Literature data demonstrate already that the presence of adenine nucleotides in the extracellular environment induces cell death that leads to several retinopathies. As said, the objective is to carry out a systematized review of the last decade, relating purinergic signaling to the outcome of cell death and retinopathies. It is possible to identify different mechanisms that occur through the activation of purinergic receptors. The exacerbated activation of the P2X7 receptor is mainly involved in the apoptotic death pathway, and this response is due to the dysregulation of some components in the intracellular environment, such as the Ca2+ ion, CD40, MiR-187, and influence of mononuclear macrophages. The A2A receptor is involved in increasing levels of cytokines and promoting inflammatory processes. The data presented can be used as a basis to better understand the mechanisms of death in retinopathies, in addition to proposing therapeutic strategies with the potential to be transposed to several other models.
- P2x7 receptor
- A2a receptor
The retina is a tissue that is located at the back of the eye and is responsible for converting light stimuli into electrical signals, a process is known as phototransduction, being responsible for the sense of vision . In this tissue, as in many others, cell death is a highly regulated process that is important for maintaining homeostasis, in addition to preserving tissue function by excluding cells whose genome is altered . According to Fricker et al. , there are some particularities in cell death in neurons. For example, complexity in nervous system circuitry during development results in programmed cell death of neurons that fail to connect properly. Furthermore, the excitability of neurons causes a high volume of adenosine triphosphate (ATP) and, due to a cytotoxic effect of this molecule in high concentrations, a sensitivity to death in several pathological models .
Damage or dysfunction in the retina, through excess cell death, can lead to several pathological conditions that interfere with the normal function of the tissue, compromising the ability to transmit visual stimuli to higher centers. A possible classification of diseases that affect the retina is hereditary retinal dystrophies, a group of pathologies caused by spontaneous genetic alterations that lead to irreversible loss of vision . These diseases have great clinical relevance since they have a high incidence, and in Brazil, the ones that stand out are Non-syndromic Retinitis Pigmentosa, Stargardt’s disease, Leber’s Congenital Amaurosis, and hereditary retinal syndromic dystrophies . Another known classification is retinopathies, a set of pathologies that affect the retina, which involve damage to the surrounding vasculature . The decrease in the number of blood vessels leads to a process called retinal neovascularization that, occurring in a disorderly way, can deregulate tissue homeostasis or cause the process of retinal detachment .
Diseases in which vascular damage occurs are referred to as ischemic retinopathies and include diabetic retinopathy, retinal vein occlusions (RVOs), retinopathy of prematurity (ROP), and sickle-cell retinopathy. Thus, following this definition, other diseases fit this classification as they present stages with vascular damage, such as age-related macular degeneration (AMD), retinal detachment, and glaucoma . Both in the case of retinopathies and retinal dystrophies, most of them remain without a cure or with inadequate treatment and, therefore, new therapeutic strategies are necessary. In this case, the manipulation of molecular pathways can change the course of cell death, and the participation of the purinergic system is of great interest in these cases.
Cell death in non-neural cells has already been described, in which extracellular ATP triggers cell death by binding to P2X7 receptors, which is the main receptor activated in these cases. Upon activation, P2X7 receptors induce large, non-selective membrane pores, which eventually lead to cell death [9, 10, 11, 12]. In addition to the evident relationship between the P2X7 receptor and the cell death process, other purinergic receptors participate in this cellular response. The P2X2 and P2X4 receptors, for example, are upregulated in squirrel ischemia models, and the improvement in cell death with the inhibition of these receptors confirms the participation of purinergic signaling .
Purinergic signaling mediated by extracellular ATP and adenosine is involved in the induction and protection of cell death in several models of retinal diseases . One of the mechanisms observed in retinal pathologies is the increased expression of purinergic receptors that contribute to high calcium concentrations. P2X receptors act as direct channels for calcium influx and as indirect activators of voltage-gated calcium channels. Meanwhile, activation of P2Y receptors induces a rapid transient release of calcium from internal stores, followed by an influx of calcium. The increased expression, mainly of the P2X7 receptor, is involved in the pathogenesis of several diseases that affect the retina, in which ATP in high concentrations is capable of inducing apoptosis through this purinergic receptor .
Studies show that ATP induces apoptosis in embryonic retinal neurons of chicks in culture through the activation of the P2X7 receptor and ionotropic glutamate receptors. . Other research also proves that the direct application of ATP to isolated retinas induces the death of cholinergic amacrine cells that express P2X7 receptors [9, 10].
Although ATP is well known for being toxic in high concentrations, and its receptors are involved in several pathologies as inducers of cell death, adenosine receptors are also worth mentioning. When it comes to cell death, the adenosine A2A receptor plays a role . It has already been seen that blocking the A2A receptor controls microglial reactivity , delays excitotoxic death of embryonic motor neurons
Considering the close connection between the release of adenine nucleotides in the extracellular environment and cell death through the activation of purinergic receptors, it is of great interest to observe the latest studies carried out on the subject. Therefore, the objective of the present review is to present the knowledge obtained from the studies of the last decade, making clear the participation of purinergic signaling in cell death induced by different models of retinopathies.
