Age-Related Macular Degeneration, Role Of RPE Cells, Autophagy, Antioxidants, Gene Modification Natural Treatment

Age-Related Macular Degeneration, Role of RPE Cells, Autophagy, Antioxidants, Gene modification Natural Treatment, Eye Health, Eye Vitamins, Ocular Health, Natural Supplements, Herbal Extracts, Eye Nutrition, Eye Care

The Main Functions of Retinal Pigment Epithelium in AMD
Age-related macular degeneration (AMD) is a prevalent retinal degenerative disorder that primarily affects the macular region of the retina. The disease is characterized by a loss of visual acuity and visual distortion (metamorphopsia). AMD primarily impairs the photoreceptors located in the macula, which are responsible for high visual discrimination and cortical integration.
In AMD, small cones in the macular region, which are sensitive to white and blue light stimuli, are predominantly affected. These wavelengths of light produce a high amount of oxidative damage. The outer retina, particularly the retinal pigment epithelium (RPE), is primarily impacted during the course of AMD. This is due to the constant exposure of RPE to reactive oxygen species (ROS) generated by direct exposure to natural light in the macula. The high oxidative metabolic activity in RPE creates a site-specific pro-oxidant environment, which contributes to the early degeneration observed in AMD. The oxidative metabolism of RPE is essential for various functions, including the recycling of photoreceptor outer segments, buffering glutamate, and converting retinoic acid into 11-cis-retinal. Given the substantial ROS burden, RPE cells rely on intense autophagy activity, even under baseline conditions in healthy individuals. The autophagy flux is consistently higher in RPE compared to the inner retinal layers.
RPE cells possess a robust antioxidant machinery that allows them to counteract retinal injuries and fulfill physiological functions crucial for sustaining vision and retinal integrity. They play a key role in melanin synthesis, neutralizing ROS generated by intense white and blue light exposure, and recycling damaged outer segments of photoreceptors.
Whether originating from within the retina or systemic sources via the bloodstream, ROS promote lipid oxidation, nucleic acid damage, protein oxidation, and the formation of misfolded molecules. Altered mitochondria, disrupted mitochondrial structure, and subsequent ROS generation are also affected by ROS. Therefore, the prompt removal of damaged mitochondria and the generation of new, healthy mitochondria are essential for the survival of RPE cells in oxidative conditions. The autophagy machinery serves these functions by degrading misfolded proteins, oxidizing lipids through lipophagy, removing damaged mitochondria through mitophagy, and promoting mitochondriogenesis.

RPE and melanin formation
The synthesis of melanin in RPE cells is crucial for their activity as it allows for the absorption of light that would otherwise spread between photoreceptors, leading to visual discrimination issues and increased oxidative stress. Melanin-rich inclusions, known as melanosomes, are abundant within the processes of RPE cells and help mitigate the effects of reactive oxygen species (ROS) generated during phototransduction. They absorb these compounds, thereby preventing oxidative damage. The presence of melanin within RPE cells plays a significant role in regulating cell shape and phenotype.
The presence of melanin within RPE cells also serves to counteract the detrimental effects of excessive white light stimulation, which would otherwise result in the production of toxic ROS and free radicals. Melanin has the ability to bind to lipids and sugars, effectively trapping advanced glycation end products (AGEs) that are prevalent during AMD. In this way, melanin acts as a buffer, trapping various toxic chemical species and forming inclusions that neutralize their damaging effects. It is worth noting that the occurrence of retinal inclusions, including drusen and other RPE aggregates, does not necessarily indicate a harmful condition. Instead, these inclusions may represent a compensatory segregation of toxic species, which can actually be less detrimental to cell survival.

