in the future.

Acknowledgments

The authors thank T. S. Gurieva, E. I. Mednikova for technical support for experimental and model animals from the Institute of medical-biological problems RAS.

Supported by the Ministry of Science and Higher Education of the Russian Federation, Project no. 122041400102-9.

Disclosure: N.B. Serejnikova, None; N.N. Trofimova, None; M.A. Yakovleva, None; A.E. Dontsov, None; P.P. Zak, None; M.A. Ostovsky, None
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