2. Age-related macular degeneration (AMD)
2.1 Participation of calcium pathways
Age-related macular degeneration (AMD) is the main cause of irreversible vision loss in the elderly in developed countries, being able to affect several cell types in the retina [21, 22]. From the observed damage, numerous studies try to understand the molecular mechanisms involved in this pathology.
The retinal pigment epithelium (RPE) is considered a site of great interest in this pathology, and it is now well known that ATP acts as a key signaling molecule in several cellular processes, including cell death . The P2X7 receptor is also involved in inflammation and oxidative stress in many cell types, and cell death, inflammation, and oxidative stress have been implicated in AMD.
Through the use of apoptotic markers, Yang et al. sought to know whether the presence of ATP, an endogenous P2X receptor agonist, increased the number of cells undergoing apoptosis in human pigment epithelium cell cultures. In cultures treated with ATP, it was possible to observe an increase in the intensity of apoptotic markers when compared to the control. This effect was blocked by the administration of the oxidized P2X7 antagonist (oATP). The selective exogenous P2X7 receptor agonist, 3’-O-(4-benzoyl) benzoyl-ATP (BzATP), was also able to increase apoptosis, but Brillant Blue G (BBG), a P2X7 receptor antagonist, and oATP reverse this effect .
Treatment with BAPTA-AM, used to decrease intracellular calcium levels, was able to decrease ATP and BzATP-induced apoptosis, which indicates that Ca2+ is an essential component for signaling the P2X7 receptor pathway and continuation of the apoptotic cascade. In summary, the study by Yang et al. provides the first evidence of the presence of functional P2X7 receptors in human pigment epithelium cell cultures and demonstrates that activation of P2X receptors, especially P2X7 receptors, induces Ca2+ signaling and apoptosis in these cells .
2.2 Participation of oxysterols
Another implication of AMD is the accumulation of drusen, which are extracellular proteolipid deposits, contributing to vision loss in the advanced stages of the disease. These deposits are located between the RPE and Bruch’s membrane (inner layer of the choroid) and contain β-amyloid peptide as the main component . Different oxysterols were found in human drusen, which suggests their involvement in AMD. Furthermore, the aggregated form of β-amyloid is well known as an inducer of oxidative stress and cell death [26, 27].
Oliver et al. aimed to highlight the β-amyloid/oxysterols relationship and describe the involvement of the P2X7-pannexin-1 receptor in oxysterol toxicity in human RPE cell cultures . A link was found between the presence of β-amyloid peptide aggregates and oxysterol levels. Two types of oxysterols, 25-OH and 7-KC seem to play a role in the pathogenesis of AMD through P2X7 activation, but only 25-OH causes pannexin-dependent pore opening in the cell membrane. This pannexin-stimulated pore opening is important in the pathological mechanism of the disease, as it promotes the extravasation of ATP to the extracellular environment, and consequent activation of the P2X7 receptor. Thus, the toxicity of this oxysterol occurs in two ways—increased P2X7 receptor activity and oxidative stress-dependent on pannexin-1, and pannexin-1-independent chromatin condensation .
The potential relationship between oxysterols and β-amyloid in AMD supports the notion that oxysterols can be considered as biomarkers of retinal degeneration. Considering the fundamental role of P2X7 receptor activation in oxysterol cytotoxicity, this may be an important target for the development of treatments for this disease .
2.3 Participation of alu RNA
Geographic atrophy (GA) is an advanced form of age-related macular degeneration characterized by central loss of vision due to confluent areas of retinal pigment epithelium loss and degeneration of overlying photoreceptors . The DICER1 processing enzyme is specifically reduced in the RPE in eyes with geographic atrophy, as its blockage results in an abundant increase in Alu RNA transcripts (an endogenous retroelement that requires reverse transcriptase for its life cycle), which in turn promotes the cell death of the RPE .
More recent studies have identified that the cytotoxicity of Alu RNA in the RPE is mediated by the activation of the NLRP3 inflammasome , and it has already been observed that reactive oxygen species (ROS) and the P2X7 receptor are involved in this process in other systems [31, 32]. Therefore, Kerur et al. investigated whether P2X7 signaling was also involved in Alu RNA-induced NLRP3 inflammasome activation, with experiments performed in mouse and human retinal pigment epithelial cell cultures .
After transfection of the Alu RNA into the culture media containing the cells of interest, it was seen that NF-kB signaling and P2X7 activation play important roles in Alu RNA-induced inflammasome initiation and activation and RPE degeneration. The authors also suggested, from cell cultures of P2X7 receptor knockout mice, that this receptor is an essential intermediate in the Alu RNA-induced activation of the NLRP3 inflammasome and consequent RPE degeneration. This suggests that manipulating this pathway may be a useful strategy for developing drugs for the treatment of geographic atrophy .