Role of RPE

The role of the retinal pigment epithelium (RPE) in AMD is crucial. Melanin, present in RPE cells, acts as a buffer by trapping various toxic chemical species and forming inclusions. This provides a neutralizing effect against cell damage. Additionally, the activity of RPE is important in removing iron accumulation, which is associated with oxidative damage in AMD. Exogenous oxidative stress can lead to ferroptosis, characterized by the accumulation of Fe2+ and lipid peroxidation in RPE cells.
Within AMD, RPE-derived reactive oxygen species (ROS) are toxic to various molecules, including proteins and lipids, by generating abnormal chemical species that mediate cell damage. Oxidative injury can lead to DNA damage and chronic inflammation. The accumulation of altered sugars, lipids, proteins, and organelles necessitates powerful systems to degrade and clear these compounds from the retina. Adequate clearance by the RPE prevents the accumulation of proteinaceous material and lipofuscins, thus inhibiting the formation of cellular debris in the form of drusen.
Role of Autophagy within AMD-RPE
To counteract the detrimental effects of oxidative species, RPE cells rely on various clearing systems, including chaperone systems, the proteasome system, antioxidant compounds, and predominantly the autophagy pathway. Autophagy is particularly prevalent within RPE cells, where it plays a significant role in removing oxidized proteins and lipids, thus counteracting AMD.
In AMD conditions where autophagy is not properly activated or its progression is impeded, misfolded proteins aggregate with sugars within stagnant lysosomes, leading to the formation of advanced glycation end-products (AGEs). These aggregates contain abundant lipids, which contribute to the formation of lipofuscins. The effects of ineffective autophagy can be observed within RPE cells as well as dispersed in the intercellular space.

The reactive oxygen species (ROS) generated by the retinal pigment epithelium (RPE) have a toxic effect on various molecules, including proteins and lipids, by producing abnormal chemical species that cause cellular damage. This oxidative injury leads to DNA damage and chronic inflammation. The accumulation of altered sugars, lipids, proteins, and organelles necessitates robust systems to degrade and clear these compounds from the retina. Adequate clearance within the RPE prevents the accumulation of proteinaceous material and lipofuscins, thus preventing the formation of cellular debris in the form of drusen.
To counteract the numerous detrimental effects caused by oxidative species, powerful clearing systems exist within RPE cells. These include chaperone systems, the proteasome system, antioxidant compounds, and, notably, the autophagy pathway. Autophagy plays a prominent role in RPE cells, where it removes oxidized proteins and lipids to counteract AMD.
In AMD conditions where autophagy cannot be properly activated or its progression is impeded, misfolded proteins aggregate with sugars within stagnant lysosomes, leading to the formation of advanced glycation end-products (AGEs). These aggregates contain an abundance of lipids, which contribute to the formation of lipofuscins. The effects of ineffective autophagy can be observed both within RPE cells and in the intercellular space.

The retinal pigment epithelium (RPE) plays a crucial role in protecting the retina from oxidative damage caused by reactive oxygen species (ROS). Failure to effectively clear ROS within RPE cells can lead to the accumulation of proteinaceous material and lipofuscin, resulting in the formation of cellular debris known as drusen.
To counteract the detrimental effects of oxidative species, RPE cells rely on various clearing systems, including chaperone systems, the proteasome system, antioxidant compounds, and most importantly, the autophagy pathway. Autophagy is particularly prominent in RPE cells and is responsible for removing oxidized proteins and lipids to prevent the development of AMD.
When autophagy is defective or impeded, misfolded proteins, lipids, and damaged organelles, particularly mitochondria, can accumulate, leading to experimental AMD. This accumulation can result in the formation of advanced glycation end-products (AGEs) within stagnant lysosomes, where misfolded proteins aggregate with sugars. Lipofuscin, consisting of lipids and autophagy substrates, may also be present in these aggregates. Noneffective autophagy can be observed within RPE cells as well as in the extracellular space, where toxic species, cellular debris, and damaged mitochondria are released, contributing to the spread of the disease process in AMD. The impairment of autophagy leads to the intracellular accumulation of autophagy substrates, which are subsequently released extracellularly when autophagy fails to clear them.
Drusen in AMD contain lipofuscin and melanosomes, which can overlap within single structures known as lipomelanofuscin. These structures can mix with autophagy substrates and residues of photopigments. During early stages of AMD, when extracellular drusen are not yet evident, their components can already be detected as aggregates within RPE cells, primarily within inert lysosomes.
Recent findings highlight the significance of defective autophagy in AMD, which fails to digest toxic species formed within RPE cells and release them extracellularly after transient intracellular accumulation. The toxicity exerted by cytosolic-free oxidized molecules or damaged mitochondria, before they aggregate into extracellular inclusions, plays a significant role in the development of AMD.
AMD symptoms appear to be more related to altered metabolism caused by oxidized structures rather than mechanical impairments induced by inclusions or aggregates. A biochemical defect resulting from defective autophagy appears to have a greater impact on visual impairment than the deleterious effects of inclusions or aggregates.
Loss of visual acuity may be driven by an upstream biochemical dysfunction in cell clearance rather than the amount of drusenoid area. Assessing the status of autophagy could better predict the severity of AMD-related visual symptoms. A biochemical alteration that impairs the clearance of oxidizing species directly contributes to the loss of visual acuity, which is subsequently associated with morphological changes such as the formation of drusen and disruption of retinal arrangement.
RPE cells produce autophagy-dependent genes that are essential for key functions, such as the digestion and recycling of intracellular and photoreceptor-derived components. The regulation of these genes responds quickly to daily light and stress conditions, ensuring autophagy’s ability to fine-tune protein expression and maintain visual processing. This is crucial for countering visual impairment in AMD patients. An effective autophagy within RPE must be able to handle the ROS generated by light exposure and the excess oxidative species arising from both focal and systemic sources.
Disruption of this delicate orchestration can impair vision in real-time while progressively leading to the formation of delayed extracellular deposits that may not directly contribute to visual deterioration. Stimulation of autophagy can suddenly improve visual acuity due to its direct effect on retinal metabolism.
Among the key proteins involved in vision and regulated by autophagy activity, the retinal-pigment-epithelium-derived factor (RPEDF) is produced by RPE cells. RPEDF restores the visual cycle while leaving retinal aggregates intact. To maintain steady visual acuity, rapid metabolic changes supported by autophagy activation within RPE cells are necessary.

