Anti-inflammatory reprogramming of microglia cells by metabolic modulators to counteract neurodegeneration; a new role for Ranolazine.

Microglia chronic activation is a hallmark of several neurodegenerative diseases, including the retinal ones, possibly contributing to their etiopathogenesis. However, some microglia sub-populations have anti-inflammatory and neuroprotective functions, thus making arduous deciphering the role of these cells in neurodegeneration. Since it has been proposed that functionally different microglia subsets also rely on different metabolic routes, we hypothesized that modulating microglia metabolism might be a tool to enhance their anti-inflammatory features. This would have a preventive and therapeutic potential in counteracting neurodegenerative diseases. For this purpose, we tested various molecules known to act on cell metabolism, and we revealed the anti-inflammatory effect of the FDA-approved piperazine derivative Ranolazine on microglia cells, while confirming the one of the flavonoids Quercetin and Naringenin, both in vitro and in vivo. We also demonstrated the synergistic anti-inflammatory effect of Quercetin and Idebenone, and the ability of Ranolazine, Quercetin and Naringenin to counteract the neurotoxic effect of LPS-activated microglia on 661W neuronal cells. Overall, these data suggest that using the selected molecules -also in combination therapies- might represent a valuable approach to reduce inflammation and neurodegeneration while avoiding long term side effects of corticosteroids.

A Window to the Brain: The Retina to Monitor the Progression and Efficacy of Saffron Repron® Pre-Treatment in an LPS Model of Neuroinflammation and Memory Impairment.

A mechanism shared by most neurodegenerative diseases, like Alzheimer's disease (AD) and Parkinson's disease (PD), is neuroinflammation. It has been shown to have a link between cognitive impairment and retinal function under neuroinflammatory conditions, confirming the essential role of the retina as a window to the brain. Here, we characterize a mouse model of LPS-induced neuroinflammation describing the parallel deterioration of both memory and visual function. Then, we demonstrate, using the Novel Object Recognition test (NOR) and electroretinogram (ERG) recordings, that preventive, chronic treatment with saffron Repron® is able to reduce the neuroinflammation process and prevent the impairment of both cognitive and visual function. The improvement in behavioral and visual function is confirmed by the pattern of expression of neuroinflammation-related genes and related proteins where pre-treatment with Repron® saffron presents a positive modulation compared with that obtained in animals treated with LPS alone. These results hold for retinal tissue and partially in the brain, where it appears that the onset of damage was delayed. This trend underlines the critical role of the retina as a most sensitive portion of the central nervous system to LPS-induced damage and could be used as a "sensor" for the early detection of neurodegenerative diseases such as Alzheimer's.

Efficacy of Hydroponically Cultivated Saffron in the Preservation of Retinal Pigment Epithelium.

Saffron treatment is a broad-spectrum therapy used for several retinal diseases, and its effectiveness depends on a particular molecular composition (REPRON® saffron). Its production requires specific crops and procedures that, together with low yields, make this spice expensive. To reduce costs, the use of hydroponic crops is gradually increasing. In this study, we tested the protective properties of a hydroponic saffron (sH) batch in models of retinal pigmented epithelium (RPE) degeneration. ARPE-19 cells were pretreated with 40 µg/mL saffron and exposed to different types of damage: excess light and retinol (LE + RET) or oxidative stress (H2O2). After analyzing the composition of all saffron types with spectroscopy, we performed cell viability and immunofluorescence analysis for both protocols. We compared the sH results with those of a validated batch of saffron REPRON® (sR) and those of a saffron non-REPRON® (sNR) batch. sH and sR, which we found had the same chemical composition, were more effective than sNR in increasing cell survival and attenuating the morphological changes related to apoptosis. In conclusion, hydroponic culturing is a suitable strategy to produce high-quality saffron to reduce costs and increase the accessibility of this promising treatment for retinal degeneration.

Targeting TSPO Reduces Inflammation and Apoptosis in an In Vitro Photoreceptor-Like Model of Retinal Degeneration.