In complete agreement with these results, Fowler et al. also investigated the relationship between the P2X7 purinergic receptor and Alu RNA-induced AMD. It was based on what was previously demonstrated, that Alu-derived RNA activates the NLRP3 inflammasome, via the P2X7 receptor, to cause cell death of the retinal pigment epithelium in geographic atrophy . As Alu RNA requires reverse transcriptase for its life cycle, the use of transcriptase inhibitors has been proposed for a definition of other therapeutic alternatives for the disease .
After injection of Alu RNA or transfection into human and mouse retinal pigment epithelium cell cultures, Alu RNA was seen to be cytotoxic, as it activates caspase-1 and activates IRAK4 (interleukin-1 receptor-associated kinase 4), whose phosphorylation in these cases leads to degeneration of the pigment epithelium). Alu and LPS, a bacterial compound known to activate inflammatory pathways, activate the NLRP3 inflammasome via activation of the P2X7 receptor. d4T (reverse transcriptase inhibitor—NRTI) acts in a protective manner, preventing caspase-1 activation and IRAK4 phosphorylation. Several approved and clinically relevant NRTIs, including lamivudine (3TC) and abacavir (ABC), prevented the activation of caspase-1, and the Alu RNA-induced inflammasome effect of NLRP3. NRTIs were effective in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation .
2.4 Abnormal vascular growth
In the neovascular form of age-related macular degeneration, visual loss commonly occurs as a result of the invasion of abnormal blood vessels from the choroidal circulation, that is, choroidal neovascularization (CNV), which induces irreversible damage to the overlying retina. CNV mainly occurs due to dysregulation in the production of endothelial growth factors in the retinal vascular network .
Photoreceptor degeneration involves the activation of several regulated cell death signaling pathways that may constitute potential therapeutic targets. ATP has already been discovered as an important extracellular messenger that may contribute to lethal signaling . Thus, Notomi et al. hypothesized that ATP acting via the P2X7 receptor is involved in the pathogenesis of photoreceptor loss in subretinal hemorrhage.
The results suggest that ATP levels in the subretinal space could potentially be higher than those detected in the vitreous because extracellular ATP diffuses into the vitreous cavity from the subretinal space. From the analysis of cell death in cultures of primary retinas in vitro and in a subretinal hemorrhage model in vivo, it was observed that a concentration of 1 mM of ATP triggered an apoptotic process in photoreceptor cells through binding to the P2X7 receptor, while the use of a selective inhibitor of the P2X7 receptor (Brilliant Blue G (BBG)) was able to prevent this effect. These results indicate that extracellular ATP can trigger apoptosis of photoreceptor cells via P2X7 receptor-dependent machinery. Thus, it is shown that pharmacological inhibition of the P2X7 receptor with BBG may result in neuroprotection of photoreceptors in cases of subretinal hemorrhage .
The study further suggests that similar severe neurodegenerative pathologies, such as subarachnoid hemorrhage or intracerebral hemorrhage, may be related to elevations in extracellular ATP. In this way, P2X7 receptor antagonists including BBG may have a neuroprotective therapeutic effect in retinal diseases as well as in Central Nervous System diseases with excessive extracellular ATP.
2.5 Infiltration and accumulation of mononuclear phagocytes
Also focusing on damage to photoreceptors in AMD, Hu et al. related this disease to the infiltration and chronic accumulation of mononuclear phagocytes, , which in excess lead to neuronal degeneration . It has also been seen that a deficiency in Cx3cr1, a transmembrane chemokine receptor involved in leukocyte adhesion and migration, leads to the accumulation of mononuclear phagocytes, but the mechanism by which this occurs has not yet been well elucidated .
From the isolation of bone marrow-derived monocytes that are recruited to the inflammatory site, the expression levels of the P2X7 receptor in these cells were evaluated by flow cytometry. The study confirmed that the accumulation of mononuclear phagocytes in cases of Cx3Cr1 deficiency leads to increased expression of the P2X7 receptor in these cells. The authors observed that, in these situations, P2X7 receptors provoke the opening of pannexin-dependent pores and release ATP to the external environment. This ATP, from the P2X7 receptor, is able to activate inflammasomes which, in turn, are responsible for the maturation and release of interleukin-1 β (IL-1β), responsible for cytotoxicity and increased cell death in photoreceptors. This was confirmed by the ELISA assay, in which IL-1β levels are increased in cases of Cx3Cr1 deficiency .
To test whether P2X7 receptor inhibition has a protective effect against death, intravitreal injection of BBG, a selective inhibitor of the P2X7 receptor, was performed. The TUNEL assay showed that the number of apoptotic cells in the photoreceptor layer was reduced after administration of BBG in cases of Cx3Cr1 deficiency. Immunostaining with Iba-1 to quantify inflammation-associated reactive microglia showed that intravitreal injection of BBG was able to protect against inflammation in these cases.
P2X7 receptor inhibitors, therefore, may be a promising therapeutic target to inhibit lesion expansion in cases of Macular Degeneration, as they may prevent RPE cell death, and IL-1β and P2X7 inhibitors may help to prevent RPE cell death. Photoreceptor loss associated with inflammation .