The activity of autophagy-dependent steps within the retinal pigment epithelium (RPE), as described by Datta et al, corresponds to specific requirements. These requirements include the rapid phagocytosis of outer segments of photoreceptors and the ability to counteract the high levels of photo-oxidative stress experienced in the retina. Additionally, the recycling of visual pigment and the turnover of receptors in photosensitive neurons depend on the autophagy status.
The pathology of retinal degeneration remains autophagy-dependent and demonstrates an ineffective antioxidant response. However, this pathology progresses slowly over time. Recent studies have shown that impaired vision in AMD does not necessarily correlate with the presence of drusen.
Light has a stimulating effect on autophagy structures. During periods of light exposure, autophagy-related molecules are upregulated, leading to the generation of more autophagy structures such as autophagosomes and lysosomes. This cycle of autophagy activation occurs rapidly and happens numerous times throughout the day, being induced by light exposure and suppressed by light deprivation. Phototransduction, the process by which light is converted into neural signals, activates at least 23 autophagy-related genes, some of which facilitate the rapid progression of autophagosomes merging with lysosomes, a crucial step in maintaining vision. This explains the abundance of autophagosomes and active lysosomes observed in the retina, particularly within the RPE, following light exposure. RPE cells are responsible for recycling the outer segments of photoreceptors through a process called LC3-associated phagocytosis (LAP). In LAP, phagocytic vacuoles containing the outer segments of rods and cones recruit LC3, generating autophagosomes specific to photoreceptors. These autophagosomes quickly digest the disk membranes, the photosensitive domain of photoreceptors. LAP represents a specialized form of autophagy/phagocytosis crucial for sustaining vision at the biochemical level.
It is likely that impaired autophagy leads to a loss of visual acuity before any detectable loss of photoreceptor integrity or the appearance of drusen in the retina. At early stages of AMD, autophagy failure may primarily manifest as a loss of visual acuity, particularly evident in visual discrimination tasks. The biochemical steps of LAP are regulated by various molecules and quickly inducible genes within the retinal pigment epithelium (RPE), including melanoregulin, rubicon, epidermal growth factor receptor (EGFR), Bcl-2-associated X protein (Bax), forkhead box O3 (FOXO3), and the mitogen-activated protein kinase (MAPK)-dependent signaling pathway.

