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Background and Purpose: Retinal ganglion cells (RGCs) are the output stage of retinal information processing, via their axons forming the optic nerve (ON). ON damage leads to axonal degeneration and death of RGCs, and results in vision impairment. Nerve growth factor (NGF) signalling is crucial for RGC operations and visual functions. Here, we investigate a new neuroprotective mechanism of a novel therapeutic candidate, a p75‐less, TrkA‐biased NGF agonist (hNGFp) in rat RGC degeneration, in comparison with wild type human NGF (hNGFwt). Experimental Approach: Both neonate and adult rats, whether subjected or not to ON lesion, were treated with intravitreal injections or eye drops containing either hNGFp or hNGFwt. Different doses of the drugs were administered at days 1, 4 or 7 after injury for a maximum of 10 days, when immunofluorescence, electrophysiology, cellular morphology, cytokine array and behaviour studies were carried out. Pharmacokinetic evaluation was performed on rabbits treated with hNGFp ocular drops. Results: hNGFp exerted a potent RGC neuroprotection by acting on microglia cells, and outperformed hNGFwt in rescuing RGC degeneration and reducing inflammatory molecules. Delayed use of hNGFp after ON lesion resulted in better outcomes compared with treatment with hNGFwt. Moreover, hNGFp‐based ocular drops were less algogenic than hNGFwt. Pharmacokinetic measurements revealed that biologically relevant quantities of hNGFp were found in the rabbit retina. Conclusions and Implications: Our data point to microglia as a new cell target through which NGF‐induced TrkA signalling exerts neuroprotection of the RGC, emphasizing hNGFp as a powerful treatment to tackle retinal degeneration. [ABSTRACT FROM AUTHOR]

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. Despite the important role that NAD plays in axon maintenance and degeneration, an understanding of how NMNAT2 expression, a terminal enzyme for NAD production, influences retinal ganglion cell degeneration is lacking. Here the authors demonstrate epigallocatechin gallate (EGCG) drives NAD production in neurons through an NMNAT2 dependent mechanism and provides neuroprotection against retinal ganglion cell injury ex vivo. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Glaucoma, the most common cause of irreversible blindness, is characterized by progressive dysfunction and loss of retinal ganglion cells. There are no neuroprotective therapies. Metabolic dysfunction is an emerging pathophysiological mechanism in glaucoma with widespread changes to energy homeostasis, yet little is known about non‐energy metabolism. We recently identified elevated retinal homocysteine as a strong early metabolomic signature in a rat model of glaucoma. We tested whether local elevation of homocysteine to the retina compounded glaucoma in the rat model by artificially rising homocysteine through intravitreal injection. Short‐term elevation of homocysteine (from 1 to 15 μM, as has been detected in the vitreous of diabetic retinopathy patients) either alone or in addition to glaucoma does not worsen retinal ganglion cell death, suggesting that local elevation of homocysteine does not contribute to retinal ganglion cell degeneration, but may be an indicator of wider metabolic dysfunction. Homocysteine is central to one‐carbon metabolism, an anabolic pathway with an essential role in generation of methionine and methyl donors (necessary for the epigenetic regulation of gene expression), and glutathione (antioxidant). In a mouse model of glaucoma, we identified early‐ and sustained‐dysregulation of genes involved in one‐carbon metabolism in whole retina. In retinal ganglion cells, a number of these genes change prior to detectable neurodegeneration. These pathways require vitamin B6 and B12 as essential cofactors, and folic acid (B9) and choline are essential precursors, for the metabolism of homocysteine. Genes involved in the transport and utilization of these cofactors and precursors were also significantly dysregulated in the retina, further supporting dysfunctional homocysteine metabolism. Dietary deficiency in these leads to homocysteine accumulation and are well associated with optic neuropathies. We hypothesize that local retinal deficiency in these may contribute to dysfunction in one‐carbon metabolism and impact wider retinal ganglion cell homeostasis. To test this hypothesis, we first supplemented the drinking water of mice with B6 (4.5 mg/kg), folic acid (B9) (1.5 mg/kg), cobalamin (B12) (20 μg/kg), and choline (750 mg/kg) as a prophylactic treatment 7 days prior to intravitreal homocysteine injection. We used a supraphysiological dose of homocysteine (500 μM) that causes spontaneous death of ~10% of retinal ganglion cell death after 7 days in untreated animals. Pre‐treated animals were completely protected from retinal ganglion cell death. Next, we pre‐treated rats in the same way for 7 days prior to induction of our glaucoma model. In untreated rats there was a ~ 40% loss of retinal ganglion cells after 14 days, and this was significantly mitigated in pre‐treated rats (only ~25% loss), demonstrating that these cofactors can provide moderate neuroprotection in a complex, multifactorial neurodegenerative context. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]