3. Diabetic retinopathy
3.1 Damage to the blood-retinal barrier
Diabetic retinopathy is a serious complication of diabetes mellitus. Breach of the blood-retinal barrier (BRB) is a hallmark of diabetic retinopathy, as well as other eye diseases . The human retina contains two BRBs, the inner and the outer, including endothelial cells and retinal pigment epithelial cells, respectively . Maintenance of the physiological structure of retinal cells requires complex cell-to-cell interactions. These interactions occur at special contact sites called cell junctions, which include tight junctions (TJs), adherent junctions (AJs), and gap junctions (GJs) .
Knowing this, Platania et al. tested the hypothesis that activation of the P2X7 receptor contributes to the degradation of the inner portion of the BRB, also interfering with the integrity of the endothelial barrier, through the disruption of TJs between endothelial cells of human retinas, in an environment with high concentrations of glucose . Using the bioinformatics program GEO2R, used to identify differentially expressed genes between two groups, P2X7 receptor expression was measured in human retinal endothelial cell cultures. The expression of the P2X7 receptor underwent a significant increase, induced by both the high concentration of glucose and the agonist BzATP, when compared to the control. Furthermore, high glucose induced the activation and release of the pro-inflammatory cytokine IL-1β via P2X7 receptor activation in human retinal endothelial cells. Glucose exposure also caused a decrease in endothelial cell viability and damage to the BRB .
It was also seen, by performing the transendothelial electrical resistance assay (TEER) to measure cell membrane integrity and cell-to-cell interactions, that blocking the P2X7 receptor with the drug JNJ47965567 was able to protect retinal endothelial cells against damage induced by high glucose concentrations and protected the blood-retinal barrier. In addition, treatment with JNJ47965567 significantly decreased the expression and release of IL-1β, induced by high glucose. These findings suggest that the P2X7 receptor plays an important role in regulating the integrity of the retinal blood barrier, and blocking this receptor was useful to counteract the damage caused by high glucose concentration in retinal endothelial cells. Thus, the use of P2X7 receptor antagonists may be useful in the treatment of diabetic retinopathy .
3.2 Participation of P2X receptors in hyperglycemic retinas
Long-term exposure to high glucose concentration, considered the main factor in the development of diabetic retinopathy, has already been shown to affect extracellular ATP levels in retinal cell cultures . Furthermore, ATP can act as a neurotransmitter in the retina [45, 46], and through activation of plasma membrane receptors, it can increase intracellular calcium concentration. Some of the inflammatory mediators and excitatory neurotransmitters seen in neuronal death in diabetic retinas are released in response to an increase in intracellular Ca2+ concentration. Considering this, Pereira et al. sought to investigate whether the exposure of retinal cells from mice grown under high glucose levels could alter the function of P2X receptors .
In this study, through the Western Blot assay, it was seen that cultures of rat retinas exposed to high glucose concentration, the following subunits of the P2X receptor were found—P2X2, P2X3, and P2X7, but these did not undergo any significant change in their content when compared to the control. It is noteworthy that in these retinas the P2X4 receptor was affected by the high concentration of glucose, and its expression was reduced .
Through the Fura-2 assay (dye used for labeling intracellular calcium) it was shown that intracellular calcium concentrations triggered by the stimulation of P2 receptors are increased in retinal cells of rats cultured at high glucose concentrations, in a model used to simulate the hyperglycemic conditions seen in diabetes. Also using Fura-2, a difference in the pattern of Ca2+ concentration based on cell type was noted. In retinal neurons, the increase in intracellular Ca2+ concentration was mainly due to the influx of Ca2+ through voltage-sensitive calcium channels. In microglial cells, Ca2+ influx occurred mainly through P2X receptor channels, although there was also a minor component of increased intracellular Ca2+ concentration dependent on calcium release from intracellular stores .
These increased calcium responses may be responsible for the increased release of neurotransmitters and/or inflammatory mediators found in diabetic retinas and therefore contribute to retinal neural cell death detected in the early stages of diabetic retinopathy. Since intracellular calcium plays a key role in cell death, inhibition of some purinergic receptors may exert protective effects against retinal neural cell dysfunction or degeneration, and therefore P2 receptors may become a potential therapeutic target for the treatment of early stages of diabetic retinopathy.
3.3 Involvement of the differentiation cluster 40 (CD40)
Capillary degeneration is a hallmark of early diabetic retinopathy. They are the result of loss of retinal endothelial cells and pericytes (perivascular cells essential in maintaining metabolic, mechanical, and signaling functions in microvessels) . Cluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the death of retinal endothelial cells . However, binding of CD40 on endothelial cells does not normally induce cell death, likely because CD40 activates PI3K/Akt-mediated pro-survival signals [50, 51]. Thus, Portillo et al. aimed to identify a mechanism by which CD40 triggers programmed cell death in human retinal endothelial cell cultures and address this apparent contradiction .