Amber light and red light have direct antioxidant effects, which are further enhanced by their ability to counteract the harmful effects of reactive oxygen species (ROS) through the activation of the autophagy machinery. These specific wavelengths of light activate multiple steps in the autophagy process. The effects extend downstream, promoting autophagy flux by facilitating the merging of autophagosomes with lysosomes and promoting lysosomal degradation. Conversely, prolonged exposure to blue light increases ROS levels within RPE cells and inhibits autophagy. It is intriguing that the effects of these different light wavelengths converge on cellular targets involved in the autophagy process.
Amber light, around a wavelength of 590 nm, activates multiple steps of autophagy and increases the levels of autophagy-related proteins. It promotes autophagy flux by facilitating the merging of autophagosomes with lysosomes and promoting lysosomal degradation. This is achieved through the interaction of amber light with the leupeptin/NH4Cl complex, which tonically inhibits lysosomal activity. Amber light removes this inhibition, resulting in rapid autophagy activation in response to pulses of amber light. This rapid autophagy activation facilitates the clearance of accumulated substrates during retinal degeneration.
Pure red light exposure has a potent antioxidant effect on its own and also activates the inducible isoform of the chaperone protein heat shock protein 70 (HSP 70). Red light also activates autophagy and aids in the removal of specific autophagy substrates, such as the misfolded isoform of the tau protein. The increase in autophagy-related proteins induced by red and amber light can be observed through transmission electron microscopy, where the increase in autophagosomes and lysosomes is evident.
Long-term exposure to blue light is known to have detrimental effects on the retina and can contribute to the development of AMD when applied continuously for extended periods. This is due to the high production of ROS and free radicals caused by blue light. Prolonged exposure to blue light leads to an excess of oxidative stress, particularly affecting the RPE layer. This results in altered mitochondria and severe oxidative stress caused by the light itself and altered mitochondrial metabolism.
Phytochemicals, natural compounds found in various sources, exhibit autophagy-inducing effects. They act as powerful antioxidants and stimulate autophagy. Some of these compounds can also exert epigenetic control over autophagy-related genes. Phytochemicals clear extracellular aggregates at the border between the retinal pigment epithelium (RPE) and the Bruch’s membrane, leading to significant improvements in visual acuity. Since oxidative stress and abnormal angiogenesis play key roles in AMD, both of which are strongly influenced by autophagy, it is worth exploring the potential disease-modifying effects of phytochemicals as autophagy inducers in AMD.

Lutein

It is remarkable that lutein has the ability to stimulate autophagy, potentially counteracting the harmful effects caused by autophagy inhibition. The antioxidant effects of lutein are largely achieved through the activation of autophagy processes, as this compound enhances various steps in the autophagy machinery. Interestingly, when lutein is administered systemically, it tends to accumulate in the retina, particularly in the macular region. This is intriguing considering that the macula is the area prone to degeneration in AMD (Age-related Macular Degeneration). In fact, research has demonstrated that administering lutein and its metabolite zeaxanthin to AMD patients improves their disease status. Due to the human body’s inability to synthesize lutein, and the evidence supporting its therapeutic efficacy, a diet rich in lutein and zeaxanthin is recommended for individuals with AMD. The effectiveness of lutein can be attributed to multiple mechanisms, summarized as follows: (i) direct antioxidant defense; (ii) activation of the autophagy/mitophagy pathway, which helps neutralize oxidative species; (iii) facilitation of the clearance of oxidative by-products; (iv) elimination of damaged mitochondria, thereby reducing oxidative stress; (v) promotion of new mitochondria synthesis; (vi) exertion of a potent anti-inflammatory effect; and (vii) inhibition of new blood vessel proliferation. Like other phytochemicals, lutein may also induce the activation of retinal stem cells, which are stimulated by autophagy activation.

