Blockade of microglial adenosine A2A receptor impacts inflammatory mechanisms, reduces ARPE-19 cell dysfunction and prevents photoreceptor loss in vitro.

Age-related macular degeneration (AMD) is characterized by pathological changes in the retinal pigment epithelium (RPE) and loss of photoreceptors. Growing evidence has demonstrated that reactive microglial cells trigger RPE dysfunction and loss of photoreceptors, and inflammasome pathways and complement activation contribute to AMD pathogenesis. We and others have previously shown that adenosine A2A receptor (A2AR) blockade prevents microglia-mediated neuroinflammatory processes and mediates protection to the retina. However, it is still unknown whether blocking A2AR in microglia protects against the pathological features of AMD. Herein, we show that an A2AR antagonist, SCH58261, prevents the upregulation of the expression of pro-inflammatory mediators and the alterations in the complement system triggered by an inflammatory challenge in human microglial cells. Furthermore, blockade of A2AR in microglia decreases the inflammatory response, as well as complement and inflammasome activation, in ARPE-19 cells exposed to conditioned medium of activated microglia. Finally, we also show that blocking A2AR in human microglia increases the clearance of apoptotic photoreceptors. This study opens the possibility of using selective A2AR antagonists in therapy for AMD, by modulating the interplay between microglia, RPE and photoreceptors.

Adenosine A2A receptor regulation of microglia morphological remodeling-gender bias in physiology and in a model of chronic anxiety.

Developmental risk factors, such as the exposure to stress or high levels of glucocorticoids (GCs), may contribute to the pathogenesis of anxiety disorders. The immunomodulatory role of GCs and the immunological fingerprint found in animals prenatally exposed to GCs point towards an interplay between the immune and the nervous systems in the etiology of these disorders. Microglia are immune cells of the brain, responsive to GCs and morphologically altered in stress-related disorders. These cells are regulated by adenosine A2A receptors, which are also involved in the pathophysiology of anxiety. We now compare animal behavior and microglia morphology in males and females prenatally exposed to the GC dexamethasone. We report that prenatal exposure to dexamethasone is associated with a gender-specific remodeling of microglial cell processes in the prefrontal cortex: males show a hyper-ramification and increased length whereas females exhibit a decrease in the number and in the length of microglia processes. Microglial cells re-organization responded in a gender-specific manner to the chronic treatment with a selective adenosine A2A receptor antagonist, which was able to ameliorate microglial processes alterations and anxiety behavior in males, but not in females.

The Adenosinergic System in Diabetic Retinopathy.

The neurodegenerative and inflammatory environment that is prevalent in the diabetic eye is a key player in the development and progression of diabetic retinopathy. The adenosinergic system is widely regarded as a significant modulator of neurotransmission and the inflammatory response, through the actions of the four types of adenosine receptors (A1R, A2AR, A2BR, and A3R), and thus could be revealed as a potential player in the events unfolding in the early stages of diabetic retinopathy. Herein, we review the studies that explore the impact of diabetic conditions on the retinal adenosinergic system, as well as the role of the said system in ameliorating or exacerbating those conditions. The experimental results described suggest that this system is heavily affected by diabetic conditions and that the modulation of its components could reveal potential therapeutic targets for the treatment of diabetic retinopathy, particularly in the early stages of the disease.

Neuropeptide Y receptors activation protects rat retinal neural cells against necrotic and apoptotic cell death induced by glutamate.