Administration of CD40 ligand in primary cultures of human retinal endothelial cells did not significantly alter the percentage of apoptotic cells. Given the close connection between these cells and Müller’s glia, we sought to determine whether Müller’s glia would indirectly influence the triggering of CD40-mediated cell death. The results showed that CD40 does not exert its effects directly on endothelial cells, but on circulating Müller’s glia. It was also seen, by measuring cytokines by the ELISA assay, that CD40 also did not provoke the secretion of IL-β or TNF-α. In fact, CD40-stimulated Müller glia releases ATP into the extracellular medium. By performing a qPCR, it was noted that CD40 also upregulated the expression of the P2X7 receptor on the surface of endothelial cells, making them susceptible to the cell death process mediated by ATP/P2X7.
In summary, these studies have uncovered a mechanism by which CD40 enhances cell death of retinal endothelial cells and suggest that CD40 signaling on Müller cells may be an important contributor to vascular injury in diabetic retinopathy. The expression of CD40 was responsible for the secretion of ATP to the extracellular medium, favoring a greater activation of the P2X7 receptor. Increased programmed cell death accompanies these disorders and the P2X7 receptor is consistently seen as pathogenic in these diseases . The findings may be relevant to other diseases caused by CD40, such as atherosclerosis and inflammatory bowel disease. Thus, new therapies can be developed to treat these diseases: blocking CD40 or the P2X7 receptor may prove to be effective alternatives in the treatment of diabetic retinopathy .
Growing evidence indicates that chronic inflammation is important for the development of diabetic retinopathy [54, 55]. TNF-α and IL-1β are pro-inflammatory molecules upregulated in this disease . In addition to macrophages/microglia, Müller’s glia (the main retinal microglia) become dysfunctional in diabetes and contribute to the development of diabetic retinopathy. Since CD40 deficiency impairs this process and prevents diabetic retinopathy [52, 57], Portillo et al. sought to elucidate the mechanisms by which this response occurs .
The study carried out showed, through real-time PCR, an increase in CD40 expression by Müller’s glia in a mouse model of diabetic retinopathy. Additionally, it was seen that CD40 binding on Müller’s glia triggered phospholipase C-dependent release of ATP. This release provoked activation of P2X7 receptors, resulting in the release of TNF-α and IL-1β by macrophages. To better prove the role of the P2X7 receptor in this process, mice that do not express the P2X7 receptor and mice treated with a P2X7 inhibitor were protected from the increase in the concentration of TNF-α, IL-1β, ICAM-1, and NOS2 induced by diabetes, thus preventing the inflammatory process and cell death .
The observed effects are relevant in vivo because TNF-α is up-regulated in microglia/macrophages of diabetic mice that express CD40 on Müller glia and mice treated with BBG (P2X7 receptor inhibitor) are protected from diabetes-induced upregulation of TNF-α and IL-1β. This protection prevents the inflammatory process that normally accompanies the release of these cytokines, alleviating the pathological effects of diabetic retinopathy. Thus, this study indicates that CD40 in Müller’s glia is sufficient to up-regulate retinal inflammatory markers. Furthermore, CD40 appears to promote experimental diabetic retinopathy and Müller’s glia orchestrates inflammatory responses in myeloid cells via a CD40-ATP-P2X7 pathway .
3.4 Therapeutic potential of the A2A receptor
Diabetic retinopathy is one of the main complications of diabetes mellitus and one of the main causes of blindness. The pathogenesis of diabetic retinopathy is accompanied by chronic low-grade inflammation. Adenosine is a neuromodulator of the central nervous system that exerts its actions through the activation of its four receptors: A1, A2A, A2B, and A3. Some reports demonstrate that the microglial cell response can be altered by A2A receptor antagonists in the different brain and retinal diseases [59, 60]. Therefore, Aires et al. sought to find out whether blocking the A2A receptor can provide protection to the retina by modulating microglial reactivity .
Through the use of specific immunomarkers, it was observed that the number of reactive microglia was increased in the retina of mice in a model of induced diabetes. Intravitreal injection of SCH 58261, the A2A receptor antagonist significantly decreased microglial reactivity in the retinas of diabetic animals. The ELISA assay confirmed that, accompanied by this decrease in microglial activity, treatment with the A2A receptor antagonist was able to decrease the levels of TNF and IL-1β cytokines, also demonstrating the ability to control inflammatory processes .
In addition to these results, the injection of SCH 58261 was able to decrease the levels of reactive oxygen species. The TUNEL assay confirmed the neuroprotective potential of this inhibitor, demonstrated in the fall of apoptotic cells in the retina of mice in vivo, and the prevention of cell death preserved the thickness of the retinal tissue. Finally, regarding the vascular damage characteristic of Diabetic Retinopathy, it was seen that the inhibition of the A2A receptor contributes to the preservation of the integrity of the blood-retinal barrier. All these data demonstrate the therapeutic potential of A2A receptor antagonists for the treatment of diabetic retinopathy .