Resveratrol
Resveratrol, like lutein, also demonstrates antioxidant effects on retinal pigment epithelium (RPE) cells. When administered to RPE cells, resveratrol reduces the levels of oxidative by-products, such as malondialdehyde, and enhances the activity of the enzyme superoxide dismutase (SOD). Moreover, resveratrol significantly activates autophagy within the RPE cells, leading to an increase in mitochondrial biogenesis. By promoting mitochondrial quality control and function, resveratrol improves the overall mitochondrial activity in the retina.
Interestingly, the autophagy-inducing effects of phytochemicals like lutein and resveratrol do not rely on inhibiting the mechanistic target of rapamycin (mTOR). Both lutein and resveratrol activate autophagy without affecting mTOR activity. Additionally, resveratrol enhances the fusion of autophagosomes with lysosomes. It exerts a potent anti-inflammatory effect by activating sirtuin 3 or directly affecting RPE cells. This anti-inflammatory effect, coupled with the inhibition of angiogenesis through the blockage of vascular endothelial growth factor (VEGF), suggests that resveratrol may be effective in both wet and dry AMD.
Similar to lutein, resveratrol may also provide beneficial effects on retinal cells by stimulating stem cell niches, as observed in the hippocampus. Specifically, through sirtuin mediation, resveratrol directs stem cells towards specific neuronal phenotypes, contributing to neuronal differentiation.
Studies have demonstrated that combining phytochemicals with pulses of amber and red light, following specific timing protocols, enhances the beneficial effects on retinal anatomy and visual acuity. In patients undergoing this combination treatment of photobiomodulation (PBM) and phytochemicals, there was a significant improvement in visual acuity, accompanied by the clearance of submacular aggregates (drusen). The combination of light and phytochemicals effectively enhances visual acuity and restores retinal anatomical integrity.
Therefore, the combination of light and phytochemicals in the treatment of retinal oxidative damage can be seen as harnessing the natural interaction that occurs in nature between light and light-sensitive natural compounds. The synergistic stimulation of autophagy induced by the combination of light and phytochemicals may provide protection against AMD and improve visual acuity. Preliminary data suggests that combining specific doses of phytochemicals (such as resveratrol and lutein) with light exposure (following specific timing and wavelengths) represents a natural approach to mitigate the progression of AMD.

Crocus sativus L., commonly known as saffron

Saffron has been used for centuries as a herbal medicine, as well as a coloring and flavoring spice. In recent years, there has been increasing evidence highlighting the pharmacological properties of saffron and its constituents, particularly in relation to potential therapeutic applications in the central nervous system. The neuroprotective activity of saffron has also been investigated in age-related macular degeneration (AMD). Studies on saffron supplementation therapy have provided valuable insights into its neuroprotective actions, with crocins, crocetin, picrocrocin, and safranal being identified as its main components with therapeutic properties.

Saffron has been shown to possess potent antioxidant activity, primarily attributed to its carotenoids. While crocin is often credited for saffron’s antioxidant effects, it is important to consider the synergistic effect of all its bioactive constituents. Furthermore, saffron components have demonstrated anti-inflammatory and antiapoptotic effects. Crocin and crocetin have also been found to enhance oxygen diffusion and improve ocular blood flow in the retina and choroid, factors that play a crucial role in AMD.

It is worth noting that saffron does not simply act as a conventional antioxidant, but exhibits complex mechanisms of action ranging from antioxidant activity to direct gene expression control. Several clinical studies have been conducted to assess the impact of oral saffron supplementation (at a dose of 20mg daily) on vision-related parameters in AMD patients. Both short-term studies and longer-term follow-ups have demonstrated improvements in visual functions with saffron supplementation.

Vitamins C and E have been extensively studied for their role in preventing and treating macular degeneration and other eye-related diseases. One of the main reasons for their effectiveness is their antioxidant properties. Vitamin C, also known as ascorbic acid, is a potent antioxidant that helps protect proteins, lipids, carbohydrates, and nucleic acids from damage caused by free radicals and reactive oxygen species (ROS). By neutralizing these harmful molecules, vitamin C helps prevent oxidative stress and the associated damage to cellular components.

Vitamin E is another important antioxidant nutrient. It exists in several forms, with alpha-tocopherol being the most biologically active form. Like vitamin C, vitamin E acts as a scavenger of free radicals and ROS, protecting cellular structures and molecules from oxidative damage. It plays a crucial role in maintaining the integrity of cell membranes, including those in the retina.

Both vitamins C and E work synergistically to combat oxidative stress and protect against macular degeneration and other eye diseases. Their antioxidant actions help preserve the health of ocular tissues, including the retina, by reducing the impact of oxidative damage on vital cellular components. It is important to ensure an adequate intake of these vitamins through a balanced diet or, if necessary, through supplementation under the guidance of healthcare professionals.

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