The 18 kDa translocator protein (TSPO) is predominantly located in the mitochondrial outer membrane, playing an important role in steroidogenesis, inflammation, survival, and cell proliferation. Its expression in the CNS, and mainly in glial cells, is upregulated in neuropathologies and brain injury. In this study, the potential of targeting TSPO for the therapeutic treatment of inflammatory-based retinal neurodegeneration was evaluated by means of an in vitro model of lipopolysaccharide (LPS)-induced degeneration in 661 W cells, a photoreceptor-like cell line. After the assessment of the expression of TSPO in 661W cells, which, to the best of our knowledge, was never investigated so far, the anti-inflammatory and cytoprotective effects of a number of known TSPO ligands, belonging to the class of N,N-dialkyl-2-arylindol-3-ylglyoxylamides (PIGAs), were evaluated, using the classic TSPO ligand PK11195 as the reference standard. All tested PIGAs showed the ability to modulate the inflammatory and apoptotic processes in 661 W photoreceptor-like cells and to reduce LPS-driven cellular cytotoxicity. The protective effect of PIGAs was, in all cases, reduced by cotreatment with the pregnenolone synthesis inhibitor SU-10603, suggesting the involvement of neurosteroids in the protective mechanism. As inflammatory processes play a crucial role in the retinal neurodegenerative disease progression toward photoreceptors' death and complete blindness, targeting TSPO might represent a successful strategy to slow down this degenerative process that may lead to the inexorable loss of vision.

Medicinal Hypervalent Tellurium Prodrugs Bearing Different Ligands: A Comparative Study of the Chemical Profiles of AS101 and Its Halido Replaced Analogues.

Ammonium trichloro (dioxoethylene-O,O') tellurate (AS101) is a potent immunomodulator prodrug that, in recent years, entered various clinical trials and was tested for a variety of potential therapeutic applications. It has been demonstrated that AS101 quickly activates in aqueous milieu, producing TeOCl3-, which likely represents the pharmacologically active species. Here we report on the study of the activation process of AS101 and of two its analogues. After the synthesis and characterization of AS101 and its derivatives, we have carried out a comparative study through a combined experimental and computational analysis. Based on the obtained results, we describe here, for the first time, the detailed reaction that AS101 and its bromido- and iodido-replaced analogues undergo in presence of water, allowing the conversion of the original molecule to the likely true pharmacophore. Interestingly, moving down in the halogens' group we observed a higher tendency to react, attributable to the ligands' effect. The chemical and mechanistic implications of these meaningful differences are discussed.

Design, Synthesis, and In Vitro Evaluation of Novel 8-Amino-Quinoline Combined with Natural Antioxidant Acids.

Overproduction of reactive oxygen species (ROS) and alterations in metallostasis are common and related hallmarks in several neurodegenerative diseases (NDDs). Nature-based derivatives always represent an attractive tool in MTDL drug design, especially against ROS in NDDs. On this notion, we designed a new series of 8-quinoline-N-substituted derivatives with a natural antioxidant portion (i.e., lipoic, caffeic, and ferulic acids). These compounds were shown to chelate copper, a metal involved in ROS-induced degeneration, and scavenger oxygen radicals in DPPH assay. Then, selected compounds 4 and 5 were evaluated in an in vitro model of oxidative stress and shown to possess cytoprotective effects in 661W photoreceptor-like cells. The obtained results may represent a starting point for the application of the proposed class of compounds in retinal neurodegenerative diseases such as retinitis pigmentosa (RP), comprising a group of hereditary rod-cone dystrophies that represent a major cause of blindness in patients of working age, where the progression of the disease is a multifactorial event, with oxidative stress contributing predominantly.

Nutraceutical Molecules Slow Down Retinal Degeneration, in Tvrm4 Mice a Model of Retinitis Pigmentosa, by Genetic Modulation of Anti-oxidant Pathway.

Rhodopsin (RHO) mutations are responsible for 25-40% of the dominant cases of retinitis pigmentosa (RP) with different severity and progression rates. The Tvrm4 mice, heterozygous for an I307N dominant mutation of RHO, display a normal retinal phenotype when raised in ambient light conditions, but undergo photoreceptor degeneration when briefly exposed to strong white light. Here, The Tvrm4 mice is pre-treated with naringenin 100 mg/kg/die, quercetin 100 mg/kg/die, naringenin 50 + quercercetin 100 mg/kg/die or vehicle dimethyl sulfoxide (DMSO 0.025%) in the drinking water for 35 days. On the 30th day, retinal degeneration was induced by exposure for 1 min to the white light of 12,000 lux intensity, and the treatment was repeated for another 5 days. At the end of the protocol retinal functionality was tested by recording an electroretinogram (ERG). The retinal tissue was collected and was used for further analyses, including immunohistochemically, biochemical, and molecular biology assays. The data obtained show that treatment with nutraceutical molecules is effective in counteracting retinal degeneration by preserving the functionality of photoreceptors and increasing the antioxidant and anti-apoptotic pathways of retinal cells. The present data confirm that nutraceutical molecules are effective in slowing photoreceptor degeneration in a mutation-independent way by modulating the antioxidant response of the retina at the gene expression level.