4. Photoreceptors’ degeneration
Photoreceptor degeneration involves the activation of several cell death pathways that may constitute potential therapeutic targets, and an alternative for the inhibition of death pathways is to intercept death, such as the activation of caspases. Among the seven mammalian P2X receptors, the P2X7 receptor has the highest affinity for ATP . Thus, extracellular ATP can induce apoptotic and/or necrotic cell death, acting on the P2X7 receptor . Taking into account the therapeutic possibility of P2X7 receptor inhibitors (such as Brillant Blue G) [11, 63], Notomi et al. decided to investigate the pathogenic implications of the P2X7 receptor in the pathological loss of photoreceptors in mice, as well as the therapeutic utility of BBG in this context .
By administering ATP or BzATP (selective P2X7 receptor agonist) in primary cultures of mouse retinal cells, it was seen that stimulation of the P2X7 receptor with these ligands could directly mediate the cell death pathway in photoreceptors. Inhibition of the P2X7 receptor with BBG or KN-62 was able to prevent photoreceptor cell death, confirming the role of this purinergic receptor in this process. The pathway followed after activation of the P2X7 receptor was also demonstrated, in which photoreceptor cell death occurred through calcium influx (observed through the use of the calcium marker Fluo-4 AM) and caspase-8 activation, suggesting a potential mechanism to an extrinsic pathway mediated by the P2X7 receptor .
Intravitreal injections of BzATP administered to mice showed that this specific P2X7 receptor agonist has the potential to induce retinal cell death in vivo. Inhibition of the P2X7 receptor proved to be effective in preventing cell death and preserving photoreceptors. Furthermore, blocking the P2X7 receptor indirectly inhibits the caspases of the mitochondrial cell death pathway in retinal cell cultures. Together, these results clarify some of the mechanisms of cell death induced by the binding of ATP to the P2X7 receptor and how antagonists, especially BBG, have clear relevant therapeutic effects that can be transferred to other models of neurodegenerative diseases, having neuroprotective potential that are also relevant. Applies to photoreceptors .
5.1 Ischemia-induced damage
As a chronic neurodegenerative condition, glaucoma is characterized by the loss of retinal ganglion cells, resulting in progressive optic neuropathy and consequent visual field loss. Reduced blood flow to the optic nerve region and consequent ischemia has been suggested as a mechanism of ganglion cell death in glaucoma [65, 66].
There is currently considerable interest in the P2X7 receptor in mediating neurodegeneration, with increasing evidence indicating its role in chronic disease [67, 68]. Some studies have also provided evidence that the P2X7 receptor may play a role in glaucoma-induced death [69, 70, 71]. Taking this into account, Niyadurupola et al. sought to determine whether stimulation of ischemia-induced death in the ganglion cell layer is mediated by P2X7 in the human retina .
As a result, it was seen that stimulation of the P2X7 receptor by the selective agonist BzATP induced cell death in ganglion layer cells in organotypic cultures of human retinas, which was inhibited by the P2X7 receptor inhibitor (BBG). In addition, the ischemia caused to cells in culture led to the loss of retinal ganglion cells, and this effect was also inhibited by BBG, which suggests the participation of the P2X7 receptor in the observed degeneration. Finally, it was possible to locate the P2X7 receptor in the outer and inner plexiform layers of the retina, and the ganglion cells also expressed the mRNA encoding the P2X7 receptor protein .
All these data confirm the great importance of this purinergic receptor in the retina and its relationship with glaucoma, since the stimulation of the P2X7 receptor can mediate the death of retinal ganglion cells and that this mechanism plays a role in ischemia-induced neurodegeneration in the human retina. In addition, the therapeutic potential of P2X7 receptor inhibitors is clear, with the aim of preventing cell death .
5.2 NMDA-induced damage
It is known that glutamate receptor stimulation by excess glutamate during hypoxia  and ischemia-reperfusion  is toxic to neurons. Activation of the N-methyl-D-aspartic acid (NMDA) receptor, a subtype of glutamate receptor, is followed by a large influx of Ca2+. This excess of intracellular Ca2+ is predominantly involved in neuronal excitotoxicity processes and is considered one of the mechanisms of glaucoma-induced neuronal cell death . Furthermore, some studies have also suggested that the P2X7 receptor plays a role in retinal ganglion cell death induced by high ocular pressure [69, 74, 75]. So, Sakamoto et al. sought to examine the role of P2X7 receptors in NMDA-induced retinal damage in mice in vivo .
The results obtained in the study demonstrate that, as expected, the intravitreal injection of the P2X7 receptor agonist (BzATP) induces cell death in the rat retina, an effect that was prevented by the administration of receptor antagonists (A438079 and Brillant Blue G). After this confirmation, it was evaluated whether the NMDA receptor produces its toxic effects through this receptor. Injections of the P2X receptor inhibitors, A438079 and BBG, were able to reduce the deleterious effects of NMDA, decreasing the number of apoptotic cells in cases of glaucoma induced through the NMDA receptor, confirming the neuroprotective effect of these drugs on the retina against toxicity of the drug. Glutamate. Finally, the immunohistochemistry technique was performed to determine the distribution pattern of the P2X7 receptor in mouse retinas. The results indicated that P2X7 receptors were expressed in the somatic region of RGCs that had DAPI-labeled nuclei in the ganglion cell layer and in the inner and outer plexiform layers. .
