RESUMO
Glaucoma, a major ocular neuropathy originating from a progressive degeneration of retinal ganglion cells, is often associated with increased intraocular pressure (IOP). Daily IOP fluctuations are physiologically influenced by the antioxidant and signaling activities of melatonin. This endogenous modulator has limited employment in treating altered IOP disorders due to its low stability and bioavailability. The search for low-toxic compounds as potential melatonin agonists with higher stability and bioavailability than melatonin itself could start only from knowing the molecular basis of melatonergic activity. Thus, using a computational approach, we studied the melatonin binding toward its natural macromolecular targets, namely melatonin receptors 1 (MT1) and 2 (MT2), both involved in IOP signaling regulation. Besides, agomelatine, a melatonin-derivative agonist and, at the same time, an atypical antidepressant, was also included in the study due to its powerful IOP-lowering effects. For both ligands, we evaluated both stability and ligand positioning inside the orthosteric site of MTs, mapping the main molecular interactions responsible for receptor activation. Affinity values in terms of free binding energy (ΔGbind) were calculated for the selected poses of the chosen compounds after stabilization through a dynamic molecular docking protocol. The results were compared with experimental in vivo effects, showing a higher potency and more durable effect for agomelatine with respect to melatonin, which could be ascribed both to its higher affinity for hMT2 and to its additional activity as an antagonist for the serotonin receptor 5-HT2c, in agreement with the in silico results.
Assuntos
Melatonina , Receptor MT1 de Melatonina , Receptores de Melatonina , Simulação de Acoplamento Molecular , Receptor MT1 de Melatonina/metabolismo , Melatonina/metabolismo , Ligantes , Receptor MT2 de Melatonina/metabolismoRESUMO
In retinopathy of prematurity (ROP), the abnormal retinal neovascularization is often accompanied by retinal neuronal dysfunction. Here, a rat model of oxygen-induced retinopathy (OIR), which mimics the ROP disease, was used to investigate changes in the expression of key mediators of autophagy and markers of cell death in the rat retina. In addition, rats were treated from birth to postnatal day 14 and 18 with 3-methyladenine (3-MA), an inhibitor of autophagy. Immunoblot and immunofluorescence analysis demonstrated that autophagic mechanisms are dysregulated in the retina of OIR rats and indicated a possible correlation between autophagy and necroptosis, but not apoptosis. We found that 3-MA acts predominantly by reducing autophagic and necroptotic markers in the OIR retinas, having no effects on apoptotic markers. However, 3-MA does not ameliorate retinal function, which results compromised in this model. Taken together, these results revealed the crucial role of autophagy in retinal cells of OIR rats. Thus, inhibiting autophagy may be viewed as a putative strategy to counteract ROP.
Assuntos
Autofagia , Oxigênio/efeitos adversos , Retina/metabolismo , Doenças Retinianas/etiologia , Doenças Retinianas/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Animais , Biomarcadores , Suscetibilidade a Doenças , Humanos , Recém-Nascido , Ratos , Retina/patologia , Doenças Retinianas/patologia , Transdução de SinaisRESUMO
Purpose: In response to hypoxia, sympathetic fibers to the retina activate ß-adrenoceptors (ß-ARs) that play an important role in the regulation of vascular and neuronal functions. We investigated the role of ß3-AR using the mouse model of oxygen-induced retinopathy (OIR). Methods: Mouse pups were exposed to 75% oxygen at postnatal day 7 (PD7) followed by a return to room air at PD12. The ß3-AR preferential agonist BRL37344 was subcutaneously administered once daily at different times after the return to room air. At PD17, the OIR mice underwent flash and pattern electroretinogram. After sacrifice, retinal wholemounts were used for vessel staining or immunohistochemistry for astrocytes, Müller cells, or retinal ganglion cells (RGCs). In retinal homogenates, the levels of markers associated with neovascularization (NV), the blood-retinal barrier (BRB), or astrocytes were determined by western blot, and quantitative reverse-transcription polymerase chain reaction was used to assess ß3-AR messenger. Administration of the ß3-AR antagonist SR59230A was performed to verify BRL37344 selectivity. Results: ß3-AR expression is upregulated in response to hypoxia, but its increase is prevented by BRL37344, which counteracts NV by inhibiting the pro-angiogenic pathway, activating the anti-angiogenic pathway, recovering BRB-associated markers, triggering nitric oxide production, and favoring revascularization of the central retina through recovered density of astrocytes that ultimately counteracts NV in the midperiphery. Vasculature rescue prevents dysfunctional retinal activity and counteracts OIR-associated retinal ganglion cell loss. Conclusions: ß3-AR has emerged as a crucial intermediary in hypoxia-dependent NV, suggesting a role of ß3-AR agonists in the treatment of proliferative retinopathies.