It has been claimed that glutamate excitotoxicity might have a role in the pathogenesis of several retinal degenerative diseases, including glaucoma and diabetic retinopathy. Neuropeptide Y (NPY) has neuroprotective properties against excitotoxicity in the hippocampus, through the activation of Y1, Y2 and/or Y5 receptors. The principal objective of this study is to investigate the potential protective role of NPY against glutamate-induced toxicity in rat retinal cells (in vitro and in an animal model), unraveling the NPY receptors and intracellular mechanisms involved. Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500 µM) for 24 h. Necrotic cell death was evaluated by propidium iodide (PI) assay and apoptotic cell death using TUNEL and caspase-3 assays. The cell types present in culture were identified by immunocytochemistry. The involvement of NPY receptors was assessed using selective agonists and antagonists. Pre-treatment of cells with NPY (100 nM) inhibited both necrotic cell death (PI-positive cells) and apoptotic cell death (TUNEL-positive cells and caspase 3-positive cells) triggered by glutamate, with the neurons being the cells most strongly affected. The activation of NPY Y2, Y4 and Y5 receptors inhibited necrotic cell death, while apoptotic cell death was only prevented by the activation of NPY Y5 receptor. Moreover, NPY neuroprotective effect was mediated by the activation of PKA and p38K. In the animal model, NPY (2.35 nmol) was intravitreally injected 2 h before glutamate (500 nmol) injection into the vitreous. The protective role of NPY was assessed 24 h after glutamate (or saline) injection by TUNEL assay and Brn3a (marker of ganglion cells) immunohistochemistry. NPY inhibited the increase in the number of TUNEL-positive cells and the decrease in the number of Brn3a-positive cells induced by glutamate. In conclusion, NPY and NPY receptors can be considered potential targets to treat retinal degenerative diseases, such as glaucoma and diabetic retinopathy.

Contribution of TNF receptor 1 to retinal neural cell death induced by elevated glucose.

Diabetic retinopathy (DR), a leading cause of vision loss and blindness among working-age adults, holds several hallmarks of an inflammatory disease. The increase in cell death in neural retina is an early event in the diabetic retina, preceding the loss of microvascular cells. Since tumor necrosis factor-α (TNF-α) has been shown to trigger the death of perycites and endothelial cells as well as the breakdown of the blood-retinal barrier, we set out to investigate whether TNF-α acting through tumor necrosis factor receptor 1 (TNFR1), the major receptor responsible for mediating TNF-induced cell death, could also be responsible for the early neuronal cell death observed in DR. We used retinal neural cell cultures exposed to high glucose conditions, to mimic hyperglycaemia, and evaluated the contribution of TNFR1 in neural cell death. TNFR1 was found to be present to a great extent in retinal neurons and the levels of this receptor were found to be altered in cells cultured in high glucose conditions. High glucose induced an early decrease in cell viability, an increase in apoptosis and a higher immunoreactivity for the cleaved caspase-3, indicating a high glucose-induced caspase-dependent cell death. These observations were correlated with an increase in TNF-α expression. Nonetheless, inhibiting the activation of TNFR1 was sufficient to prevent the decrease in cell viability and the increase in retinal cell death by apoptosis. In conclusion, our data indicate that TNF-α acting through TNFR1 is responsible for the high glucose-induced cell death and that blocking the activity of this receptor is an adequate strategy to avoid cell loss in such conditions.

High glucose changes extracellular adenosine triphosphate levels in rat retinal cultures.

Diabetic retinopathy (DR) is the leading cause of blindness in adults. In diabetes, there is activation of microglial cells and a concomitant release of inflammatory mediators. However, it remains unclear how diabetes triggers an inflammatory response in the retina. Activation of P2 purinergic receptors by adenosine triphosphate (ATP) may contribute to the inflammatory response in the retina, insofar as it has been shown to be associated with microglial activation and cytokine release. In this work, we evaluated how high glucose, used as a model of hyperglycemia, considered the main factor in the development of DR, affects the extracellular levels of ATP in retinal cell cultures. We found that basal extracellular ATP levels were not affected by high glucose or mannitol, but the extracellular elevation of ATP, after a depolarizing stimulus, was significantly higher in retinal cells cultured in high glucose compared with control or mannitol-treated cells. The increase in the extracellular ATP was prevented by application of botulinum neurotoxin A or by removal of extracellular calcium. In addition, degradation of exogenously added ATP was significantly lower in high-glucose-treated cells. It was also observed that, in retinal cells cultured under high-glucose conditions, the changes in the intracellular calcium concentrations were greater than those in control or mannitol-treated cells. In conclusion, in this work we have shown that high glucose alters the purinergic signaling system in the retina, by increasing the exocytotic release of ATP and decreasing its extracellular degradation. The resulting high levels of extracellular ATP may lead to inflammation involved in the pathogenesis of DR.

High glucose induces caspase-independent cell death in retinal neural cells.