Retinal Neurodegeneration: Correlation between Nutraceutical Treatment and Animal Model.

Retinal diseases can be induced by a variety of factors, including gene mutations, environmental stresses and dysmetabolic processes. The result is a progressive deterioration of visual function, which sometimes leads to blindness. Many treatments are under investigation, though results are still mostly unsatisfactory and restricted to specific pathologies, particularly in the case of gene therapy. The majority of treatments have been tested in animal models, but very few have progressed to human clinical trials. A relevant approach is to study the relation between the type of treatments and the degenerative characteristics of the animal model to better understand the effectiveness of each therapy. Here we compare the results obtained from different animal models treated with natural compounds (saffron and naringenin) to anticipate the potentiality of a single treatment in different pathologies.

Myriocin Effect on Tvrm4 Retina, an Autosomal Dominant Pattern of Retinitis Pigmentosa.

Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsin gene that can be activated by brief exposure to very intense light. Here, we test the possibility of an anatomical, metabolic, and functional recovery by delivering to degenerating Tvrm4 animals, Myriocin, an inhibitor of ceramide de novo synthesis previously shown to effectively slow down retinal degeneration in rd10 mutants (Strettoi et al., 2010; Piano et al., 2013). Different routes and durations of Myriocin administration were attempted by using either single intravitreal (i.v.) or long-term, repeated intraperitoneal (i.p.) injections. The retinal function of treated and control animals was tested by ERG recordings. Retinas from ERG-recorded animals were studied histologically to reveal the extent of photoreceptor death. A correlation was observed between Myriocin administration, lowering of retinal ceramides, and preservation of ERG responses in i.v. injected cases. Noticeably, the i.p. treatment with Myriocin decreased the extension of the retinal-degenerating area, preserved the ERG response, and correlated with decreased levels of biochemical indicators of retinal oxidative damage. The results obtained in this study confirm the efficacy of Myriocin in slowing down retinal degeneration in genetic models of RP independently of the underlying mutation responsible for the disease, likely targeting ceramide-dependent, downstream pathways. Alleviation of retinal oxidative stress upon Myriocin treatment suggests that this molecule, or yet unidentified metabolites, act on cellular detoxification systems supporting cell survival. Altogether, the pharmacological approach chosen here meets the necessary pre-requisites for translation into human therapy to slow down RP.

Oxy-imino saccharidic derivatives as a new structural class of aldose reductase inhibitors endowed with anti-oxidant activity.

Aldose reductase is a key enzyme in the development of long term diabetic complications and its inhibition represents a viable therapeutic solution for people affected by these pathologies. Therefore, the search for effective aldose reductase inhibitors is a timely and pressing challenge. Herein we describe the access to a novel class of oxyimino derivatives, obtained by reaction of a 1,5-dicarbonyl substrate with O-(arylmethyl)hydroxylamines. The synthesised compounds proved to be active against the target enzyme. The best performing inhibitor, compound (Z)-8, proved also to reduce both cell death and the apoptotic process when tested in an in vitro model of diabetic retinopathy made of photoreceptor-like 661w cell line exposed to high-glucose medium, counteracting oxidative stress triggered by hyperglycaemic conditions.

The Citrus Flavonoid Naringenin Protects the Myocardium from Ageing-Dependent Dysfunction: Potential Role of SIRT1.