Assuntos
Agonistas de Receptores Adrenérgicos beta 3 , Modelos Animais de Doenças , Eletrorretinografia , Camundongos Endogâmicos C57BL , Oxigênio , Receptores Adrenérgicos beta 3 , Neovascularização Retiniana , Animais , Camundongos , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/prevenção & controle , Neovascularização Retiniana/patologia , Oxigênio/toxicidade , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Receptores Adrenérgicos beta 3/metabolismo , Animais Recém-Nascidos , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Etanolaminas/farmacologia , Vasos Retinianos/efeitos dos fármacos , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/tratamento farmacológico , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Imuno-Histoquímica , AngiogêneseRESUMO
Maintenance of NAD pools is critical for neuronal survival. The capacity to maintain NAD pools declines in neurodegenerative disease. We identify that low NMNAT2, the critical neuronal NAD producing enzyme, drives retinal susceptibility to neurodegenerative insults. As proof of concept, gene therapy over-expressing full length human NMNAT2 is neuroprotective. To pharmacologically target NMNAT2, we identify that epigallocatechin gallate (EGCG) can drive NAD production in neurons through an NMNAT2 and NMN dependent mechanism. We confirm this by pharmacological and genetic inhibition of the NAD-salvage pathway. EGCG is neuroprotective in rodent (mixed sex) and human models of retinal neurodegeneration. As EGCG has poor drug-like qualities, we use it as a tool compound to generate novel small molecules which drive neuronal NAD production and provide neuroprotection. This class of NMNAT2 targeted small molecules could have an important therapeutic impact for neurodegenerative disease following further drug development.
Assuntos
Catequina , NAD , Neurônios , Fármacos Neuroprotetores , Nicotinamida-Nucleotídeo Adenililtransferase , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/genética , NAD/metabolismo , Humanos , Animais , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Catequina/análogos & derivados , Catequina/farmacologia , Fármacos Neuroprotetores/farmacologia , Masculino , Camundongos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/genética , Feminino , Retina/metabolismo , Retina/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Ratos , Modelos Animais de Doenças , Terapia Genética/métodosRESUMO
Nutraceuticals are natural substances whose anti-oxidant and anti-inflammatory properties may be used to treat retinal pathologies. Their efficacy is limited by poor bioavailability, which could be improved using nanocarriers. Lisosan G (LG), a fermented powder from whole grains, protects the retina from diabetic retinopathy (DR)-induced damage. For this study, we tested whether the encapsulation of LG in liposomes (LipoLG) may increase its protective effects. Diabetes was induced in mice via streptozotocin administration, and the mice were allowed to freely drink water or a water dispersion of two different doses of LG or of LipoLG. Electroretinographic recordings after 6 weeks showed that only the highest dose of LG could partially protect the retina from diabetes-induced functional deficits, while both doses of LipoLG were effective. An evaluation of molecular markers of oxidative stress, inflammation, apoptosis, vascular endothelial growth factor, and the blood-retinal barrier confirmed that the highest dose of LG only partially protected the retina from DR-induced changes, while virtually complete prevention was obtained with either dose of LipoLG. These data indicate that the efficacy of LG in contrasting DR is greatly enhanced by its encapsulation in liposomes and may lay the ground for new dietary supplements with improved therapeutic effects against DR.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Camundongos , Animais , Lipossomos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Retinopatia Diabética/metabolismo , ÁguaRESUMO
Retinal ganglion cells are highly metabolically active requiring strictly regulated metabolism and functional mitochondria to keep ATP levels in physiological range. Imbalances in metabolism and mitochondrial mechanisms can be sufficient to induce a depletion of ATP, thus altering retinal ganglion cell viability and increasing cell susceptibility to death under stress. Altered metabolism and mitochondrial abnormalities have been demonstrated early in many optic neuropathies, including glaucoma, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy. Pyrroloquinoline quinone (PQQ) is a quinone cofactor and is reported to have numerous effects on cellular and mitochondrial metabolism. However, the reported effects are highly context-dependent, indicating the need to study the mechanism of PQQ in specific systems. We investigated whether PQQ had a neuroprotective effect under different retinal ganglion cell stresses and assessed the effect of PQQ on metabolic and mitochondrial processes in cortical neuron and retinal ganglion cell specific contexts. We demonstrated that PQQ is neuroprotective in two models of retinal ganglion cell degeneration. We identified an increased ATP content in healthy retinal ganglion cell-related contexts both in in vitro and in vivo models. Although PQQ administration resulted in a moderate effect on mitochondrial biogenesis and content, a metabolic variation in non-diseased retinal ganglion cell-related tissues was identified after PQQ treatment. These results suggest the potential of PQQ as a novel neuroprotectant against retinal ganglion cell death.