Diabetic retinopathy is a leading cause of blindness among adults in the western countries. It has been reported that neurodegeneration may occur in diabetic retinas, but the mechanisms underlying retinal cell death are poorly understood. We found that high glucose increased the number of cells with condensed nuclei and the number of TUNEL-positive cells, and caused an increase in the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, indicating that high glucose induces apoptosis in cultured retinal neural cells. The activity of caspases did not increase in high glucose-treated cells, but apoptosis-inducing factor (AIF) levels decreased in the mitochondria and increased in the nucleus, indicating a translocation to the nucleus where it may cause DNA fragmentation. These results demonstrate that elevated glucose induces apoptosis in cultured retinal neural cells. The increase in apoptosis is not dependent on caspase activation, but is mediated through AIF release from the mitochondria.

Increase of the intracellular Ca2+ concentration mediated by transport of glutamate into rat hippocampal synaptosomes: characterization of the activated voltage sensitive Ca2+ channels.

The changes in the intracellular free Ca2+ concentration, [Ca2+]i, mediated by glutamate and D-aspartate into rat hippocampal synaptosomes was studied. Glutamate increased the [Ca2+]i in a dose-dependent manner with an EC50 of 1.87 microM and a maximal increase of 31.5 +/- 0.9 nM. We also observed that stimulation of the synaptosomes with 100 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), 100 microM kainate, or 100 microM D-aspartate increased the synaptosomal [Ca2+]i. The effect of either of these non-NMDA receptor agonists and of D-aspartate was additive, suggesting the activation of two different components (the ionotropic non-NMDA receptors or the glutamate transporters). Stimulation of synaptosomes with 100 microM glutamate increased the [Ca2+]i and prevented the effect of either non-NMDA receptor agonists and the effect of D-aspartate. We also observed that incubation of the synaptosomes with D-aspartate induced the Ca(2+)-independent release of glutamate, possibly through the reversal of the glutamate carrier. The aim of incubating the synaptosomes with D-aspartate was to avoid undesirable secondary activation of glutamate receptors. After incubating the synaptosomes with 100 microM D-aspartate (10 min at 37 degrees C), the subsequent stimulation with D-aspartate increased the [Ca2+]i due to glutamate transport. This increase in [Ca2+]i induced by 100 microM D-aspartate was insensitive to 1 microM nitrendipine, but was inhibited by about 50% by the presence of both 500 nM omega-CgTx GVIA and 100 nM omega-Aga IVA or by 500 nM omega-CgTx MVIIC. We clearly identified two different processes by which glutamate increased the [Ca2+]i in rat hippocampal synaptosomes: activation of non-NMDA receptors and activation of the glutamate transporters. We also characterized the voltage sensitive Ca2+ channels (VSCC) activated as a consequence of the glutamate transport, and determined that class B (N-type) and class A (P or Q-type) Ca2+ channels were responsible for about 50% of the signal.

Modulation of glutamate release from rat hippocampal synaptosomes by nitric oxide.

We used hippocampal synaptosomes to study the effect of NO originating from NO donors and from the activation of the NO synthase on the Ca2+-dependent release of glutamate due to 4-aminopyridine (4-AP) depolarization. We distinguished between the effects of NO on the exocytotic and on the carrier-mediated release of glutamate, which we found to be related to an increase in cGMP content and to a reduction of the ATP/ADP ratio, respectively. The NO donor hydroxylamine, at concentrations < or = 0.3 mM, inhibited the Ca2+-dependent glutamate release evoked by 4-AP, and addition of the NO donor, NOC-7, had a similar effect, which was reversed by the NO scavenger, carboxy-PTIO. Increasing the activity of NO synthase by addition of L-arginine also led to a decrease in the Ca2+-dependent release of glutamate induced by 4-AP, and this effect was reversed by inhibiting NO synthase with NG-nitro-L-arginine. This depression of the exocytotic release of glutamate was accompanied by an increase in cGMP levels due to the stimulation of soluble guanylyl cyclase by NO, produced either by the NO donors (hydroxylamine <0.3 mM) or by the endogenous NO synthase, but no significant decrease in ATP/ADP ratio was observed. However, at concentrations > or = 0.3 mM, hydroxylamine drastically increased the basal release and completely inhibited the Ca2+-dependent release of glutamate (IC50 = 168 microM). At these higher levels of NO, cGMP levels dropped to about 40% of the maximal values obtained at lower concentrations, and the ATP/ADP ratio decreased to about 50% (at 0.3 mM hydroxylamine). The large increase in the basal release could be partially inhibited by L-trans-2,4-PDC, previously loaded into the synaptosomes, suggesting that the nonexocytotic basal release occurred by reversal of the glutamate carrier. Therefore, the increase in cGMP induced by NO stimulation of the guanylyl cyclase decreases the exocytotic release of glutamate, but higher NO levels reduce the ATP/ADP ratio by inhibiting mitochondrial function, which therefore causes the massive release of cytosolic glutamate through the glutamate carrier.