These results then showed for the first time that P2X7 receptor activation is, at least in part, involved in NMDA-induced retinal damage. In summary, the study authors demonstrate the possibility that P2X7 receptor antagonists are effective in preventing retinal diseases caused by glutamate excitotoxicity .
5.3 Participation of miR-187
MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate transcription and translation of target genes by interacting with the 3′-untranslated region of the target gene (3’-UTR), thus mediating the pathogenesis of multiple human diseases . Previous studies confirmed that miR-187 promoted retinal ganglion cell survival and decreased apoptosis of these cells in human ganglion cell culture incubated with TGF-β, suggesting a protective role of miR-187 against glaucoma . Given the role of miR-187 and the P2X7 receptor in glaucoma, Zhang et al. sought to know whether there is a functional correlation between miR-187 and the P2X7 receptor in apoptosis in a mouse retinal ganglion cell culture-induced model of glaucoma .
The results showed that high pressure-induced oxidative stress in retinal ganglion cells was accompanied by a decrease in miR-187 expression and an increase in P2X7 receptor expression. It was also found that miR-187 down-regulated P2X7 receptor expression in ganglion cells, and this inhibition was able to inhibit oxidative stress and apoptosis in these cells . These data demonstrated that miR-187/P2X7 signaling is involved in retinal cell apoptosis, at least in part, through oxidative stress activation. In vitro experiments showed that both miR-187 and the P2X7 receptor were upregulated in the retina of mouse models of chronic ocular hypertension . Thus, miR-187 and the P2X7 receptor promise to be a new target for the prevention and treatment of ophthalmic neurodegenerative diseases.
6. Retinopathy of prematurity (ROP)
Retinopathy of prematurity (ROP) is a disease that can cause blindness in very low birth weight babies and remains a leading cause of childhood blindness in many parts of the world [81, 82]. As a disease that mainly affects the retinal vasculature, existing treatments for this disease, such as anti-VEGF therapy, can have adverse effects, compromising the development of blood vessels and leading to peripheral blindness . The therapeutic potential provided by the antagonism of the purinergic A2A receptor has already been verified, and it may represent a new and promising pharmacological strategy to control pathological retinal angiogenesis under ROP conditions. This strategy avoids the onset of negative effects observed in the anti-VEGF strategy, as it alters molecular mechanisms without compromising the maintenance of the vasculature or the formation of new blood vessels . That said, Zhou et al. sought to extend this potential of A2A receptor inhibition, using it as a therapeutic strategy to selectively control pathological retinal neovascularization, in a model of oxygen-induced retinopathy leading to ROP .
To verify whether the use of the A2A receptor inhibitor would alter the retinal vasculature under physiological conditions, KW6002 (A2A receptor inhibitor) was administered intraperitoneally in mice of the C57BL/6 strain. Repeated exposure to KW6002 did not alter the mice’s normal retinal vasculature, showing that the treatment has no unwanted effects. After that, immunohistochemistry and immunofluorescence assays showed that, in a model of induced RDP in mice, the administration of KW6002 reduced avascular areas and neovascularization, with apoptosis and cell proliferation also reduced, and astrocyte functions increased. Thus, KW6002 treatment increased astrocyte participation and reduced cell proliferation and apoptosis to confer protection against pathological angiogenesis in ROP .
7. Retina detachment
Retinal detachment involves the separation of the sensorineural retina (responsible for receiving and conducting light stimuli to the higher centers of vision) from the retinal pigment epithelium. Direct contact between the retina and the pigment epithelium is essential for its normal function, and detachment can lead to irreversible vision loss . A2A adenosine receptor signaling has been shown to be neuroprotective in some models of retinal damage, but its role in neuronal survival during retinal detachment is unclear. Therefore, Gao et al. sought to modulate the A2A receptor-dependent signaling cascade and observe whether there would be changes in the rate of photoreceptor apoptosis .
In a mouse model of retinal detachment, A2A receptor expression was determined from real-time PCR and Western blot assays. It was found that A2A receptor protein was detected in the ganglion cell layer, the inner and outer plexiform layers, and the inner nuclear layer after the retinal detachment protocol. It is worth noting that this receptor was predominantly expressed in microglia and in Müller’s glia .
The role of the A2A receptor in different models of pathologies is quite controversial, and the cellular response followed may favor cell death or neuroprotection. However, the effect caused by this receptor in cases of retinal detachment had not been well elucidated so far. Thus, in this study, intravitreal injection of the drug ZM241385, a selective antagonist of the A2A receptor, was performed. Through immunofluorescence assays using specific markers, it was seen that blockade of the A2A receptor inhibited microglia reactivity after the triggering of retinal detachment, accompanied by a reduction of microglial proliferation. The drug also decreased the expression of GFAP (reactive gliosis marker) and decreased the expression of the inflammatory cytokine IL-1β. Furthermore, by performing a specific measurement assay for oxidative stress, it was seen that inhibition of the A2A receptor reduced the amount and production of reactive oxygen species in detached retinas .