Sirtuin 1 (SIRT1) enzyme plays a pivotal role in the regulation of many physiological functions. In particular, it is implicated in ageing-related diseases, such as cardiac hypertrophy, myocardial infarct, and endothelial dysfunction; moreover, its expression decreases with age. Therefore, an effective strategy to extend the lifespan and improve cardiovascular function is the enhancement of the expression/activity of SIRT1 with exogenous agents. The Citrus flavonoid naringenin (NAR) presents structural similarity with the natural SIRT1 activator resveratrol. In this study, we demonstrate through in vitro assays that NAR significantly activates SIRT1 enzyme and shows antisenescence effects. The binding mode of NAR into SIRT1 was detailed investigated through in silico studies. Moreover, chronic administration (for six months) of NAR (100 mg/kg/day) to 6-month-old mice leads to an enhancement of SIRT1 expression and a marked reduction of reactive oxygen species production in myocardial tissue. Furthermore, at the end of the treatment, the plasma levels of two well-known markers of cardiovascular inflammation, TNF-α and IL6, are significantly reduced in 12-month-old mice treated with NAR, as well as the cardiovascular risk (total cholesterol/HDL ratio) compared to control mice. Finally, the age-associated fibrotic remodeling, which is well detected through a Mallory trichrome staining in the vehicle-treated 12-month-old mice, is significantly reduced by the chronic treatment with NAR. Moreover, an improvement of myocardium functionality is highlighted by the enhancement of citrate synthase activity and stabilization of the mitochondrial membrane potential after NAR treatment. Taken together, these results suggest that a nutraceutical approach with NAR may have positive impacts on many critical hallmarks of myocardial senescence, contributing to improve the cardiac performance in aged subjects.

Retinal Phenotype in the rd9 Mutant Mouse, a Model of X-Linked RP.

Retinal degeneration 9 (rd9) mice carry a mutation in the retina specific "Retinitis Pigmentosa GTPase Regulator (RPGR)" Open Reading Frame (ORF) 15 gene, located on the X chromosome and represent a rare model of X-linked Retinitis Pigmentosa (XLRP), a common and severe form of retinal degeneration (Wright et al., 2010; Tsang and Sharma, 2018). The rd9 RPGR-ORF15 mutation in mice causes lack of the protein in photoreceptors and a slow degeneration of these cells with consequent decrease in Outer Nuclear Layer (ONL) thickness and amplitude of ERG responses, as previously described (Thompson et al., 2012). However, relative rates of rod and cone photoreceptor loss, as well as secondary alterations occurring in neuronal and non-neuronal retinal cell types of rd9 mutants remain to be assessed. Aim of this study is to extend phenotype analysis of the rd9 mouse retina focusing on changes occurring in cells directly interacting with photoreceptors. To this purpose, first we estimated rod and cone survival and its degree of intraretinal variation over time; then, we studied the morphology of horizontal and bipolar cells and of the retinal pigment epithelium (RPE), extending our observations to glial cell reactivity. We found that in rd9 retinas rod (but not cone) death is the main cause of decrease in ONL thickness and that degeneration shows a high degree of intraretinal variation. Rod loss drives remodeling in the outer retina, with sprouting of second-order neurons of the rod-pathway and relative sparing of cone pathway elements. Remarkably, despite cone survival, functional defects can be clearly detected in ERG recordings in both scotopic and photopic conditions. Moderate levels of Muller cells and microglial reactivity are sided by striking attenuation of staining for RPE tight junctions, suggesting altered integrity of the outer Blood Retina Barrier (BRB). Because of many features resembling slowly progressing photoreceptor degeneration paradigms or early stages of more aggressive forms of RP, the rd9 mouse model can be considered a rare and useful tool to investigate retinal changes associated to a process of photoreceptor death sustained throughout life and to reveal disease biomarkers (e.g., BRB alterations) of human XLRP.

Synthesis and investigation of polyhydroxylated pyrrolidine derivatives as novel chemotypes showing dual activity as glucosidase and aldose reductase inhibitors.

Diabetes is a multi-factorial disorder that should be treated with multi-effective compounds. Here we describe the access to polyhydroxylated pyrrolidines, belonging to the d-gluco and d-galacto series, through aminocyclization reactions of two differentially protected d-xylo-hexos-4-ulose derivatives. The prepared compounds proved to inhibit both alpha-glucosidase, responsible for the emergence of hyperglycemic spikes, and aldose reductase, accountable for the development of abnormalities in diabetic tissues. Accordingly, they show the dual inhibitory profile deemed as ideal for diabetes treatment. Significantly, compound 17b reduced the process of cell death and restored the physiological levels of oxidative stress when tested in the photoreceptor-like 661w cell line, thus proving to be effective in an in vitro model of diabetic retinopathy.