Assuntos
Neuroproteção , Fármacos Neuroprotetores , Células Ganglionares da Retina , Cofator PQQ/farmacologia , Fármacos Neuroprotetores/farmacologia , Trifosfato de AdenosinaRESUMO
Purpose: Glaucoma is an eye-brain axis disorder characterized by loss of retinal ganglion cells (RGCs). Although the role of intraocular pressure (IOP) elevation in glaucoma has been established, the reduction of oxidative stress and inflammation has emerged as a promising target for neuronal tissue-supporting glaucoma management. Therefore, we evaluated the effect of a proprietary spearmint extract (SPE) on RGC density, activity, and neuronal health markers in a rat model of hypertensive glaucoma. Methods: Animals were divided in four groups: untreated healthy control and three glaucomatous groups receiving orally administered vehicle, SPE-low dose, or SPE-high dose for 28 days. Ocular hypertension was induced through intracameral injection of methylcellulose at day 15. At day 29, rats underwent electroretinogram (ERG) recordings, and retinas were analyzed for RGC density and markers of neural trophism, oxidative stress, and inflammation. Results: SPE exerted dose-dependent response benefits on all markers except for IOP elevation. SPE significantly improved RGC-related ERG responses, cell density, neurotrophins, oxidative stress, and inflammation markers. Also, in SPE-high rats, most of the parameters were not statistically different from those of healthy controls. Conclusions: SPE, a plant-based, polyphenolic extract, could be an effective nutritional support for neuronal tissues. Translational Relevance: These results suggest that SPE not only may be a complementary approach in support to hypotensive treatments for the management of glaucoma but may also serve as nutritional support in other ocular conditions where antioxidant, anti-inflammatory, and neuroprotective mechanism are often disrupted.
Assuntos
Glaucoma , Mentha spicata , Hipertensão Ocular , Animais , Ratos , Glaucoma/tratamento farmacológico , Hipertensão Ocular/tratamento farmacológico , Retina , Inflamação/tratamento farmacológicoRESUMO
In diabetic retinopathy (DR), high blood glucose drives chronic oxidative stress and inflammation that trigger alterations of the neurovascular balance finally resulting in vascular abnormalities and retinal cell death, which converge towards altered electroretinogram (ERG). In the last years, a growing body of preclinical evidence has suggested that nutrients with anti-inflammatory/antioxidant properties can be able to hamper DR progression since its very early stages. In the present study, we used a streptozotocin-induced rat model of DR, which mimics most aspects of the early stages of human DR, to test the preventive efficacy of a novel compound containing cyanidin-3-glucoside (C3G), verbascoside and zinc as nutrients with antioxidant and anti-inflammatory properties. Western blot, immunofluorescence and electroretinographic analyses demonstrated a dose-dependent inhibition of oxidative stress- and inflammation-related mechanisms, with a significant counterpart in preventing molecular mechanisms leading to DR-associated vasculopathy and its related retinal damage. Preventive efficacy of the compound on dysfunctional a- and b-waves was also demonstrated by electroretinography. The present demonstration that natural compounds, possibly as a consequence of vascular rescue following ameliorated oxidative stress and inflammation, may prevent the apoptotic cascade leading to ERG dysfunction, adds further relevance to the potential application of antioxidants as a preventive therapy to counteract DR progression.