Finally, through the TUNEL assay, the rate of apoptotic cells in the retina after the induction of retinal detachment was evaluated. The administration of ZM241385 was able to prevent the loss of photoreceptors caused by the high concentration of reactive oxygen species triggered after retinal detachment, further reinforcing the role of A2A receptor inhibition in the control of neuroinflammation. Thus, the involvement of the purinergic A2A receptor in the pathogenesis of retinal detachment was confirmed, making it a promising therapeutic target in the treatment of the pathology .
8. Transitory retina ischemia
Transient retinal ischemia refers to a pathological condition that involves loss of blood supply to the retina, resulting in cell damage and death from lack of oxygen supply . It has already been seen that a mechanism for triggering this pathological condition is microglia-mediated neuroinflammation, raising the hypothesis that the control of microglial reactivity may provide neuroprotection. Furthermore, it has already been seen that inhibition of the A2A receptor led to neuroprotection from microglial control in cases of ischemia . Taking this into account, Boia et al. aimed to investigate the therapeutic potential of oral administration of the A2A receptor antagonist and the effects of caffeine ingestion (adenosine receptor antagonist) against neuroinflammation and cell death in a model of ischemia caused by intraocular pressure. in mice .
Knockout mice for the A2A receptor were used to evaluate the effects of the absence of this receptor in relation to the control in cases of ischemia. From the quantification of TNF and IL-1β levels by the ELISA assay, it was seen that IL-1β levels were not altered, but TNF levels were significantly reduced in A2A knockout retinas when compared to Wild-Type animals. Likewise, the use of the A2A receptor antagonist (KW6002) caused a reduction in TNF levels. In addition, labeling of reactive microglial activity was also found to be decreased when the A2A receptor was inhibited by the drug KW6002. The TUNEL assay confirmed the neuroprotection caused by the use of the A2A receptor inhibitor since the number of apoptotic cells was found to be reduced in relation to the control group .
Focusing on caffeine, which is an adenosine receptor antagonist, we quantified the expression of the same cytokines seen previously through qPCR and ELISA assay. In ischemic retinas, the acute administration of the substance was not able to change the levels of TNF or IL-1β; however, the transcriptional levels of the two cytokines were found to be elevated after 24 hours of administration. Regarding microglial activity, caffeine showed dichotomous results: after 24 hours, caffeine increased microglial reactivity, inflammatory response, and ischemia-induced cell death compared to the control group. However, at 7 days of reperfusion, caffeine administration decreased microglia reactivity and reduced levels of pro-inflammatory cytokines and cell death. This indicates that prolonged treatment with caffeine induces the beneficial effects presented .
Purinergic signaling has already been shown to be important for several cellular processes in different organs and systems. The present study showed the relevance of purinergic receptors in signaling cell death pathways in the retina, and the cytotoxic effects can be applied in the various retinopathies addressed (Figure 1). When it comes to cell death, the participation of the P2X7 receptor in cytotoxic and inflammatory processes is clear. Thus, despite the search to encompass the entire purinergic system, P2X7 and A2A receptors were the most found when it comes to cell death in the retina.
In the studies analyzed in this review, it is possible to recognize different cellular mechanisms that occur through the activation of the P2X7 receptor. Dependence on the influx of Ca2+ ions after receptor activation was present, since the lack of these ions prevents the apoptotic cascade from occurring, and this pattern was present in models of Macular Degeneration and Diabetic Retinopathy. In AMD models, other mechanisms by which the activation of the P2X7 receptor can act can also be observed, such as the increase in the levels of oxysterols, Alu RNA and the infiltration of mononuclear macrophages. All these factors contribute to the onset of cytotoxic effects and the initiation of cell death.
Cell death induced by purinergic signaling also extends to other pathologies: in Diabetic Retinopathy, it is noted that, in addition to calcium-dependent signaling, the P2X7 receptor is also involved in damage to the blood-retinal barrier (BRB), and damage mediated by the differentiation cluster 40 (CD40); in cases of glaucoma, the ischemic injury induced by NMDA or involving the MicroRNA MiR-187 has its toxic effects also mediated by this same receptor.
We can also see that the adenosine A2A receptor plays an important role in triggering several diseases, mainly in alterations of vascular integrity. This receptor has been seen to be involved in increasing cytokine levels in pathological conditions such as TGF and IL-1B; also in triggering microglial reactivity and promoting inflammatory processes.
With the results obtained from this analysis, it is clear that more and more studies claim that the exacerbated activation of the P2X7 receptor by extracellular ATP is highly involved mainly in the apoptotic pathway of cell death. When it comes to possible therapeutic targets for the diseases addressed, it is interesting to consider P2X7 receptor inhibitors for this purpose, as some of the studies show the effectiveness of these inhibitors in improving the effects observed in each model. Thus, the data presented here can be taken as a basis to better understand the mechanisms of death in various retinopathies, in addition to proposing therapeutic strategies with the potential to be transposed to several other models.
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