A Nutraceutical Strategy to Slowing Down the Progression of Cone Death in an Animal Model of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by progressive degeneration of the visual cells and abnormalities in retinal pigment epithelium, the vision is lost slowly, and the final outcome is total blindness. RP primarily affects rods, but cones can also be affected as a secondary effect. Photoreceptor cell death is usually triggered by apoptosis, however the molecular mechanisms linking the rod degeneration to the secondary cone death are poorly understood. Possible causes of the secondary cone death are oxidative stress and/ or the release of toxic factors from dying rods. The aim of this study is to analyze the effect of nutraceutical molecules with antioxidant properties, on the progression of the disease in an established animal model of RP, and rd10 mice. We show that chronic treatment per os with a flavanone (naringenin) or a flavonol (quercetin) present in citrus fruits, grapes and apples, preserves retinal morphology, and ameliorates functionality. These actions are associated with a significant reduction of stress-oxidative markers, such as the detoxifying enzymes Sod1 and Sod2. In addition, naringenin and quercetin treatment reduces the levels of acrolein staining associated with a reduction of ROS in the cellular environment. The study demonstrates the beneficial effects of naringenin and quercetin, two molecules that possess antioxidant properties, limiting neurodegeneration, and thus preventing cone damage.

Retinal defects in mice lacking the autism-associated gene Engrailed-2.

Defective cortical processing of visual stimuli and altered retinal function have been described in autism spectrum disorder (ASD) patients. In keeping with these findings, anatomical and functional defects have been found in the visual cortex and retina of mice bearing mutations for ASD-associated genes. Here we sought to investigate the anatomy and function of the adult retina of Engrailed 2 knockout (En2-/-) mice, a model for ASD. Our results showed that En2 is expressed in all three nuclear layers of the adult retina. When compared to age-matched En2+/+ controls, En2-/- adult retinas showed a significant decrease in the number of calbindin+ horizontal cells, and a significant increase in calbindin+ amacrine/ganglion cells. The total number of ganglion cells was not altered in the adult En2-/- retina, as shown by Brn3a+ cell counts. In addition, En2-/- adult mice showed a significant reduction of photoreceptor (rhodopsin) and bipolar cell (Pcp2, PKCα) markers. Functional defects were also present in the retina of En2 mutants, as indicated by electroretinogram recordings showing a significant reduction in both a-wave and b-wave amplitude in En2-/- mice as compared to controls. These data show for the first time that anatomical and functional defects are present in the retina of the En2 ASD mouse model.

Removal of clock gene Bmal1 from the retina affects retinal development and accelerates cone photoreceptor degeneration during aging.

The mammalian retina contains an autonomous circadian clock system that controls many physiological functions within this tissue. Previous studies on young mice have reported that removal of the key circadian clock gene Bmal1 from the retina affects the circadian regulation of visual function, but does not affect photoreceptor viability. Because dysfunction in the circadian system is known to affect cell viability during aging in other systems, we compared the effect of Bmal1 removal from the retina on visual function, inner retinal structure, and photoreceptor viability in young (1 to 3 months) and aged (24 to 26 months) mice. We found that removal of Bmal1 from the retina significantly affects visual information processing in both rod and cone pathways, reduces the thickness of inner retinal nuclear and plexiform layers, accelerates the decline of visual functions during aging, and reduces the viability of cone photoreceptors. Our results thus suggest that circadian clock dysfunction, caused by genetic or other means, may contribute to the decline of visual function during development and aging.

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes.

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N1 (1,2) or N10 (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A1 and A2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A1 and A2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs. Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A1 AR and A2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A1 and A2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.

Heteromeric MT1/MT2 melatonin receptors modulate the scotopic electroretinogram via PKCζ in mice.