RESUMO
Light-induced retinal damage (LD) is characterized by the accumulation of reactive oxygen species leading to oxidative stress and photoreceptor cell death. The use of natural antioxidants has emerged as promising approach for the prevention of LD. Among them, lutein and cyanidin-3-glucoside (C3G) have been shown to be particularly effective due to their antioxidant and anti-inflammatory activity. However, less is known about the possible efficacy of combining them in a multicomponent mixture. In a rat model of LD, Western blot analysis, immunohistochemistry and electroretinography were used to demonstrate that lutein and C3G in combination or in a multicomponent mixture can prevent oxidative stress, inflammation, gliotic and apoptotic responses thus protecting photoreceptor cells from death with higher efficacy than each component alone. Combined efficacy on dysfunctional electroretinogram was also demonstrated by ameliorated rod and cone photoreceptor responses. These findings suggest the rationale to formulate multicomponent blends which may optimize the partnering compounds bioactivity and bioavailability.
RESUMO
During retinal development, a physiologic hypoxia stimulates endothelial cell proliferation. The hypoxic milieu warrants retina vascularization and promotes the activation of several mechanisms aimed to ensure homeostasis and energy balance of both endothelial and retinal cells. Autophagy is an evolutionarily conserved catabolic system that contributes to cellular adaptation to a variety of environmental changes and stresses. In association with the physiologic hypoxia, autophagy plays a crucial role during development. Autophagy expression profile was evaluated in the developing retina from birth to post-natal day 18 of rat pups, using qPCR, western blotting and immunostaining methodologies. The rat post-partum developing retina displayed increased active autophagy during the first postnatal days, correlating to the hypoxic phase. In latter stages of development, rat retinal autophagy decreases, reaching a normalization between post-natal days 14-18, when the retina is fully vascularized and mature. Collectively, the present study elaborates on the link between hypoxia and autophagy, and contributes to further elucidate the role of autophagy during retinal development.
Assuntos
Autofagia , Regulação da Expressão Gênica , Retina/crescimento & desenvolvimento , Animais , Ratos , Ratos WistarRESUMO
Pyrroloquinoline quinone is a quinone described as a cofactor for many bacterial dehydrogenases and is reported to exert an effect on metabolism in mammalian cells/tissues. Pyrroloquinoline quinone is present in the diet being available in foodstuffs, conferring the potential of this compound to be supplemented by dietary administration. Pyrroloquinoline quinone's nutritional role in mammalian health is supported by the extensive deficits in reproduction, growth, and immunity resulting from the dietary absence of pyrroloquinoline quinone, and as such, pyrroloquinoline quinone has been considered as a "new vitamin." Although the classification of pyrroloquinoline quinone as a vitamin needs to be properly established, the wide range of benefits for health provided has been reported in many studies. In this respect, pyrroloquinoline quinone seems to be particularly involved in regulating cell signaling pathways that promote metabolic and mitochondrial processes in many experimental contexts, thus dictating the rationale to consider pyrroloquinoline quinone as a vital compound for mammalian life. Through the regulation of different metabolic mechanisms, pyrroloquinoline quinone may improve clinical deficits where dysfunctional metabolism and mitochondrial activity contribute to induce cell damage and death. Pyrroloquinoline quinone has been demonstrated to have neuroprotective properties in different experimental models of neurodegeneration, although the link between pyrroloquinoline quinone-promoted metabolism and improved neuronal viability in some of such contexts is still to be fully elucidated. Here, we review the general properties of pyrroloquinoline quinone and its capacity to modulate metabolic and mitochondrial mechanisms in physiological contexts. In addition, we analyze the neuroprotective properties of pyrroloquinoline quinone in different neurodegenerative conditions and consider future perspectives for pyrroloquinoline quinone's potential in health and disease.