Inhibition of N-,P/Q- and other types of Ca2+ channels in rat hippocampal nerve terminals by the adenosine A1 receptor.

The effects of the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), on both the increase in intracellular free Ca2+ concentration ([Ca2+]i) and on the release of endogenous glutamate in rat hippocampal synaptosomes were studied. The inhibitory effect of CPA on the increase in [Ca2+]i stimulated with 4-aminopyridine was neutralized by the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The inhibitory effect of CPA was greater in synaptosomes from the CA1 subregion than in whole hippocampal synaptosomes. The inhibitory effects of both CPA and of the Ca2+ channel blockers, omega-conotoxin GVIA, omega-conotoxin MVIIC or omega-conotoxin GVIA plus omega-conotoxin MVIIC, were greater than those caused by the Ca2+ channel blockers. The release of endogenous glutamate was inhibited by 41% by CPA. The inhibition observed when CPA and omega-conotoxin GVIA or CPA and omega-conotoxin MVIIC were present was also greater than the inhibition by the Ca2+ channel blockers alone. The presence of both omega-conotoxin GVIA and omega-conotoxin MVIIC did not completely inhibit the release of glutamate, and CPA significantly enhanced this inhibition. The membrane potential and the accumulation of [3H]tetraphenylphosphonium of polarized or depolarized synaptosomes was not affected by CPA, suggesting that adenosine did not increase potassium conductances. The present results suggest that, in hippocampal glutamatergic nerve terminals, adenosine A1 receptor activation partly inhibits P/Q- and other non-identified types of Ca2+ channels.

Modulation of Ca2+ channels by activation of adenosine A1 receptors in rat striatal glutamatergic nerve terminals.

We determined that activation of adenosine A1 receptors in striatal synaptosomes with 100 nM N6-cyclopentyladenosine (CPA) inhibited both the release of endogenous glutamate and the increase of intracellular free Ca2+ concentration ([Ca2+]i), due to 4-aminopyridine (4-AP) stimulation, by 28 and 19%, respectively. Furthermore, CPA enhanced the inhibition of endogenous glutamate release due to omega-conotoxin GVIA (omega-Cgtx GVIA), omega-Cgtx MVIIC or omega-Cgtx GVIA plus omega-Cgtx MVIIC. Similar effects were observed in the [Ca2+]i signal. The inhibitory effects of CPA and omega-Cgtx GVIA were additive, but the effects of CPA and omega-Cgtx MVIIC were only partially additive. These results suggest that P/Q-type Ca2+ channels and other type(s) of Ca2+ channel(s), coupled to glutamate release, are inhibited subsequently to activation of adenosine A1 receptors.

Involvement of class A calcium channels in the KCl induced Ca2+ influx in hippocampal synaptosomes.

Using Ca2+ channel toxins, we determined the types of voltage-sensitive calcium channels activated by two levels of KCl depolarization in hippocampal synaptosomes. The increase in the intracellular Ca2+ concentration ([Ca2+]i) induced by 30 mM KCl was equally sensitive to either omega-agatoxin IVA (omega-Aga IVA) or to omega-conotoxin MVIIC (omega-CgTx MVIIC), and the inhibition produced by these two peptides was not additive. The present results indicate that omega-Aga IVA and omega-CgTx MVIIC do not distinguish between two different VSCC in hippocampal synaptosomes and that they both inhibit a channel with the alpha 1A subunit which is present in the rat hippocampus.