Melatonin plays an important role in the regulation of retinal functions, and previous studies have also reported that the action of melatonin on photoreceptors is mediated by melatonin receptor heterodimers. Furthermore, it has been reported that the melatonin-induced increase in the amplitude of the a- and b-wave is significantly blunted by inhibition of PKC. Previous work has also shown that PKCζ is present in the photoreceptors, thus suggesting that PCKζ may be implicated in the modulation of melatonin signaling in photoreceptors. To investigate the role PKCζ plays in the modulation of the melatonin effect on the scotopic ERG, mice were injected with melatonin and with specific inhibitors of different PKC isoforms. PKCζ knockout mice were also used in this study. PKCζ activation in photoreceptors following melatonin injection was also investigated with immunocytochemistry. Inhibition of PKCζ by PKCζ-pseudosubstrate inhibitor (20 µM) significantly reduced the melatonin-induced increase in the amplitude of the a- and b-wave. To further investigate the role of different PKCs in the modulation of the ERGs, we tested whether intra-vitreal injection of Enzastaurin (a potent inhibitor of PCKα, PKCß, PKCγ, and PKCε) has any effect on the melatonin-induced increase in the a- and b-wave of the scotopic ERGs. Enzastaurin (100 nM) did not prevent the melatonin-induced increase in the amplitude of the a-wave, thus suggesting that PCKα, PKCß, PKCγ, and PKCε are not involved in this phenomenon. Finally, our data indicated that, in mice lacking PKCζ, melatonin injection failed to increase the amplitude of the a- and b-waves of the scotopic ERGs. An increase in PKCζ phosphorylation in the photoreceptors was also observed by immunocytochemistry. Our data indicate that melatonin signaling does indeed use the PKCζ pathway to increase the amplitude of the a- and b-wave of the scotopic ERG.

The A2B Adenosine Receptor Modulates the Epithelial- Mesenchymal Transition through the Balance of cAMP/PKA and MAPK/ERK Pathway Activation in Human Epithelial Lung Cells.

The epithelial-mesenchymal transition (EMT) is a complex process in which cell phenotype switches from the epithelial to mesenchymal one. The deregulations of this process have been related with the occurrence of different diseases such as lung cancer and fibrosis. In the last decade, several efforts have been devoted in understanding the mechanisms that trigger and sustain this transition process. Adenosine is a purinergic signaling molecule that has been involved in the onset and progression of chronic lung diseases and cancer through the A2B adenosine receptor subtype activation, too. However, the relationship between A2BAR and EMT has not been investigated, yet. Herein, the A2BAR characterization was carried out in human epithelial lung cells. Moreover, the effects of receptor activation on EMT were investigated in the absence and presence of transforming growth factor-beta (TGF-ß1), which has been known to promote the transition. The A2BAR activation alone decreased and increased the expression of epithelial markers (E-cadherin) and the mesenchymal one (Vimentin, N-cadherin), respectively, nevertheless a complete EMT was not observed. Surprisingly, the receptor activation counteracted the EMT induced by TGF-ß1. Several intracellular pathways regulate the EMT: high levels of cAMP and ERK1/2 phosphorylation has been demonstrated to counteract and promote the transition, respectively. The A2BAR stimulation was able to modulated these two pathways, cAMP/PKA and MAPK/ERK, shifting the fine balance toward activation or inhibition of EMT. In fact, using a selective PKA inhibitor, which blocks the cAMP pathway, the A2BAR-mediated EMT promotion were exacerbated, and conversely the selective inhibition of MAPK/ERK counteracted the receptor-induced transition. These results highlighted the A2BAR as one of the receptors involved in the modulation of EMT process. Nevertheless, its activation is not enough to trigger a complete transition, its ability to affect different intracellular pathways could represent a mechanism at the basis of EMT maintenance/inhibition based on the extracellular microenvironment. Despite further investigations are needed, herein for the first time the A2BAR has been related to the EMT process, and therefore to the different EMT-related pathologies.

Melatonin partially protects 661W cells from H2O2-induced death by inhibiting Fas/FasL-caspase-3.

Purpose: Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MT1 and MT2) in 661W cells, then whether MEL can prevent H2O2-induced cell death, and last, through which pathway MEL confers protection. Methods: The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with H2O2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H2O2-mediated cell death, a Fas/FasL antagonist was used before the exposure to H2O2. Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H2O2 and/or MEL. Cell viability was analyzed by using Trypan Blue. Results: Both MEL receptors (MT1 and MT2) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H2O2-mediated cell death (20-25%). This effect was replicated with IIK7 (a melatonin receptor agonist) when used at a concentration of 1 µM. Preincubation with luzindole (a melatonin receptor antagonist) blocked MEL protection. Kp7-6, an antagonist of Fas/FasL, blocked cell death caused by H2O2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H2O2, and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H2O2. Conclusions: The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H2O2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.